Nanobody vaccine compositions

Abstract

Disclosed herein are protein-based compositions and conjugates thereof that can take advantage of beneficial delivery properties to improve vaccine efficacy. An example protein-based composition includes an albumin-binding nanobody and a peptide antigen domain. An example conjugate includes the protein-based composition attached to a drug through a linker. Also disclosed are methods of making and using the protein-based compositions and conjugates thereof.

Description

Attorney Docket No.093386-0008-WO02 NANOBODY VACCINE COMPOSITIONS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 589,838 filed on October 12, 2023, U.S. Provisional Patent Application No.63 / 678,924 filed on August 2, 2024, and U.S. Provisional Patent Application No.63 / 678,932 filed on August 2, 2024, each of which are incorporated fully herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with Government support under Federal Grant no. 1R01CA266767-01A1 awarded by the National Institutes of Health. The Federal Government has certain rights to this invention. TECHNICAL FIELD

[0003] This disclosure relates to nanobody vaccine compositions and their use in biomedical applications, such as immune therapy. INTRODUCTION

[0004] Improved delivery of vaccine compositions can be useful in treating many diseases, including autoimmune diseases and cancer. In addition, designing vaccine compositions that can effectively modulate the innate immunity in subjects with these diseases can improve therapeutic results. SUMMARY

[0005] In one aspect, disclosed are protein-based compositions comprising: a nanobody domain, the nanobody domain comprising an albumin-binding nanobody, wherein the albumin- binding nanobody is capable of specifically binding albumin; and a peptide antigen domain.

[0006] In another aspect, disclosed are conjugates, or a pharmaceutically acceptable salt thereof, comprising: the protein-based composition as disclosed herein; a drug; a second linker attaching the peptide antigen domain at its C-terminal end to the drug.Attorney Docket No.093386-0008-WO02

[0007] In another aspect, disclosed are pharmaceutical compositions comprising: a protein- based composition as disclosed herein or a conjugate as disclosed herein; and a pharmaceutically acceptable excipient.

[0008] In another aspect, disclosed are methods of treating a disease or a disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the protein-based composition as disclosed herein, optionally in combination with a pharmaceutically acceptable excipient.

[0009] In another aspect, disclosed are methods of treating a disease or a disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the conjugate as disclosed herein, optionally in combination with a pharmaceutically acceptable excipient.

[0010] In another aspect, disclosed are methods of modulating an immune system of a subject in need thereof, the method comprising administering to the subject an effective amount of the protein-based composition as disclosed herein or the conjugate as disclosed herein, optionally in combination with a pharmaceutically acceptable excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0012] FIG.1 is a scheme depicting the series of sortase and strain-promoted azide-alkyne cycloaddition (SPAAC) reactions used to label the C-terminus of a protein-based composition with small molecules, including Cy5, diABZI, dexamethasone, and / or synthetic peptide antigens.

[0013] FIG.2 shows electrospray ionization mass spectrometry (ESI-MS) spectra demonstrating nanobody conjugate purity and molecular weight.

[0014] FIG.3 shows a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) gel demonstrating nanobody conjugate purity and molecular weight.

[0015] FIG.4 shows ESI-MS spectra demonstrating nanobody-antigen fusion conjugate purity and molecular weight.

[0016] FIG.5 shows ESI-MS spectra demonstrating nanobody-antigen fusion purity and molecular weight.Attorney Docket No.093386-0008-WO02

[0017] FIG.6 shows ESI-MS spectra demonstrating nanobody-antigen chemically conjugated purity and molecular weight when the first reactive group contains a bicyclononyne and the second reactive group contains an azide.

[0018] FIG.7 shows an SDS-PAGE gel demonstrating nanobody-Cy5 conjugate purity and molecular weight when the primary ligand contains a bicyclononyne and the secondary ligand contains an azide.

[0019] FIG.8 shows an SDS-PAGE gel demonstrating nanobody-antigen chemically conjugated purity and molecular weight.

[0020] FIG.9 graphically depicts the serum half-life of indicated nanobody-Cy5 conjugates following a 2 nmol injection of nanobody-Cy5 administered subcutaneously (S.C.) at the left base-of-tail in healthy C57BL / 6 mice (n = 7). Curves were fit to these data using the model ^ = ^ି^^כ^) C was the initial concentration of Cy5 in plasma, k is the0 aabsorption constant, and kcis the elimination constant.

[0021] FIG.10 shows bar graphs quantifying the average radiant efficiency per cm2of tissue of excised organs 24 h following S.C. administration of 2 nmol nGFP-Cy5 or nAlb-Cy5 in healthy C57BL / 6 mice (n = 7) as measured using an in vivo imaging system (IVIS). P values were determined by Student’s two-tailed t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0022] FIG.11 shows representative IVIS images of excised draining inguinal lymph nodes (iLNs) 24 h after SC injection of 2 nmol nGFP-Cy5 or nAlb-Cy5 in healthy C57BL / 6 mice (n = 7).

[0023] FIG.12 shows flow cytometric analysis of nanobody-Cy5 uptake by major immune cell populations in several lymphoid organs including the spleen, draining inguinal lymph node (iLN), or irrelevant axillary lymph node (aLN). The percentage of Cy5+cells within each cell type was plotted and analyzed via Student’s two-tailed t-test between the two treatment groups; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0024] FIG.13 is a schematic depicting the treatment timeline for prophylactic, tolerogenic nAlb-MOG35-55vaccination and subsequent disease challenge using an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS).

[0025] FIG.14 shows the clinical experimental autoimmune encephalomyelitis (EAE) disease severity scoring as a function of time for female C57BL / 6 mice prophylactically treated with vehicle (PBS), nAlb-MOG35-55, or an equivalent dose of free MOG35-55peptide (n = 10). P- values were determined by one-way ANOVA with post-hoc Tukey’s correction for multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0026] FIG.15 depicts several bar graphs of the cumulative clinical experimental autoimmune encephalomyelitis (EAE) disease severity scores for each individual mouse overAttorney Docket No.093386-0008-WO02 the course of the 28-day study following treatment with vehicle (PBS), nAlb-MOG35-55, or an equivalent dose of free MOG35-55peptide (n = 10). P-values were determined by one-way ANOVA with post-hoc Tukey’s correction for multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0027] FIG.16 is a schematic depicting the treatment timeline for activating nanobody- antigen-diABZI conjugate vaccination and subsequent analysis of circulating peripheral blood mononuclear cells (PBMCs) and splenocytes.

[0028] FIG.17 depicts several bar graphs of the H-2Kb / SIINFEKL-Pe Tetramer+populations on Day 14 as a percentage of CD8+T cells within the peripheral blood mononuclear cells (PBMCs) following treatment with vehicle (PBS), a full nAlb-OVA251-270-diABZI nanobody- antigen-drug conjugate, a mixture of the nAlb-OVA251-270antigen fusion and the nAlb-diABZI conjugate, a mixture of the nAlb-OVA251-270antigen fusion and free diABZI, and a mixture of a synthetic long peptide (SLP) variant of the SIINFEKL (SEQ ID NO:110) antigen (OVA251-264) and free diABZI (n = 8). P-values were determined by one-way ANOVA with post-hoc Tukey’s correction for multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0029] FIG.18 depicts several bar graphs of the H-2Kb / SIINFEKL-Tetramer+populations on Day 21 as a percentage of CD8+T cells within the spleen following treatment with vehicle (PBS), a full nAlb-OVA251-270-diABZI nanobody-antigen-drug conjugate, a mixture of the nAlb- OVA251-270antigen fusion and the nAlb-diABZI conjugate, a mixture of the nAlb-OVA251-270antigen fusion and free diABZI, and a mixture of a synthetic long peptide (SLP) variant of the SIINFEKL (SEQ ID NO:110) antigen (OVA251-264) and free diABZI (n = 8). P-values were determined by one-way ANOVA with post-hoc Tukey’s correction for multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0030] FIG.19 depicts several bar graphs of the PD-1+CD8+double-positive populations on Day 21 as a percentage of Tcr-ȕ+cells within the spleen following treatment with vehicle (PBS), a full nAlb-OVA251-270-diABZI nanobody-antigen-drug conjugate, a mixture of the nAlb-OVA251-270antigen fusion and the nAlb-diABZI conjugate, a mixture of the nAlb-OVA251-270antigen fusion and free diABZI, and a mixture of a synthetic long peptide (SLP) variant of the SIINFEKL (SEQ ID NO:110) antigen (OVA251-264) and free diABZI (n = 8). P-values were determined by one-way ANOVA with post-hoc Tukey’s correction for multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001, 0.0001.

[0031] FIG.20 depicts several bar graphs of the central memory (TCM), CD62L+CD44+double-positive populations (top), and of the effector memory (TEM), CD62L-CD44+single- positive populations (bottom), populations on Day 21 as a percentage of Tetramer+cells withinAttorney Docket No.093386-0008-WO02 the spleen following treatment with a full nAlb-OVA251-270-diABZI nanobody-antigen-drug conjugate, a mixture of the nAlb-OVA251-270antigen fusion and the nAlb-diABZI conjugate, a mixture of the nAlb-OVA251-270antigen fusion and free diABZI, and a mixture of a synthetic long peptide (SLP) variant of the SIINFEKL (SEQ ID NO:110) antigen (OVA251-264) and free diABZI (n = 8). P-values were determined by one-way ANOVA with post-hoc Tukey’s correction for multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0032] FIG.21 is a schematic depicting the treatment timeline for prophylactic vaccination S.C. at the left base-of-tail with nanobody-antigen-diABZI conjugates and subsequent challenge with B16.F10-OVA murine melanoma.

[0033] FIG.22 depicts several bar graphs of the CD8+H-2Kb / SIINFEKL-Tetramer+double- positive cells as a percentage of CD3İ+cells within the peripheral blood mononuclear cells (PBMCs) on Day 14 (left) and Day 21 (right) following treatment with vehicle (PBS), an nAlb- OVA251-270nanobody-antigen fusion alone, a mixture of the nAlb-OVA251-270antigen fusion and the nAlb-diABZI conjugate, and an equimolar dose of full ovalbumin (OVA) mixed with free diABZI (n = 5). P-values were determined by one-way ANOVA with post-hoc Tukey’s correction for multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0034] FIG.23 shows tumor growth curves for mice treated with indicated formulations plotted from the day of tumor inoculation and until the death of the first mouse in each group. Statistical analyses were performed on Day 17, following the death of the first mouse in any group. P-values were determined by one-way ANOVA with post-hoc Tukey’s correction for multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0035] FIG.24 shows the Kaplan-Meier survival curves for mice with B16.F10-OVA tumors prophylactically treated with indicated vaccine formulations. P-values were determined via Mantel-Cox log-rank test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0036] FIG.25 is a schematic depicting the treatment timeline for C57BL / 6 mice therapeutically vaccinated S.C. at the left base-of-tail with nanobody-antigen-diABZI conjugates once average B16.F10-OVA melanoma tumor volumes reached ~50-75 mm3.

[0037] FIG.26 shows the tumor growth curves for mice treated with indicated formulations plotted from the day of first treatment until the death of the first mouse in each group. Statistical analyses were performed on Day 8, following the death of the first mouse in any group. P- values were determined by one-way ANOVA with post-hoc Tukey’s correction for multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.Attorney Docket No.093386-0008-WO02

[0038] FIG.27 shows the Kaplan-Meier survival curves for mice with B16.F10-OVA tumors therapeutically treated with indicated vaccine formulations. P-values were determined via Mantel-Cox log-rank test; *P < 0.05, **P < 0.01, ***P < 0.001,< 0.0001.

[0039] FIG.28 is a schematic of an nAlb-nPD-L1-OVA251-270protein fusion with a C-terminal eSrtA conjugation motif for the generation of a nAlb-nPD-L1-OVA251-270-diABZI protein-based composition immune checkpoint targeted cancer vaccine.

[0040] FIG.29 is an image of an SDS-PAGE gel demonstrating bispecific nanobody-antigen- diABZI conjugate molecular weight and purity.

[0041] FIG.30 is an ESI-MS spectra demonstrating nAlb-nPD-L1-OVA251-270molecular weight and purity.

[0042] FIG.31 is a schematic depicting the treatment timeline for C57BL / 6 mice therapeutically vaccinated S.C. at the left base-of-tail with bispecific nanobody-antigen-diABZI conjugates sometimes with concurrent intraperitoneal (I.P.) administration of anti-PD-L1 (aPD- L1) immune checkpoint blockade (ICB) once average B16.F10-OVA melanoma tumor volumes reached ~50-75 mm3.

[0043] FIG.32 shows the tumor growth curves for mice treated with indicated formulations plotted from the day of first treatment until the death of the first mouse in each group. Statistical analyses were performed on Day 10, following the death of the first mouse in any group. P- values were determined by one-way ANOVA with post-hoc Tukey’s correction for multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0044] FIG.33 shows the Kaplan-Meier survival curves for mice with B16.F10-OVA tumors therapeutically treated with indicated vaccine formulations. P-values were determined via Mantel-Cox log-rank test; *P < 0.05,< 0.01,< 0.001, ****P < 0.0001. DETAILED DESCRIPTION 1. Definitions

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. Methods and materials similar or equivalent to those described herein can be used in practice or testing of the disclosed technology. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.Attorney Docket No.093386-0008-WO02

[0046] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0047] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1. Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.

[0048] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are contemplated, and for the range 1.5-2, the numbers 1.5, 1.6, 1.7, 1.8, 1.9, and 2 are contemplated.

[0049] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0050] “Amino acid” as used herein refers to naturally occurring and non-natural synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a mannerAttorney Docket No.093386-0008-WO02 similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code. Amino acids can be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Amino acids include the side chain and polypeptide backbone portions.

[0051] The term “C-terminal end,” as used herein, refers to a fragment of a polypeptide that begins at any amino acid in the C-terminal half of the polypeptide and ends at the last amino acid of the polypeptide. The term “C-terminus,” as used herein, refers to the last amino acid of a polypeptide.

[0052] The term “drug” refers to a substance that can act on a cell, virus, tissue, organ, organism, or the like, to create a change in the functioning of the cell, virus, tissue, organ, or organism. Examples of drugs include, but are not limited to, chemotherapeutics, anti- inflammatory drugs, and immunomodulating drugs. A drug is capable of treating and / or ameliorating a condition or disease, or one or more symptoms thereof, in a subject. Drugs of the present disclosure also include prodrug forms of the agent.

[0053] The term “effective dosage” or “therapeutic dosage” or “therapeutically effective amount” or “effective amount,” as used herein, refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results, to modulate a biological process, and / or treat a disease or one or more of its symptoms and / or to prevent or reduce the risk of the occurrence or reoccurrence of the disease or disorder or symptom(s) thereof. A therapeutically effective amount is also one in which any toxic or detrimental effects of substance are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. In reference to treating a disease or disorder an effective or therapeutically effective amount can include an amount sufficient to, among other things, improve survival from a disease, such as cancer, and improving conditions associated with an autoimmune disease, such as multiple sclerosis.

[0054] The term “expression vector,” as used herein indicates a plasmid, a virus or another medium, known in the art, into which a nucleic acid sequence for encoding a desired protein can be inserted or introduced.

[0055] The term “fusion,” as used herein refers to a single protein or polypeptide that is produced by joining two or more originally separate genes into a single gene.Attorney Docket No.093386-0008-WO02

[0056] The term “heterologous” as used herein refers to nucleic acid comprising two or more subsequences that are not found in the same relationship to each other in nature. For instance, a nucleic acid that is recombinantly produced typically has two or more sequences from unrelated genes synthetically arranged to make a new functional nucleic acid, for example, a promoter from one source and a coding region from another source. The two nucleic acids are thus heterologous to each other in this context. When added to a cell, the recombinant nucleic acids would also be heterologous to the endogenous genes of the cell. Thus, in a chromosome, a heterologous nucleic acid would include a non-native (non-naturally occurring) nucleic acid that has integrated into the chromosome, or a non-native (non-naturally occurring) extrachromosomal nucleic acid. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (for example, a “fusion protein,” where the two subsequences are encoded by a single nucleic acid sequence).

[0057] The term “host cell,” as used herein is a cell that is susceptible to transformation, transfection, transduction, conjugation, and the like with a nucleic acid construct or expression vector. Host cells can be derived from plants, bacteria, yeast, fungi, insects, animals, etc. In some embodiments, the host cell includes Escherichia coli.

[0058] The terms “identity,” “identical,” “percent identity,” and / or “percent identical,” as used herein as applicable to one or more particular polynucleotide or amino acid sequences, refer to the proportion of identical residues between a particular reference sequence and another sequence, as calculated by a pairwise alignment using the Needleman-Wunsch algorithm using a generally available alignment program, e.g., the Needle (EMBOSS) program. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of the single sequence are included in the denominator but not the numerator for the purposes of calculating identity. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent.

[0059] The term “nanobody,” as used herein, refers to an antibody fragment including a single monomeric variable antibody domain, such as a single variable domain of a heavy chain. Nanobodies typically have molecular weights of between 12 kDa and 15 kDa and can include peptide chains of from 90 to 120 amino acids. Nanobodies can be obtained from heavy-chain antibodies found in camelid species and from cartilaginous fish species (e.g., sharks). Nanobodies can also be obtained from variable domains of common IgG derived from humans or mice. It should be understood that while most nanobodies are derived from heavy-chainAttorney Docket No.093386-0008-WO02 variable domains of antibodies, nanobodies can also be derived from light-chain antibody domains.

[0060] To create, e.g., a camelid nanobody immune library, camelids can be immunized against a molecule of interest (albumin, checkpoint immune ligand, etc.). mRNA of the camelids’ peripheral blood mononuclear cells can then be converted into cDNA. PCR can then be employed to amplify the VHH genes. These immune VHH genes can then be cloned into a phage display vector. Phages can then be generated using E. coli strains such as TG1. Phage libraries can then be panned against immobilized antigens to select for nanobodies that selectively bind the antigen with high affinity. The panned libraries can be used for reinfection of E. coli to obtain specific clones. Further description of nanobodies can be found in S. Muyldermans, A guide to: generation and design of nanobodies, FEBS J.2021 Apr; 288(7): 2084–2102, which is incorporated by reference herein in its entirety.

[0061] The terms “nucleic acid,” “oligonucleotide” or “polynucleotide” as used herein refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form and complements thereof. In some embodiments, the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Any combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine are expressly contemplated herein.

[0062] The term “N-terminal end,” as used herein, refers to a fragment of a polypeptide that begins at the first amino acid of the polypeptide and ends at any amino acid in the N-terminal half of the polypeptide. The term “N-terminus,” as used herein, refers to the first amino acid of a polypeptide.

[0063] A “peptide” or “polypeptide,” as used herein, refers to a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The terms “polypeptide”, “protein,” and “peptide” are used interchangeably herein. “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains,Attorney Docket No.093386-0008-WO02 for example, enzymatic domains, extracellular domains, transmembrane domains, nanobody domains, and peptide antigen domains. “Domains” are portions of a polypeptide that form a compact unit of the polypeptide and can be 8 to 1,500 amino acids in length, such as 8 to 500 amino acids in length or 8 to 50 amino acids in length. Example domains include domains with enzymatic activity or ligand binding activity. Typical domains can be made up of sections of lesser organization such as stretches of beta-sheet and alpha-helices. “Tertiary structure” refers to the complete three-dimensional structure of a polypeptide. “Quaternary structure” refers to the three-dimensional structure formed by the noncovalent association of independent tertiary units. A “motif” is a portion of a polypeptide sequence and includes at least two amino acids. A motif may be 2 to 20, 2 to 15, or 2 to 10 amino acids in length. In some embodiments, a motif includes 3, 4, 5, 6, or 7 sequential amino acids. A domain may be comprised of a series of the same type of motif or of different motifs.

[0064] “Recombinant” when used with reference to, e.g., a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed, or not expressed at all.

[0065] “Sortase” refers to an enzyme that recognizes a sortase recognition site in a protein and cleaves a peptide bond therein, forming a stable intermediate that joins the catalytic thiol of sortase to the carboxyl group of an amino acid within the recognition site via a thioester bond. An example sortase is Sortase A (SrtA).

[0066] As used herein, the term “specifically binds” is generally meant that a molecule (e.g., a nanobody, a protein-based composition thereof, or a conjugate thereof) binds to a target molecule when it binds to that target molecule more readily than it would bind to a random, unrelated target. For example, nanobodies disclosed herein can specifically bind to a target molecule with nanomolar affinity. “Specific binding” does not necessarily require (although it can include) exclusive binding to a target molecule or epitope thereof.

[0067] “Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal that wants or is in need of the herein described protein- based compositions, conjugates thereof, or methods. The subject may be a human or a non- human. The subject may be a vertebrate. The subject may be a mammal. The mammal may be a primate or a non-primate. The mammal can be a non-primate such as, for example, cow, pig,Attorney Docket No.093386-0008-WO02 camel, llama, hedgehog, anteater, platypus, elephant, alpaca, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse. The mammal can be a primate such as a human. The mammal can be a non-human primate such as, for example, monkey, cynomolgous monkey, rhesus monkey, chimpanzee, gorilla, orangutan, and gibbon. The subject may be of any age or stage of development, such as, for example, an adult, an adolescent, or an infant. The subject may be male. The subject may be female. In some embodiments, the subject has a specific genetic marker. The subject may be undergoing other forms of treatment.

[0068] “Substantially identical” can mean that a first and second amino acid or polynucleotide sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 1100 amino acids or nucleotides, respectively.

[0069] “T cells” are a type of white blood cell of the immune system and play a central role in the adaptive immune response. T cells express a T-cell receptor (TCR) on their cell surface. The T cell receptor (TCR) of a T cell is able to interact with immunogenic peptides (epitopes) bound to major histocompatibility complex (MHC) molecules and presented on the surface of target cells. Specific binding of the TCR triggers a signal cascade inside the T cell leading to proliferation and differentiation into a maturated effector T cell. T cells may differentiate into different types of T cells. T cells may include, for example, CD8+ T cells (“killer T cells” or “cytotoxic T cells) and CD4+ T cells (“helper T cells”). CD8+ T cells and CD4+ T cells may further differentiate into other types of T cells including, for example, regulatory T cells (“suppressor T cells”) and memory T cells.

[0070] In some embodiments, the T cell is a memory T cell. An antigen-naïve T cell expands and differentiates into a memory T cell after encountering the cognate antigen within the context of a major histocompatibility complex (MHC) molecule on the surface of an antigen presenting cell. Memory T cells may be CD8+ or CD4+. Memory T cells are long-lived and can quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen. Tissue-resident memory T cells (TRMcells) are a subset of a long-lived memory T cells that occupy epithelial, mucosal, and other tissues such as skin, mucosa, lung, brain, pancreas, and gastrointestinal tract, without recirculating. TRMcells may be transcriptionally, phenotypically, and functionally different from central memory (TCM) and effector memory (TEM) T cells that recirculate between blood, the T cell zones of secondary lymphoid organ, lymph tissues, and nonlymphoid tissues. TRMcells can develop from circulating effector memory T cell precursorsAttorney Docket No.093386-0008-WO02 in response to an antigen. TRMcells may be CD103+. TRMcells may provide superior protection against infection in extralymphoid tissues. A T cell detailed herein may be a TRMcell.

[0071] The terms “treatment” or “treating” refer to the medical management of a patient with the intent to heal, cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0072] “Vector” as used herein means a nucleic acid sequence containing an origin of replication. A vector may be capable of directing the delivery or transfer of a polynucleotide sequence to target cells, where it can be replicated or expressed. A vector may contain an origin of replication, one or more regulatory elements, and / or one or more coding sequences. A vector may be a viral vector, bacteriophage, bacterial artificial chromosome, plasmid, cosmid, or yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be a self- replicating extrachromosomal vector. Viral vectors include, but are not limited to, adenovirus vector, adeno-associated virus (AAV) vector, retrovirus vector, or lentivirus vector. A vector may be an adeno-associated virus (AAV) vector. The vector may encode a recombinant protein.

[0073] For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. 2. Protein-Based Compositions

[0074] Provided herein are protein-based compositions that can advantageously bind albumin. The protein-based composition includes a nanobody domain and a peptide antigen domain. The nanobody domain can include a nanobody that is capable of specifically binding albumin, where this nanobody can be referred to as an albumin-binding nanobody. The ability to bind albumin can provide advantageous benefits to the protein-based composition andAttorney Docket No.093386-0008-WO02 conjugate thereof including, but not limited to, improved pharmacokinetics and pharmacodynamics. For example, by being able to bind albumin, the peptide antigen domain of the protein-based composition can localize to specific biological locations, such as lymph nodes, which can be beneficial for immunity / vaccine applications. The nanobody domain can also include a second nanobody that is capable of specifically binding to an immune checkpoint ligand and / or an antigen presenting cell ligand. The inclusion of the second nanobody can further increase the efficacy and / or potency of the protein-based composition and conjugate thereof in e.g., immune therapy applications.

[0075] The protein-based composition can include the nanobody domain and the peptide antigen domain arranged in different ways. For example, the nanobody domain can be positioned N-terminal to the peptide antigen domain. In some embodiments, the protein-based composition includes, in a N-terminus to a C-terminus direction, the albumin-binding nanobody and the peptide antigen domain. In embodiments that include a second nanobody, the albumin- binding nanobody can be N-terminal to the second nanobody, and the peptide antigen domain can be C-terminal to the second nanobody. In other words, the second nanobody can be in between the albumin-binding nanobody and the peptide antigen domain. In some embodiments, the protein-based composition includes, in a N-terminus to a C-terminus direction, the albumin- binding nanobody, the second nanobody, and the peptide antigen domain.

[0076] The nanobody domain can be attached (e.g., covalently) to the peptide antigen domain through a first linker. The nanobody domain can be attached at its C-terminal end to the N-terminal end of the peptide antigen domain through the first linker. The first linker can include a peptide linker, a sortase moiety, or a combination thereof. The first linker can also be one that is formed from biorthogonal chemistry. In some embodiments, the peptide antigen domain is flanked on either side by a first linker, such as a peptide linker.

[0077] The peptide linker can provide flexibility between the different domains. The peptide linker can also be cleavable. For example, the peptide linker can be cleavable by cathepsins and / or proteasomes. Example peptide linkers include, but are not limited to, GGGS (SEQ ID NO:99), SGSETPGTSESA (SEQ ID NO:100), SLVR (SEQ ID NO:101), SLVRYLL (SEQ ID NO:102), valine–citrulline linkers, and alanine-based linkers (e.g., dialanine AA). In some embodiments, the peptide linker is 2 to 30 amino acids in length, such as 2 to 25, 3 to 30, 4 to 25, or 2 to 20.

[0078] The sortase moiety can include a sortase recognition site. For example, the sortase moiety can include an amino acid sequence of LPXT (SEQ ID NO:1) (e.g., a sortase recognition site), wherein X is any amino acid. In some embodiments, the sortase moiety includes an aminoAttorney Docket No.093386-0008-WO02 acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some embodiments, the sortase moiety and / or amino acid sequence thereof is described as part of a sequence encoding a nanobody. The sortase moiety may also include a hydrophilic moiety, such as polyethylene glycol as described herein.

[0079] How the protein-based composition is made can dictate the type of linker used to attach the nanobody domain to the peptide antigen domain. For example, in some embodiments, the nanobody domain and the peptide antigen domain are made as a fusion protein. In these embodiments, the nanobody domain and the peptide antigen domain can be expressed within the same protein. Thus, in these embodiments, the nanobody domain can be attached to the peptide antigen domain by a first linker that includes a peptide linker as described herein. In some embodiments, the albumin-binding nanobody is attached to the peptide antigen domain by a first linker that includes a peptide linker as described herein.

[0080] Alternatively, the nanobody domain and the peptide antigen domain can be attached by chemical methods. For example, the nanobody domain or nanobody therein can be designed to include a sortase recognition site. An example sortase recognition site is LPXT (SEQ ID NO:1), wherein X is any amino acid. In some embodiments, the sortase recognition site includes an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The sortase recognition site can be at the C-terminus of the nanobody domain and can allow for site-specific conjugation of a primary amine reagent via sortase. The result of the reaction can be referred to as a reactive nanobody reagent.

[0081] The primary amine reagent can include a first reactive group. In some embodiments, the reactive amine reagent is of formula (I): (I), wherein: X1is the first reactive group (e.g., BCN) and n’ is 2 to 20. In some embodiments, n’ is 2 to 4, 2 to 6, 2 to 10, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, or 10 to 20. In some embodiments, n’ is greater than 2, greater than 3, greater than 4, greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, or greater than 10.

[0082] In some embodiments, the reactive nanobody reagent is of formula (II):Attorney Docket No.093386-0008-WO02 wherein: X1is the first reactive group, X is any amino acid, and n’ is 2 to 20. The description for n’ of the reactive amine reagent can also be applied to the reactive nanobody reagent.

[0083] After the nanobody domain is conjugated to the amine reagent the resultant reactive nanobody reagent can be coupled to a reactive peptide antigen reagent. The reactive peptide antigen reagent can include a peptide antigen domain and a second reactive group (e.g., azide). The reaction between the reactive nanobody reagent and the reactive peptide antigen reagent can form a protein-based composition, the protein-based composition including the nanobody domain attached to the peptide antigen domain by a first linker, the first linker comprising a sortase moiety. Thus, when using chemical methods to attach the peptide antigen domain to the nanobody domain, the first linker can include a sortase moiety as described herein.

[0084] Further description of a sortase mediated conjugation can be found below under the synthesis of the conjugates section, as well as International Patent Application No. PCT / US2023 / 079885, which is fully incorporated by reference herein in its entirety. For example, the description below regarding the reactive groups can be applied to the methods of making the protein-based composition.

[0085] Being able to conjugate a peptide antigen domain to the nanobody domain via, e.g., bioorthogonal chemistry, can allow for quick production of protein-based compositions and provide flexibility of being able to quickly interchange many different peptide antigens as needed. This could be especially useful in personalized vaccines, where peptide antigens will / could be different for different subjects and trying to create fusions of each different peptide antigen could be difficult and time consuming.

[0086] In some embodiments, the protein-based composition includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO:84 to SEQ ID NO:87, SEQ ID NO:89 to SEQ ID NO:91, SEQ ID NO:93, and SEQ ID NO:109. A. Nanobody Domains

[0087] The nanobody domain includes at least one nanobody. In some embodiments, the nanobody domain includes a first nanobody and a second nanobody. The first nanobody and the second nanobody can specifically bind to different target molecules. A nanobody domain including a first nanobody and a second nanobody can be considered bivalent, and as a result can instill bivalency to the protein-based composition. Bivalency of the protein-based composition can aid in overall targeting of the protein-based composition and can aid in inhibition of signaling pathways associated with a target molecule.

[0088] The first nanobody can be an albumin-binding nanobody. The albumin-binding nanobody is capable of specifically binding albumin, and thus can instill in the protein-basedAttorney Docket No.093386-0008-WO02 composition the ability to specifically bind albumin. The albumin-binding nanobody can have a binding affinity (Kd) to albumin of less than or equal to 100 nM at a pH of about 7 to about 8, such as less than or equal to 75 nM, less than or equal to 50 nM, less than or equal to 25 nM, less than or equal to 10 nM, or less than or equal to 1 nM at a pH of about 7 to about 8. In some embodiments, the albumin-binding nanobody has a Kdto albumin of greater than or equal to 0.1 nM, greater than or equal to 0.2 nM, greater than or equal to 0.4 nM, greater than or equal to 0.5 nM, greater than or equal to 0.6 nM, greater than or equal to 0.7 nM, greater than or equal 0.8 nM, greater than or equal to 0.9 nM, or greater than or equal to 1 nM at a pH of about 7 to about 8. In some embodiments, the albumin-binding nanobody has a Kdto albumin of about 0.1 nM to about 100 nM, such as about 0.2 nM to about 90 nM, about 1 nM to about 100 nM, or about 0.1 nM to about 50 nM. In some embodiments, the albumin-binding nanobody does not covalently bind to albumin. Binding affinity of nanobodies can be measured via techniques known within the art, such as isothermal calorimetry (ITC).

[0089] The albumin-binding nanobody can include any nanobody suitable for specifically binding albumin as described herein. In some embodiments, the albumin-binding nanobody includes an amino acid sequence selected from the group consisting of SEQ ID NO:5 to SEQ ID NO:81. In some embodiments, the albumin-binding nanobody includes an amino acid sequence of SEQ ID NO:80.

[0090] The second nanobody can specifically bind to an immune checkpoint ligand, an antigen presenting cell ligand, or both. In some embodiments, the second nanobody is capable of specifically binding to an immune checkpoint ligand or an antigen presenting cell ligand. In some embodiments, the immune checkpoint ligand and the antigen presenting cell ligand are the same ligand. For example, MMR can be both an immune checkpoint ligand and an antigen presenting cell ligand.

[0091] In some embodiments, the second nanobody is capable of specifically binding to an immune checkpoint ligand. An “immune checkpoint ligand” refers to ligand-receptor pairs expressed on immune cells that inhibit or stimulate the immune response. The immune checkpoint ligand can be present on a cell surface. Example immune checkpoint ligands include, but are not limited to, CTLA-4, PD-1, PD-L1, B7-H3, B7-H4, HVEM, GITRL, CD80 / 86, CD155, PD-L2, Galectin 9, LAG3, TIM3, VISTA, TIGIT, PD1, MMR, and GITR. In some embodiments, the immune checkpoint ligand is CTLA-4, PD-1, PD-L1, B7-H3, or B7-H4. In some embodiments, the immune checkpoint ligand is PD-1, PD-L1, B7-H3, or B7-H4. In some embodiments, the immune checkpoint ligand is PD-L1 or B7-H3. In some embodiments, theAttorney Docket No.093386-0008-WO02 immune checkpoint ligand is PD-L1. The second nanobody can have a Kdto an immune checkpoint ligand as described above for the albumin-binding nanobody to albumin.

[0092] In some embodiments, the second nanobody is capable of specifically binding to an antigen presenting cell ligand. An “antigen presenting cell” is an immune cell that is capable of processing and presenting antigens for recognition by T cells to initiate the adaptive cellular immune response, classically considered dendritic cells, macrophages, and B cells. An “antigen presenting cell ligand” refers to a ligand that an antigen presenting cell can include. The antigen presenting cell ligand can be present on a surface of the antigen presenting cell. Example antigen presenting cell ligands include, but are not limited to, PD-L1, PD-L2, B7-H3, B7-H4, MMR, MHC-II, CD11b, CD11c, C-type lectin receptors, Fc receptors, complement receptors (e.g., CR1, CR3, CR4), scavenger receptors (e.g., SR-A, CD36), PS receptors (e.g., MerTK, TIM-4), B cell receptor, folate receptor, CD40, CD80, CD86, TLR2, TLR4, and TLR5. In some embodiments, the antigen presenting cell ligand is MMR, CD11b, or CD11c. The second nanobody can have a Kdto an antigen presenting cell ligand as described above for the albumin-binding nanobody to albumin.

[0093] In some embodiments, the second nanobody includes an amino acid sequence selected from the group consisting of SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:106, SEQ ID NO:107, and SEQ ID NO:108. In some embodiments, the second nanobody includes an amino acid sequence of SEQ ID NO:94.

[0094] The first nanobody can be attached to the second nanobody through a peptide linker. The peptide linker attaching the first and the second nanobody can be any of the peptide linkers described for the first linker (e.g., between the nanobody domain and the peptide antigen domain). In some embodiments, the peptide antigen domain is flanked on either side by a peptide linker. For the purpose of brevity, the description of the peptide linkers of the first linker will not be repeated here.

[0095] In some embodiments, the nanobody domain includes an albumin-binding nanobody that is capable of specifically binding to albumin and a second nanobody that is capable of specifically binding to an immune checkpoint ligand. In some embodiments, the nanobody domain includes an albumin-binding nanobody including an amino acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 81, and a second nanobody including an amino acid sequence selected from the group consisting of SEQ ID NO:94 and SEQ ID NO:95. In some embodiments, the nanobody domain includes an amino acid sequence selected from the group consisting of SEQ ID NO:80, SEQ ID NO: 94, and a combination thereof.Attorney Docket No.093386-0008-WO02

[0096] In some embodiments, the nanobody domain includes an albumin-binding nanobody that is capable of specifically binding to albumin and a second nanobody that is capable of specifically binding to an antigen presenting cell ligand. In some embodiments, the nanobody domain includes an albumin-binding nanobody including an amino acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 81, and a second nanobody including an amino acid sequence selected from the group consisting of SEQ ID NO:106 to SEQ ID NO:108. In some embodiments, the nanobody domain includes an amino acid sequence selected from the group consisting of SEQ ID NO:80, SEQ ID NO:106 to SEQ ID NO:108, and a combination thereof.

[0097] In some embodiments, the nanobody domain includes an amino acid sequence of SEQ ID NO:5 to SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:88, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:106 to SEQ ID NO:108, or a combination thereof.

[0098] The nanobodies disclosed herein can be commercially purchased or provided by recombinant expression. B. Peptide Antigen Domains

[0099] The peptide antigen domain includes a peptide antigen. A “peptide antigen” refers to a peptide capable of being bound by an antibody or a T cell receptor. The term “peptide antigen” also encompasses T-cell epitopes. A peptide antigen is additionally capable of being recognized by the immune system and / or being capable of inducing a humoral immune response and / or cellular immune response leading to the activation of B-lymphocytes and / or T-lymphocytes. In some embodiments, the peptide antigen contains or is linked to a Th cell epitope. A peptide antigen can have one or more epitopes (B-epitopes and T-epitopes). Accordingly, the peptide antigen can facilitate modulating an immune response in a subject. In some embodiments, the peptide antigen domain consists of a peptide antigen.

[0100] The peptide antigen can have a molecular weight of about 1 kiloDalton (kDa) to about 10 kDa, such as about 2 kDa to about 9 kDa, about 3 kDa to about 8 kDa, about 1 kDa to about 5 kDa, about 3 kDa to about 10 kDa, or about 5 kDa to about 10kDa. In some embodiments, the peptide antigen has a molecular weight of greater than 1 kDa, greater than 2 kDa, greater than 3 kDa, or greater than 4 kDa. In some embodiments, the peptide antigen has a molecular weight of less than 10 kDa, less than 9 kDa, less than 8 kDa, or less than 7 kDa. Molecular weight of the peptide antigen can be measured by techniques known within the art, such as, but not limited to, mass spectrometry.

[0101] The peptide antigen domain can include any suitable peptide antigen that would benefit from the improved pharmacokinetics and pharmacodynamics associated with theAttorney Docket No.093386-0008-WO02 disclosed protein-based compositions. The peptide antigen can also include any number of different immunogenic peptides that can elicit a desired immune response as described herein. In other words, the peptide antigen is sequence agnostic. Example peptide antigens include, but are not limited to, an autoantigen, a foreign antigen, a xenoantigen, an alloantigen, a tumor antigen, and combinations thereof.

[0102] An autoantigen refers to a normal, self-antigen, or a peptide derived from a self- antigen, that is recognized by the immune system to cause autoimmune disease. Examples include, but are not limited to, myelin basic protein (MBP), Type II Collagen, glutamic acid decarboxylase (GAD65), insulin, TSH receptor, and tissue transglutaminase.

[0103] A foreign antigen refers to a peptide that is not derived from the host (that is, non-self) capable of generating an immune response. Examples include, but are not limited to, viral antigens (e.g., HIV Gag, Pol, Env; influenza hemagglutinin and neuraminidase; SARS-CoV-2 spike and nucleocapsid protein), bacterial antigens (e.g., mycobacterium tuberculosis antigens such as Ag85, ESAT-6, and CFP-10), parasitic antigens (e.g., from Plasmodium falciparum such as circumsporozoite protein and merozoite surface protein-1).

[0104] A xenoantigen refers to a peptide antigen from another species that the immune system of the host recognizes as foreign. Examples include, but are not limited to, alpha- Galactosyl, porcine major histocompatibility complex, and N-glycolylneuraminic acid.

[0105] An alloantigen refers to a peptide antigen present in some subjects of the same species (e.g. humans) but not in others, leading to an immune response when cells, tissues, or organs are transferred between genetically different individuals. Examples include, but are not limited to, human leukocyte antigens, ABO blood group antigens, and paternal alloantigens expressed by the fetus during pregnancy.

[0106] A tumor antigen refers to a peptide that is expressed on the surface of tumor cells but is absent, or present at much lower levels, on normal cells. These antigens can be recognized by the immune system as abnormal or foreign, leading to an immune response against the tumor. Examples include, but are not limited to, tumor-specific antigens, such as neoantigens that are derived from mutations in the tumor, as mutations in KRAS, p53, or EGFRvIII, and tumor-associated antigens, such as Her2 / neu, prostate-specific antigen, MUC1, gp100, EGFR, MAGE-A1 / 3, and tyrosinase.

[0107] A “neoantigen” refers a novel antigen that is specific and unique to an individual patient’s tumor, due to genetic mutations that have occurred in the tumor DNA. As the tumor genome is unique to each individual patient, novel neoantigens can be identified using whole exome sequencing, advanced immunopeptidomic methods then can be employed to determineAttorney Docket No.093386-0008-WO02 which mutations are most likely to generate neoepitopes. Finally, neoantigenic peptides can be synthesized and administered to the patient as a personalized vaccine product. This approach has been tried clinically, using both peptide antigens (Hu et al., Nat Medicine, 2021, Keskin et al., Nature, 2019, as well as mRNA encoding for the neoantigen (Rojas et al., Nature, 2023) – each of which is incorporated fully herein by reference in their entirety).

[0108] Further example peptide antigens can be found in Table 1 below. Table 1: Summary Table of Peptide Antigens and HLA Associations:

[0109] In some embodiments, the peptide antigen domain includes ovalbumin, myelin oligodendrocyte glycoprotein, or patient / tumor-specific neoantigens. In some embodiments, the peptide antigen domain includes an amino acid sequence of SEQ ID NO:96 to SEQ ID NO:98. C. Example Protein-Based CompositionsAttorney Docket No.093386-0008-WO02

[0110] In some embodiments, the protein-based composition includes, in a N-terminus to a C-terminus direction: the nanobody domain, the nanobody domain comprising the albumin- binding nanobody, wherein the albumin-binding nanobody is capable of specifically binding albumin, and the second nanobody, wherein the second nanobody is capable of specifically binding to an immune checkpoint ligand; and the peptide antigen domain, the peptide antigen domain comprising a tumor antigen.

[0111] In some embodiments, the protein-based composition includes, in a N-terminus to a C-terminus direction: the nanobody domain, the nanobody domain comprising the albumin- binding nanobody, wherein the albumin-binding nanobody is capable of specifically binding albumin, and the second nanobody, wherein the second nanobody is capable of specifically binding to an antigen presenting cell ligand; and the peptide antigen domain, the peptide antigen domain comprising a tumor antigen. D. Polynucleotides

[0112] Further provided are polynucleotides encoding the protein-based compositions detailed herein, including the nanobody domain, the albumin-binding nanobody, the second nanobody, and the peptide antigen domain. A vector may include the polynucleotide encoding the protein-based composition or domains thereof detailed herein. To obtain expression of a polypeptide, one may subclone the polynucleotide encoding the polypeptide into an expression vector that contains a promoter to direct transcription, a transcription / translation terminator, and if for a nucleic acid encoding a protein, a ribosome binding site for translational initiation. An example of a vector is pet24. Suitable bacterial promoters are well known in the art. Further provided is a host cell transformed or transfected with an expression vector comprising a polynucleotide encoding the protein-based composition or domains thereof as detailed herein. Bacterial expression systems for expressing the protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Paiva et al., Gene 1983, 22, 229-235; Mosbach et al., Nature 1983, 302, 543-545 – both of which are incorporated by reference in their entirety herein). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. Retroviral expression systems can also be used. 3. Conjugates

[0113] The protein-based compositions can be included as part of a conjugate, e.g., a drug conjugate. The conjugate can include the protein-based composition as disclosed herein and a drug attached to the protein-based composition by a second linker. Because the protein-basedAttorney Docket No.093386-0008-WO02 composition is able to bind to albumin, the conjugate thereof can also specifically bind to albumin. The ability to bind albumin can provide advantageous benefits to the conjugate including, but not limited to, improved pharmacokinetics and pharmacodynamics of the attached drug.

[0114] The disclosed conjugates may exist as salts, such as pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” refers to salts or zwitterions of the conjugates which are water or oil-soluble or dispersible, suitable for administration to a subject (e.g., treatment of disorders) without undue toxicity, irritation, and allergic response, commensurate with a reasonable benefit / risk ratio and effective for their intended use. The salts may be prepared during the final isolation and purification of the conjugates or separately by reacting an amino group of the conjugates with a suitable acid. For example, the conjugate may be dissolved in a suitable solvent and treated with at least one equivalent of an acid, like hydrochloric acid. The resulting salt may precipitate out and be isolated by filtration and dried under reduced pressure. Alternatively, the solvent and excess acid may be removed under reduced pressure to provide a salt. Representative salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, glutamate, para-toluenesulfonate, undecanoate, hydrochloric, hydrobromic, sulfuric, phosphoric and the like. Amino groups of the conjugates may also be quaternized with alkyl chlorides, bromides and iodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl and the like.

[0115] Basic addition salts may be prepared during the final isolation and purification of the disclosed conjugates by reaction of a carboxyl group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation such as lithium, sodium, potassium, calcium, magnesium, or aluminum, or an organic primary, secondary, or tertiary amine. Quaternary amine salts can be prepared, such as those derived from methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine and N,N’-dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like. A. Second LinkerAttorney Docket No.093386-0008-WO02

[0116] The conjugate includes a linker attaching (e.g., covalently) the protein-based composition to the drug, which can be referred to as a second linker. The second linker can attach the protein-based composition to the drug through the peptide antigen domain, e.g., at its C-terminal to the drug. The second linker can include a sortase moiety. The sortase moiety can be attached to the peptide antigen domain. In some embodiments, the sortase moiety is located at the C-terminus of the peptide antigen domain. The sortase moiety can include a sortase recognition site. For example, the sortase moiety can include an amino acid sequence of LPXT (SEQ ID NO:1), wherein X is any amino acid. In some embodiments, the sortase moiety includes an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some embodiments, the sortase moiety and / or amino acid sequence thereof is described as part of a sequence encoding a protein-based compositions or domains thereof. The sortase moiety may also include a hydrophilic moiety, such as polyethylene glycol as disclosed herein.

[0117] The second linker can also include a hydrophilic moiety. The hydrophilic moiety can attach the sortase moiety to the drug. Due to the hydrophobicity of the drug, such as diABZI, the second linker can advantageously aid in solubilizing the drug. An example hydrophilic linker includes a hydrophilic polymer such as polyethylene glycol (PEG). Accordingly, in some embodiments, the second linker includes a PEG moiety that, e.g., attaches the sortase moiety to the drug. The PEG moiety can include a varying number of ethylene glycol repeats. For example, the PEG moiety can include 3 to 20 ethylene glycol repeats, such as 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, or 10 to 20. In some embodiments, the PEG moiety includes greater than 2 ethylene glycol repeats, greater than 3 ethylene glycol repeats, greater than 4 ethylene glycol repeats, greater than 5 ethylene glycol repeats, greater than 6 ethylene glycol repeats, greater than 7 ethylene glycol repeats, greater than 8 ethylene glycol repeats, greater than 9 ethylene glycol repeats, or greater than 10 ethylene glycol repeats.

[0118] In some embodiments, the PEG moiety is of formula (a):wherein: n is 2 to 20. In formula (a) as depicted, the drug would be attached to the right side of the PEG moiety of formula (a). Further description of how the second linker and the PEG moiety are attached can be found herein and in the Examples.

[0119] In some embodiments, n is 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, or 10 to 20. In some embodiments, n is greater than 2, greater than 3, greater than 4, greater than 5,Attorney Docket No.093386-0008-WO02 greater than 6, greater than 7, greater than 8, greater than 9, or greater than 10. In some embodiments, n is 11.

[0120] The second linker can also be one that is formed from biorthogonal chemistry without the presence of a sortase moiety. Further description on biorthogonal chemistry can be found below under the synthesis of the conjugate section. B. Drugs

[0121] Any suitable drug can be used in the disclosed conjugates. Example drugs include, but are not limited to, an immunomodulator, an agonist, an antagonist, an inhibitor, or a hormone. In some embodiments, the drug comprises an immunomodulator or a hormone. In some embodiments, the drug comprises an immunomodulator. In some embodiments, the drug is an immunomodulator.

[0122] The immunomodulator can be a STING agonist. Example STING agonists include, but are not limited to, a dimeric amidobenzimidazole (diABZI), 2’3’ cGAMP, 2’2’ cGAMP, 3’2’ cGAMP, 3’3’ cGAMP, c-di-GMP, c-di-AMP, ADU-S100, cIAMP 2-5, and ML RR-S2 CDA. In some embodiments, the STING agonist is a non-nucleotide STING agonist. In some embodiments, the STING agonist is diABZI.

[0123] In some embodiments, diABZI is:Attorney Docket No.093386-0008-WO02

[0124] In some embodiments, Rbis methyl. Further description of diABZI can be found in International Patent Application No. PCT / US2023 / 076732, which is incorporated fully herein by reference. C. Synthesis of the Conjugates

[0125] Also provided herein are methods of synthesizing the conjugates. The method follows similar steps as described for attaching the nanobody domain to the peptide antigen domain via sortase as it relates to the protein-based composition. Thus, the disclosed methods can similarly take advantage of an enzymatic conjugation that can provide site-specific conjugation of the drug. Briefly, the method can include reacting a sortase with the protein-based composition. The protein-based composition can include a peptide antigen domain having a sortase recognition site as described herein. Accordingly, sortase can react with a protein-based composition having a sortase recognition site to form a sortase-protein reagent, wherein the sortase-protein reagent can include the protein-based composition and the sortase moiety including an amino acid sequence of LPXT (SEQ ID NO:1), wherein X is any amino acid.

[0126] In some embodiments, the sortase is sortase A (SrtA) or an engineered variant thereof. SrtA may be any SrtA, such as Staphylococcus aureus SrtA. SrtA may be from a Gram-positive bacterium, such as, for example, bacteria in a genus selected from Staphylococcus, Streptococcus, Enterococcus, Bacillus, Corynebacterium, Nocardia, Clostridium, Actinobacteria, and Listeria. In some embodiments, SrtA is from S. aureus. The SrtA may be wild-type SrtA or a variant (e.g., engineered) thereof.

[0127] The method can further include reacting the sortase-protein reagent with a reactive amine reagent. The reactive amine reagent can include a first reactive group. The reaction between the sortase-protein reagent and the reactive amine reagent can form a reactive protein reagent, where the reactive protein reagent includes the protein-based composition, the sortase moiety, and a first reactive group attached to the sortase moiety.

[0128] In some embodiments, the reactive amine reagent is of formula (I):wherein: X1is the first reactive group and n’ is 2 to 20.

[0129] In some embodiments, n’ is 2 to 4, 2 to 6, 2 to 10, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, or 10 to 20. In some embodiments, n’ is greater than 2, greater than 3, greater than 4, greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, or greater than 10.Attorney Docket No.093386-0008-WO02

[0130] In some embodiments, the reactive protein reagent is of formula (III):wherein: X1is the first reactive group, X is any amino acid, and n’ is 2 to 20. The description for n’ of the reactive amine reagent can also be applied to the reactive protein reagent.

[0131] The method can also include coupling the reactive protein reagent with a reactive drug reagent. The reactive drug reagent can include a drug and a second reactive group. The reaction between the reactive protein reagent and the reactive drug reagent can form a conjugate, the conjugate including the protein-based composition attached to the drug by a linker (e.g., second linker), the linker comprising the sortase moiety.

[0132] In some embodiments, the reactive drug reagent is of formula (IV-a):wherein: A1is the drug, X2is the second reactive group, and n is 2 to 20. The description for n of the linker (e.g., second linker) herein can also be applied to the reactive drug reagent.

[0133] In some embodiments, the reactive drug reagent is of formula (IV-a) and n is 11.

[0134] The first reactive group and the second reactive group can include functional groups that are complimentary to each other in that they can form a covalent bond between the functional groups under appropriate conditions. Representative complimentary functional groups that can form a covalent bond include, but are not limited to, an amine and an activated ester, an amine and an isocyanate, an amine and an isothiocyanate, an amine and a carbonate, thiols for formation of disulfides, an aldehyde and amine for enamine formation, and an azide for formation of an amide via a Staudinger ligation. Functional groups suitable for conjugation also include bioorthogonal functional groups. Bioorthogonal functional groups can selectively react with a complementary bioorthogonal functional group through, e.g., Click-chemistry reactions. Bioorthogonal functional groups include, but are not limited to, an azide and alkyne for formation of a triazole, an azide with bicyclononyne (BCN) and DBCO (e.g., strain promoted azide-alkyne cycloaddition), trans-cyclooctene (TCO) and tetrazine (Tz) (e.g., 1,2,4,5-tetrazine), thiols with maleimide groups via Michael addition, and others.

[0135] In some embodiments, the first reactive group (e.g., X1) includes an azide, an alkyne, an alkene, a 1,2,4,5-tetrazine, BCN, or a thiol. In some embodiments, the first reactive group includes an azide or BCN.Attorney Docket No.093386-0008-WO02

[0136] In some embodiments, the second reactive group (e.g., X2) includes an alkyne, an azide, an alkene, a 1,2,4,5-tetrazine, BCN, or a thiol. In some embodiments, the second reactive group includes an alkyne or an azide. In some embodiments, the second reactive group is.

[0137] The drug (e.g., A1) can be any suitable drug as disclosed herein. In some embodiments, the drug is diABZI as disclosed herein. By using site-specific conjugation techniques disclosed herein, the conjugate can include the drug in a precise manner. For example, the conjugate can include the drug and the protein-based composition at a 1:1 molecular ratio (e.g., 1 drug molecule: 1 protein-based composition).

[0138] The description of the protein-based composition, nanobody domain, peptide antigen domain, drug, and linkers above may be applied to the disclosed methods of synthesizing the conjugate. 4. Compositions including the Protein-Based Composition and / or Conjugate Thereof

[0139] Also disclosed herein are compositions that include the protein-based composition and / or conjugate thereof and a pharmaceutically acceptable excipient, where such compositions can also be referred to as a pharmaceutical composition. The description of the protein-based composition, nanobody domain, peptide antigen domain, drug, conjugate, and linkers above can be applied to the disclosed compositions.

[0140] The term “pharmaceutically acceptable excipient,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable excipients are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, citrate buffers, and phosphate buffer solutions, as well asAttorney Docket No.093386-0008-WO02 other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The route by which the composition is administered, and the form of the composition can dictate the type of excipient to be used.

[0141] In some embodiments, the pharmaceutically acceptable excipient includes buffering agents (e.g., phosphate buffered saline), carbohydrates (e.g., glucose, trehalose, starch, etc.) solubilizers, solvents, antimicrobial preservatives, antioxidants, suspension agents, or a combination thereof. The compositions and pharmaceutical compositions can be used in the methods disclosed herein.

[0142] General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006, which is incorporated by reference herein in its entirety. Conventional excipients and accessory ingredients may be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory ingredient may be incompatible with one or more components of a protein-based composition or conjugate thereof. An excipient or accessory ingredient may be incompatible with a component of a protein-based composition or conjugate thereof if its combination with the component may result in any undesirable biological effect or otherwise deleterious effect. 5. Uses of the Protein-Based Composition and / or the Conjugate Thereof A. Administration

[0143] The protein-based composition, conjugate thereof, and composition thereof may be suitable for administration to a subject (such as a patient, which may be a human or non- human) well known to those skilled in the pharmaceutical art. The protein-based composition, conjugate thereof, and composition thereof may be prepared for administration to a subject. Such protein-based composition, conjugate thereof, and composition thereof can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration.

[0144] The protein-based composition, conjugate thereof, and composition thereof can be administered prophylactically or therapeutically. In prophylactic administration, the protein- based composition, conjugate thereof, and composition thereof can be administered in anAttorney Docket No.093386-0008-WO02 amount sufficient to induce a response. In therapeutic applications, the protein-based composition, conjugate thereof, and composition thereof can be administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition of the regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.

[0145] The protein-based composition, conjugate thereof, and composition thereof can be administered via a variety of routes. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular or subcutaneous delivery. Other routes include oral administration, intranasal, intravaginal, transdermal, intravenous, intraarterial, intratumoral, intraperitoneal, and epidermal routes. In some embodiments, the protein-based composition, conjugate thereof, or composition thereof is administered intravenously, subcutaneously, intradermally, intramuscularly, or intraperitoneally. In some embodiments, protein-based composition, conjugate thereof, or composition thereof is administered intravenously or subcutaneously.

[0146] The protein-based composition, conjugate thereof, and composition thereof may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations. The protein- based compositions and conjugates thereof can also be administered as combination doses. For example, a protein-based composition and a conjugate of a different or the same protein- based composition can be administered in combination. Varying combinations can be used as described herein. In addition, the protein-based composition and / or conjugate thereof can be administered in combination with conjugates as described in International Patent Application No. PCT / US2023 / 079885, which is fully incorporated by reference herein in its entirety.

[0147] As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight, the severity of the affliction, and subjects treated, the particular drugs employed, and the specific use for which these drugs are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine methods, for example, human clinical trials, in vivo studies and in vitro studies.Attorney Docket No.093386-0008-WO02

[0148] Dosage amount and interval may be adjusted individually to provide plasma levels of the biologically active agent which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC). The MEC will vary for each agent but can be estimated from in vivo and / or in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, assays well known to those in the art can be used to determine plasma concentrations. Dosage intervals can also be determined using MEC value. Compositions can be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, such as between 30-90% or between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

[0149] It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the symptoms to be treated and the route of administration. Further, the dose, and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine. B. Methods of Treating a Disease or a Disorder

[0150] Disclosed herein are methods of treating a disease or a disorder in a subject (e.g., in need thereof). The method can include administering to the subject an effective amount of the protein-based composition, the conjugate, and / or the composition as disclosed herein. The protein-based composition, conjugate and / or composition thereof can be administered optionally with a pharmaceutically acceptable excipient as disclosed herein.

[0151] The protein-based composition and the conjugate thereof can be beneficial for treating a number of different diseases and disorders. Example diseases and disorders include, but are not limited to, multiple sclerosis, Type I diabetes, organ transplantation, celiac disease, rheumatoid arthritis, lupus, inflammatory bowl disease, psoriasis, graves’ disease, a cancer, a viral infection, and infectious diseases mediated by T-cell immunity. Example cancers include, but are not limited to, melanoma, breast cancer, neuroblastoma, renal cell carcinoma, colon cancer, lung cancer, glioma, glioblastoma, and pancreatic cancer. Example viral infections include, but are not limited to, HIV, Hep B / C, flu, Herpes, chronic viral infection, and COVID. Example infectious diseases mediated by T-cell immunity include, but are not limited to, malaria, TB, zika, CMV, and Ebola.Attorney Docket No.093386-0008-WO02

[0152] In some embodiments, the protein-based composition can be used as a vaccine for immune tolerance applications. A tolerance vaccine application can be used for any suitable autoimmune disease. Examples diseases and disorders that an immune tolerance vaccine could be beneficial for include, but are not limited to, multiple sclerosis, Type I diabetes, organ transplantation, celiac disease, rheumatoid arthritis, lupus, inflammatory bowl disease, psoriasis, and graves’ disease.

[0153] In some embodiments, the conjugate can be used as a vaccine for activating the immune system of the subject. Example diseases and disorders that an activating vaccine could be beneficial for include, but are not limited to, a cancer, a viral infection, and an infectious disease mediated by T-cell immunity.

[0154] In some embodiments, the subject is human. C. Methods of Modulating an Immune System

[0155] Further disclosed herein are methods of modulating an immune system in a subject (e.g., in need thereof). The method can include administering to the subject an effective amount of the protein-based composition, the conjugate, and / or the composition as disclosed herein. The protein-based composition, the conjugate, and / or the composition can further include a pharmaceutically acceptable excipient.

[0156] Because the protein-based composition and the conjugate thereof can act as a vaccine, they can modulate the subject’s immune system in any way that a vaccine can traditionally modulate an immune system. Example ways that the protein-based composition and conjugate thereof can modulate an immune system include, but are not limited to, increasing an amount of regulatory T cells in the subject specific to the peptide antigen domain, increasing an amount of CD8+ T cells (which includes cytotoxic T cells (CTLs)) in the subject, increasing the amount of CD4+ Th cells, or a combination thereof. Example, Th cells include, but are not limited to, CD4+ Th1, Th17 cells, CD4+ Th2 cells, and CD4+ Th9 cells.

[0157] The method can further include personalizing the protein-based composition and conjugate thereof to a specific subject. This can be done by finding a peptide antigen that is specific to the subject and then modifying the protein-based composition to include said subject- specific peptide antigen. For example, the method can further include identifying, isolating, or both a peptide antigen from the subject and including the peptide antigen within the peptide antigen domain of the protein.

[0158] The description of the protein-based composition, nanobody domain, peptide antigen domain, drug, and linkers above may be applied to the disclosed methods. The disclosed technology has multiple aspects, illustrated by the following non-limiting examples.Attorney Docket No.093386-0008-WO02 6. Examples Materials and Methods for Example 1

[0159] Cell Lines and Materials. All chemicals involved in synthesis of target compounds were reagent grade unless stated otherwise. DNase, isopropyl thiogalactoside (IPTG), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. Azido-PEG3-Amine and DBCO- PEG12-NHS Ester were purchased from Broadpharm. Magnesium sulfate, sodium hydroxide, sodium azide, sodium acetate, sodium azide, sodium chloride, sodium bicarbonate, sodium hydroxide, 2xYT media, kanamycin, Nickel NTA resin, and all other organic solvents were purchased from Thermo Fisher Scientific. All DNA block segments involved in cloning protein inserts were purchased from Integrated DNA Technologies (IDT) with standard desalting as means of purification. A 44 pET28-b(+) expression vector, Q5 Hot Start Master Mix 2x, T4 DNA ligase, Golden Gate Master 45 Mix (BsaI-HF v2), DH5Į E. coli, and T7 Shuffle Express were used. E. coli chemically competent cells were purchased from New England Biolabs (NEB). Qiaprep Miniprep Spin kits were purchased from Qiagen. THP1-Dual and A549-Dual cell lines were purchased from InvivoGen. The murine melanoma cell line B16.F10-OVA was cultured in DMEM supplemented with 2 mM L-glutamine, 4.5 g / L glucose, 10% HI-FBS, and 100 U*ml-1penicillin / 100 ^g*mL-1streptomycin. Ovalbumin (OVA) expression was maintained through continuous selection using Geneticin (G418; Gibco) after every cell passage at a concentration of 380 ^g*mL-1. All cell types used in the study were grown in a humidified atmosphere at 37 °C in 5% CO2.

[0160] Cloning of Proteins. Gene cassette was purchased from IDT in the form of a gene block, with cloning restriction sites placed on both flanking regions (BsaI – GGTCTC) (SEQ ID NO: 111). In the case of a direct nanobody-antigen fusion expression, a sequence was placed between the nanobody and antigen (GGGS) (SEQ ID NO:99). For sortase mediated bioconjugation of nanobodies, a C-terminal sequence was incorporated (LPETGGHHHHHHEPEA) (SEQ ID NO:4). The gene fragment was digested with BsaI-HF v2 in a golden gate master mix (New England Biolabs) and ligated into a pET28-b(+) plasmid. The construct was transformed into chemically competent DH5Į E. coli and plated on LB agar with Kanamycin. The sequence verified nanobody was transformed in pET28b into T7 Shuffle Express (New England Biolabs) with E. coli as the expression strain.

[0161] Sequence details.Attorney Docket No.093386-0008-WO02 Engineered Sortase A (eSrtA) Sequence (No Start Codon). eSrtA – HisTag QAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLNRGVSFAEENESLDDQNISIAGHTFIDR PNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVEVLDEQKGKDKQLTLITCDDYN EETGVWETRKIFVATEVKLE – HHHHHH (SEQ ID NO:82) Formula: C785H1234N220O242S3Molecular Weight: 17721.94 Da İ280 = 14440 M-1cm-1Anti-Albumin (nAlb) Sequence (No Start Codon). nAlb – Ligation Tag MEVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS – LPETGGHHHHHHEPEA (SEQ ID NO:81) Formula: C611H948N176O199S5Molecular Weight: 14103.61 Da İ280 = 17085 M-1cm-1Anti-GFP (nGFP) Sequence (No Start Codon). nGFP – Ligation Tag MQVQLQESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSS YEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS – LPETGGHHHHHHEPEA (SEQ ID NO:83) Formula: C636H964N190O201S6Molecular Weight: 14680.16 Da İ280 = 27055 M-1cm-1Anti-Albumin-OVA251-270(nAlb-OVA251-270) Sequence (No Start Codon). nAlb-OVA251-270– Ligation Tag MEVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS – GGGSGLEQLESIINFEKLTEWTSSGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:84) Formula: C733H1136N208O244S5Molecular Weight: 16926.63 Da İ280 = 22585 M-1cm-1Anti-Albumin-MOG35-55(nAlb-MOG35-55) Sequence (No Start Codon). nAlb-MOG35-55– Ligation Tag MEVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS – GGGSMEVGWYRSPFSRVVHLYRNGKGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:85) Formula: C747H1151N219O237S6Molecular Weight: 17184.04 Da İ280 = 25565 M-1cm-1Anti-GFP-OVA251-270(nGFP-OVA251-270) Sequence (No Start Codon). nGFP-OVA251-270– Ligation Tag MQVQLQESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSS YEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS – GGGSGLEQLESIINFEKLTEWTSSGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:86) Formula: C758H1152N222O246S6Attorney Docket No.093386-0008-WO02 Molecular Weight: 17503.19 Da İ280 = 32555 M-1cm-1Anti-GFP-MOG35-55(nGFP-MOG35-55) Sequence (No Start Codon). nGFP-MOG35-55– Ligation Tag MQVQLQESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSS YEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS – GGGSMEVGWYRSPFSRVVHLYRNGKGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:87) Formula: C772H1167N233O239S7Molecular Weight: 17760.60 Da İ280 = 35535 M-1cm-1

[0162] Expression and Purification of Proteins.5 ^L of Kanamycin (stocked at 50 mg / mL) was added to a culture tube containing 5 mL 2xYT media and inoculated with a stab of protein (cloned into a NEB T7 Shuffle Express cell line). The culture was incubated at 30 ºC, with shaking at 250 RPM, for 16 hours. Each culture was transferred to a 2 L baffled flask containing 500 mL of 2xYT media and 500 ^L of Kanamycin (25 mg) and shaken at 30 ºC in an Innova 42R (New Brunswick Scientific) incubator for 4.5-5 hours (until the OD600 reached ~0.8). The cultures were then induced with IPTG (2.5 mM final concentration). The induced cultures were shaken overnight (20-24 hours) at 30 ºC. The bacteria were harvested the next day by centrifugation (3900 rpm for 10 min) and the pellet was reconstituted in 1x PBS with Dnase with a tablet of protease inhibitor cocktail (EDTA free). The cells were lysed by sonication on an ice bath in 5 second increments over 10 minutes. The resulting bacterial lysate was centrifuged (11000 rpm for 20 min) to remove cellular debris. The lysate was added to a 50 mL Kontes Flex column (Kimbal Kontes Glassware) containing 3 mL of Nickel NTA histidine binding resin that was preequilibrated with 1x PBS buffer. This column was placed on a rotating shaker at room temperature for 1-2 hrs. After this period, the supernatant was drained from the column using gravity and the column washed with 1x PBS buffer twice. Weakly bound proteins were first washed off of the resin using a low concentration elution buffer (2x 10 mL, 10 mM imidazole, 0.02% NaN3, 1x PBS pH 7.4 at 25 ºC). The bound protein was then eluted from the resin using elution buffer (15 mL, 150 mM imidazole, 0.02% NaN3, 1x PBS pH 7.4 at 25 ºC). The eluate was then concentrated to 0.5 mL in a 15 mL Microcon 10 kDa Centrifugal Filter Unit (Millipore) and subsequently purified by size exclusion chromatography (SEC) via an Akta FPLC (Cytiva), on a Hi-Load 16 / 60 Superdex 200 column using 1x PBS and 0.02% NaN3, pH 7.4 at 4 ºC as the running buffer. Pure fractions were determined by SDS-PAGE, pooled together with buffer exchange to 1x PBS not containing NaN3, and stocked at either -20 ºC or 4 ºC.

[0163] Enzymatic Bioconjugation and Click Chemistry Reactions. Bioconjugation reactions occurred in mild conditions (20 mM HEPES at pH 7.4, 150 mM NaCl, and 10 mM CaCl2)Attorney Docket No.093386-0008-WO02 between eSrtA (100 ^M) and a nanobody containing a C-terminal ligation tag (75 ^M) using a primary amine containing functional group (20 mM). Reactions occurred with mixing by a rotary shaker overnight (16 h) and were quenched by the addition of a 1:1 volume of a chelating agent EDTA containing solution (20 mM HEPES at pH 7.4, 300 mM NaCl, and 10 mM EDTA) for one hour. After the reaction was stopped, the solution was concentrated, and buffer exchanged to 1x PBS (without NaCl or MgCl2) three times by centrifugal dialysis. The protein solution was then immobilized to Nickel NTA histidine binding resin over 2 hours, and unbound protein was collected by washing the resin with 1x PBS. For nanobodies that contain a histidine in the native sequence, proteins were eluted in mild conditions (10 mM Imidazole in 1x PBS). Collected protein was concentrated and buffer exchanged to 1x PBS by centrifugal dialysis and verified by ESI-MS and SDS-PAGE. Click chemistry reactions proceeded by the addition of 5 eq. (molar) of the complementary handle (e.g., if an azide was placed on the nanobody, the click chemistry reaction would proceed with the addition of 5 eq. of DBCO-containing moiety). After 48 hours of reaction between the protein azide and the DBCO-moiety, the mixture was purified by centrifugal dialysis four times, and verified for purity by UV-VIS, ESI-MS, and SDS-PAGE.

[0164] Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Protein samples were diluted in 1x PBS to 10 ^M before analysis.10 ^L of the protein sample was mixed with 10 ^L of reducing Laemmli buffer. Samples were boiled at 95 ºC for 5 minutes, 170 ^L and 15 ^L of each sample was loaded into a 15-well, 4-15% Tris-glycine precast SDS-PAGE (Biorad) and ran at a constant 150 V with 343 mA for 45 minutes. The gel was then directly stained using Coomassie-B-250 and imaged using a GelDoc EZ Imager (BioRad).

[0165] Electrospray Ionization Liquid Chromatography Mass Spectrometry (ESI-MS). Proteins were buffer exchanged into ammonium acetate (pH 5.5) and concentrated to approximately 100 ^M. ESI-MS data were collected using an Agilent 6210A time-of-flight (TOF) mass spectrometer at a range of 50-20,000 m / z over a period of two minutes. Data were analyzed with Agilent MassHunter IM-MS Acquisition Data software to reveal m / z data, where files were condensed across the two-minute run. These m / z data were deconvoluted using a maximum entropy deconvolution calculation using UniDec to give the deconvoluted mass spectra using background subtraction between a range of 1,000-5,000 m / z and with an export range of 5,000-50,000 Da.

[0166] Synthesis and NMR Verification of DBCO-PEG12-diABZI. First, a STING agonist was generated that features a reactive amine handle, which was synthesized in four steps. Briefly, aryl amination of an aryl chloride 1 with an amine 2 gave a dinitro analog, compound 3. The di- nitro compound 3 was subjected to reduction using sodium dithionite in methanol, generating aAttorney Docket No.093386-0008-WO02 di-amine moiety 4. Compound 4 was then treated with isothiocyanate, followed by EDC coupling, to reveal a boc-protected analog, compound 5. Next, the boc-group from compound 5 was deprotected by treating with TFA:DCM. To a stirred solution of amine 6 (100 mg, 0.089 mmol, 1 eq.) in 5 mL DMF was added Hunig’s base (77 ^L, 0.44 mmol, 5 eq.) under argon atmosphere, at room temperature. After stirring for 5 min, a solution of activated NHS ester (98 mg, 0.098 mmol, 1.1 eq.) in DMF (5 mL) was added dropwise and stirred overnight (16 h). The solvent was evaporated to get crude product 7, which was purified by silica gel column chromatography using a mixture of methanol / dichloromethane as an eluent (5% to 25% MeOH) to get the desired product as a solid (70 mg, 0.042 mmol, yield 43%). (Rf= 0.5 in 20% MeOH in DCM).1H NMR (400 MHz, DMSO) į 8.01 – 7.93 (m, 2H), 7.88 (t, J = 5.7 Hz, 1H), 7.75 (t, J = 5.7 Hz, 1H), 7.67 – 7.60 (m, 4H), 7.49 – 7.42 (m, 3H), 7.38 – 7.27 (m, 7H), 6.49 (d, J = 7.1 Hz, 2H), 5.88 – 5.79 (m, 2H), 5.01 (d, J = 13.9 Hz, 1H), 4.91 (dd, J = 29.6, 4.2 Hz, 4H), 4.53 – 4.49 (m, 4H), 3.98 (t, J = 6.0 Hz, 2H), 3.72 (s, 3H), 3.60 – 3.57 (m, 2H), 3.54 (t, J = 6.5 Hz, 2H), 3.47 (broads, 46H), 3.30 – 3.26 (m, 2H), 3.14 – 3.05 (m, 4H), 2.26 (t, J = 6.5 Hz, 2H), 2.09 (s, 3H), 2.08 (s, 3H), 2.01 – 1.96 (m, 1H), 1.78 – 1.72 (m, 1H), 1.68 (p, J = 6.5 Hz, 2H), 1.28 – 1.27 (m,6H).13C NMR (151 MHz, DMSO) į 171.57, 171.50, 170.52, 168.06, 167.33, 152.50, 152.45, 152.06, 148.88, 145.50, 145.28, 144.65, 140.37, 140.33, 132.87, 130.54, 130.49, 130.07, 129.37, 128.62, 128.58, 128.44, 128.25, 128.13, 127.24, 125.60, 122.99, 121.86, 120.11, 120.04, 114.67, 109.72, 108.61, 106.00, 105.83, 105.58, 70.21, 70.14, 70.10, 70.00, 69.94, 69.45, 67.26, 56.45, 55.34, 53.85, 46.05, 45.05, 42.12, 38.95, 36.57, 35.61, 30.80, 30.17,29.13, 18.46, 17.17, 16.58, 13.57, 12.74. HRMS (ESMS) Calculated for C84H111N15O21[M+Na]+:1688.7977, found 1688.7982. This synthesis is further described in International Patent Application No. PCT / US2023 / 079885, which is fully incorporated by reference herein in its entirety.

[0167] Synthesis of Synthetic Peptide Antigens with N-Terminus Azides. The peptide antigens N3-GGGS-GLEQLESIINFEKLTEWTSS (N3-OVA251-270) (SEQ ID NO:103), N3-GGGS- ISQAVHAAHAEINEAGR (N3-OVA323-339) (SEQ ID NO:104), and N3-GGGS- MEVGWYRSPFSRVVHLYRNGK (N3-MOG35-55) (SEQ ID NO:105) were synthesized using a LibertyBlue 2.0 automated microwave peptide synthesizer (CEM Corporation). A polystyrene resin pre-loaded with the C-terminal amino acid residue following a cleavable Wang linker was purchased for each peptide sequence (CEM Corporation). The appropriate amino acids with Fmoc protected amine groups (CEM Corporation) were conjugated to the exposed primary amines on the polystyrene resin via N,N'-Diisopropylcarbodiimide (DIC) / Oxyma catalyzed coupling. Fmoc groups were then deprotected via a solution of 10% piperdine, and the processAttorney Docket No.093386-0008-WO02 was repeated as necessary. Following deprotection of the final primary amine at the N-terminus of the peptide, azido acetic acid (N3-CH2-COOH) was conjugated to the N-terminal amine of the peptide using DIC / Oxyma coupling to generate a reactive azide at the N-terminus of the peptide. The peptide was then cleaved from the resin using a cleavage cocktail of 92.5% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIPS), 2.5% dodecanethiol (DODT), and 2.5% DI H2O for 3 h at RT. The cleaved peptide was then precipitated in cold diethyl ether, dried under vacuum. Dried crude peptide was redissolved in H2O and / or acetonitrile (AN), purified via semi-preparative high performance liquid chromatography (HPLC) (Waters) using an H2O:AN gradient and a C18 column. Collected fractions were pooled, and AN was removed using a rotary evaporator. The water-dissolved peptide was flash frozen, and water was removed via lyophilization (Labconco). The lyophilized peptide was characterized using analytical HPLC and LC-MS and stored at -20 °C.

[0168] Evaluation of Nanobodies in Tumor Models.6–8-week C57BL / 6 mice (The Jackson Laboratory) were inoculated with B16.F10-OVA tumors via subcutaneous (S.C.) administration of 5 x 105cancer cells, suspended in 100 ^L PBS, into the right flank of the mouse. For prophylactic treatment models, mice were administered S.C. injections at the left base-of-tail of indicated compounds on days -21, -14, and -7 prior to tumor inoculation. For therapeutic treatment models, treatments began on Day 0 when the average tumor volume reached ~50-75 mm3. These mice were administered three injections on Days 0, 4, and 8. Tumor volume calculations were calculated using Vtumor= L × W2× 0.5, in which Vtumoris tumor volume, L is tumor length, and W is tumor width. Tumor volume, total murine mass, and murine well-being were recorded for the duration of the study. The endpoint for maximum tumor volume (i.e. survival) during studies was 1500 mm3.

[0169] Evaluation of Antigen-Specific T Cell Responses in Blood and Spleen. For studies using peripheral blood mononuclear cells (PBMCs), approximately 100 ^L of blood was collected from each mouse using a cheek bleed into an EDTA-coated collection tube. Blood was then transferred to 5 mL of ACK lysis buffer to induce red blood cell (RBC) lysis. After 5 min, ACK buffer was quenched via addition of 10 mL PBS, and cells were pelleted via centrifugation at 1500 rpm for 5 min. Cell pellets were resuspended in an additional 5 mL ACK lysis buffer, and the quenching and centrifugation process was repeated to further eliminate any remaining RBCs. PBMCs were resuspended in 200 ^L flow buffer (FB, 2% FBS in PBS) and transferred to a 96-well round bottom plate for staining. Cells were pelleted via centrifugation at 1500 rpm for 5 min, and resuspended in 25 ^L Fc shield (Anti-Mouse CD16 / CD32, 1:500 in FB) for 15 min at 4 °C. The following antibodies were then added to the cells in a volume of 75 ^L (final volume =Attorney Docket No.093386-0008-WO02 100 ^L) and cells were incubated for 60 min at 4 °C. Following incubation, the cells were washed 1X with FB, resuspended in 100 ^L of H2-Kb / SIINFEKL-Pe tetramer (1:33 in FB), and incubated for 2 hours at 4 °C. Following incubation, cells were washed 1X with FB and resuspended in FB containing 10 ng / mL DAPI as a viability dye and analyzed via flow cytometry using a CellStream Flow Cytometer (Cytek) (Table 2). Table 2. PBMC Tetramer Flow Cytometry Panel.

[0170] For studies using splenocytes, mice were asphyxiated using CO2and whole spleens were collected via dissection. Splenocytes were passed through a 70 ^m cell strainer to generate a single-cell suspension, and then centrifuged at 380 x g for 5 min to pellet. Pellets were resuspended in 5 mL ACK lysis buffer for 5 min to induce RBC lysis. Lysis buffer was quenched via the addition of 10 mL PBS and cells were pelleted again. Cell pellets were resuspended in 1 mL FB, and approximately 4 million splenocytes were transferred to a 96-well round bottom plate for staining. Cells were resuspended in 50 ^L Fc shield (Anti-Mouse CD16 / CD32, 1:500 in FB) for 15 min at 4 °C.50 ^L H2-Kb / SIINFEKL-Pe tetramer (1:17 in FB) was then added and incubated for 60 min at 4 °C. Following incubation, the cells were washed 1X with FB, resuspended in 100 ^L the following antibodies, and incubated for 30 min at 4 °C. Cells were then pelleted at 380 x g for 5 min, washed 1X with FB, pelleted again, and then resuspended in 200 ^L 2% paraformaldehyde (PFA) in FB for 10 min at RT to fix the cells. Following fixation, cells were pelleted at 650 x g for 5 min, washed 1X with FB, and then resuspended in 100 ^L FB for flow cytometric analysis using an Aurora Flow Cytometer (Cytek) (Table 3). Table 3. Splenocyte Tetramer Flow Cytometry PanelAttorney Docket No.093386-0008-WO02

[0171] Evaluation of Nanobodies in Experimental Autoimmune Encephalomyelitis (EAE) Model of Multiple Sclerosis (MS).9–12-week C57BL / 6 mice (The Jackson Laboratory) were prophylactically treated with indicated compounds on Days -7, -5, and -3 prior to model induction. On Day 0, the model was induced according to the manufacturer’s (Hooke Laboratories) instructions. In brief, on Day 0 mice were administered two 100 ^L S.C. injections of an emulsion containing Complete Freund’s adjuvant (CFA) and the myelin oligodendrocyte glycoprotein (MOG)35-55antigen expressed on the myelin sheaths of neuronal axons and presented by the Class II Major Histocompatibility Complex (MHC) (I-A / I-E) of C57BL / 6 mice. Approximately 2-3 hours later, mice were administered 110 ng of pertussis toxin (PTX) in 100 ^L PBS injected intraperitoneally (I.P.) to induce permeabilization of the blood-brain-barrier (BBB) to facilitate the invasion of autoreactive CD4+T cells into the central nervous system (CNS). The following day, mice were administered a second I.P. injection of 110 ng of PTX in 100 ^L PBS. On Day 8, mice were monitored bi-daily and scored according to an established EAE scoring guideline. In brief, the scoring guideline is summarized as follows: 0) Healthy, 1) Limp Tail, 2) Hind Limb Weakness, 3) Hind Limb Paralysis, 4) Complete Paralysis, and 5) Death. Once the first mouse in any treatment group reached a score of 1, the frequency of monitoring and scoring was increased to daily. Mice were euthanized on Day 28.

[0172] Pharmacokinetics and Ex Vivo Imaging Experiments. Healthy 6–8-week C57BL / 6 mice (The Jackson Laboratory) were injected with a dose of 2 nmol Cy5 (conjugated to either nAlb or an irrelevant nGFP control) in 100 ^L PBS S.C. at the left base-of-tail. Blood draws were taken using heparinized capillary tubes (DWK Life Sciences) at discrete time points up to 24 hours after injection.1 ^L of blood was mixed with 50 ^L of PBS, centrifuged, and the diluted plasma was collected for analysis. Prescence of Cy5 was determined by fluorescence intensity using a plate reader, with an excitation wavelength of 645 nm and an emission wavelength of 675 nm. Pharmacokinetic analysis was performed in GraphPad Prism (V10) using a two- compartmental one-phase absorption and one-phase decay model of S.C. injection, in whichAttorney Docket No.093386-0008-WO02 the reported half-life is the decay phase (elimination). Biodistribution studies were performed by excising the hearts, lungs, livers, spleens, kidneys, draining inguinal lymph nodes (iLNs), and irrelevant axillary lymph nodes (aLNs). Tissue were washed in 1x PBS and transferred to the stage of the IVIS Lumina III (PerkinElmer). Fluorescence (radiant efficiency) was measured and areas were drawn for organs to generate average radiant efficiency values (per cm2) using the Living Image software (version 4.5).

[0173] Evaluation of Nanobody Uptake by Immune Cell Populations in Lymphoid Tissues. Healthy 6–8-week C57BL / 6 mice (The Jackson Laboratory) were injected with a dose of 2 nmol Cy5 (conjugated to either nAlb or an irrelevant nGFP control) in 100 ^L PBS S.C. at the left base-of-tail.24 h post injection, mice were euthanized via CO2asphyxiation and lymphoid tissues including the spleen, draining inguinal lymph node (iLN), and irrelevant axillary lymph node (aLN) were excised. Tissues were passed through a 70 ^m cell strainer to generate a single cell suspension, and cells were pelleted via centrifugation at 1500 rpm for 5 min. Following centrifugation, cells were suspended in 5 mL ACK lysis buffer for 5 min to induce red blood cell (RBC) lysis. ACK was quenched via addition of 10 mL PBS, and cells were again pelleted via centrifugation. Cells were resuspended in 1 mL flow buffer (FB, 2% FBS in PBS) and approximately 250,000 lymph node cells and 4 million splenocytes were transferred to a 96- well round bottom plate for staining. Cells were resuspended in 50 ^L Fc shield (Anti-Mouse CD16 / CD32, 1:500 in FB) for 15 min at 4 °C. Following incubation, the cells were washed 1X with FB, resuspended in 50 ^L the following antibodies (final volume = 100 ^L), and incubated for 30 min at 4 °C. Cells were then pelleted at 380 x g for 5 min, washed 1X with FB, pelleted again, and then resuspended in 200 ^L 2% paraformaldehyde (PFA) in FB for 10 min at RT to fix the cells. Following fixation, cells were pelleted at 650 x g for 5 min, washed 1X with FB, and then resuspended in 100 ^L FB for flow cytometric analysis using an Aurora Flow Cytometer (Cytek) (Table 4). Table 4. Nanobody-Cy5 Uptake Flow Cytometry PanelAttorney Docket No.093386-0008-WO02

[0174] Statistics. All data were plotted, and statistical analysis performed using Prism 10 (GraphPad) software. Unless indicated in the figures, all data are presented as mean ± SEM. For comparisons between two groups, unpaired two-tailed Student’s t-tests were performed 374 as indicated. For multiple comparisons a one-way ANOVA was performed with post-hoc Tukey’s correction for multiple comparisons. For tumor volume, statistically significance was examined through a two-way ANOVA followed by Tukey’s adjustment for multiple comparisons. A Logrank (Mantel-Cox) test was used to compare Kaplan-Meyer survival data. Example 1 Monovalent Proteins and Conjugates Thereof

[0175] Synthesis of albumin-hitchhiking nanobody-antigen fusions and conjugates thereof. It was hypothesized, without being bound by a particular theory, that the fusion of a peptide antigen to an albumin-binding chaperone could increase antigen retention in the interstitial fluid compartment following S.C. injection, as well as extend blood circulation half-life and enhance absolute bioavailability once the peptide antigen enters circulation, which could ultimately lead to increased antigen drainage and delivery to lymphoid tissues, thus improving the antigenicity of the peptide antigen and enhancing adaptive immune responses. While several albumin- binding molecules have been described, the experimental platform described below was built from a nanobody with high affinity for albumin because nanobodies are highly modular and programmable via genetic engineering, are molecularly well-defined, are amenable to scalable industrial manufacturing, and are components of approved and clinically advanced therapeutics, including ozoralizumab, which contains an anti-albumin nanobody domain. A nanobody domain – termed nAlb – that binds with nanomolar affinity to serum albumin was recombinantly expressed. At the C-terminus of the nanobody domain, a peptide antigen, flanked by two flexible spacers (GGGS) (SEQ ID NO:99), was cloned to generate an nAlb-antigen fusion. Additionally, after the C-terminal GGGS (SEQ ID NO:99) spacer, a selective ligation tag (LPETGGHHHHHHEPEA) (SEQ ID NO:4) was cloned to include a site-specific substrate for an enzymatic engineered sortase pentamutant A (eSrtA) mediated ligation of any primary amine containing small molecule to the C-terminal of the nAlb-antigen fusion. It was demonstrated that this approach could be used to facilitate the ligation of an NH2-PEG3-N3linker to the C-terminusAttorney Docket No.093386-0008-WO02 of the nanobody, which was able to modularly ligate other cargos via strain-promoted azide- alkyne cycloaddition (SPAAC) to a dibenzocyclooctyne (DBCO) containing molecule. For biodistribution and pharmacokinetic studies, sulfo-Cy5-DBCO (BroadPharm) was ligated to the nanobody (FIG.1). For immunostimulatory vaccine applications, a variant of the STING agonist diABZI was synthesized that was functionalized with an azide-reactive DBCO group and a PEG12spacer (DBCO-PEG12-diABZI). Conjugation of the NH2-PEG3-N3, sulfo-Cy5-DBCO, and / or DBCO-PEG12-diABZI was confirmed via SDS PAGE and / or ESI-MS (FIG.2, FIG.3, FIG. 4, and FIG.5). A list of antigens is provided in Table 5. Table 5. Antigens incorporated into clonally expressed nAlb-antigen fusion vaccines.

[0176] Synthesis of albumin-hitchhiking nanobody-synthetic azide-antigen conjugates to facilitate rapid neoantigen (neoAg) vaccine manufacturing. For some applications, the need to generate a novel Shuffle T7 E. coli clone for multiple novel antigens may prove overly time consuming and / or impractical. For example, mix-and-go cancer vaccine pipelines, in which a patient’s tumor is biopsied, novel antigen targets are identified, and personalized vaccines are rapidly developed, require the rapid addition of the newly identified antigen into the formulation. Additionally, some nanobody-antigen fusions may not express well, and it could prove burdensome to test out various expression systems. To this end, a series of eSrtA and SPAAC reactions were designed to load various chemically synthesized synthetic antigens onto an albumin-targeting nanobody (nAlb) that lacks a clonally incorporated antigen. The nAlb nanobody was designed to include the eSrtA selective ligation tag (LPETGGHHHHHHEPEA) (SEQ ID NO:4) at the C-terminus to facilitate the site-specific conjugation of a primary amine containing small molecule following the LPET motif. It was demonstrated that this approach could be used to ligate the azide reactive endo-BCN-PEG3-NH2to the C-terminus of nAlb. Synthetic peptide antigens were then synthesized using solid phase peptide synthesis (SPPS). These antigens were designed with a reactive azide group at the N-terminus, which was achieved via coupling of azido acetic acid to the N-terminal primary amine using the microwave peptide synthesizer. Azide functionalized peptides could be ligated in a 1:1 ratio to nAlb-BCN via SPAAC reaction. Conjugation of endo-BCN-PEG3-NH2, sulfo-Cy5-N3, and N3-OVA323-339Attorney Docket No.093386-0008-WO02 was confirmed via SDS PAGE and / or ESI-MS (FIG.6, FIG.7, and FIG.8). A list of chemically synthesized azide-antigens is provided in Table 6. Table 6. Synthetic azide-antigens loaded onto nAlb-BCN via SPAAC reaction.

[0177] nAlb-Antigen fusions enhance absolute bioavailability and enhance delivery to the draining inguinal lymph node (iLN). As discussed previously, it was hypothesized, without being bound by a particular theory, that fusing peptide antigens to an albumin-binding nanobody would improve peptide accumulation in the local draining lymph node by increasing residence time in the interstitial space following S.C. injection, thus limiting peptide absorption into circulation and subsequent renal clearance. Additionally, the enhanced circulation time achieved through albumin-binding was hypothesized to improve antigen delivery to distal lymphoid organs by increasing the absolute bioavailability of the peptide antigen. To test this, mice were administered 2 nmol of nAlb-Cy5 or nGFP-Cy5 S.C. at the left base-of-tail. Blood was collected at t = 5, 15, 45 min and 1.5, 3, 6, 12, and 24 h to determine the serum half-life of the constructs (FIG.9). Plasma Cy5 concentrations were fit to a two-compartment model of S.C. injection, and calculated serum half-lives (t1 / 2), absorption constants (kabs), elimination constants (kclear), and areas under the curves (AUC) are provided in Table 7. Fusion to nAlb enhanced peptide antigen half-life, showed both its absorption into and elimination from circulation, and enhanced the absolute bioavailability of the peptide antigen. Table 7. Calculated pharmacokinetic parameters of Cy5 conjugated nanobodies.

[0178] Additionally, mice were euthanized at t = 24 h, and livers, lungs, kidneys, hearts, spleens, and draining inguinal lymph nodes (iLNs) were collected and imaged ex vivo using IVISAttorney Docket No.093386-0008-WO02 imaging. The average radiant efficiency of the Cy5 signal per unit area of tissue was calculated and indicated that conjugation to nAlb significantly increased Cy5 accumulation in all organs collected (FIG.10), most notably in the iLN (FIG.11), as lymphoid tissues are the major sites of T cell priming by antigen presenting cells (APCs) – a necessary process during the elicitation of a vaccine response. Following IVIS imaging, lymphoid tissues including the spleen, draining iLN, and irrelevant axillary lymph node (aLN) were processed and stained for flow cytometry. Cy5 uptake by major immune cell populations – dendritic cells (DCs), macrophages (Mĭs), B cells, neutrophils, and T cells – was quantified. Analysis indicated that most uptake occurred in the iLN, likely due to the localized S.C. injection, and that, across all tissues, Cy5 was most readily taken up by the highly phagocytic antigen-presenting cell (APC) types Mĭs and DCs (FIG.12). This is an important finding as APCs are efficient at presenting antigen to T cells, and antigen presentation is necessary to educate and induce a desired T cell response, which is the desired effect of many peptide vaccines.

[0179] Prophylactic vaccination with nAlb-MOG35-55inhibits disease onset and progression in an Experimental Autoimmune Encephalomyelitis (EAE) model of Multiple Sclerosis (MS). Given that fusion to nAlb can induce robust antigen delivery to multiple lymphoid tissues, and that systemic antigen presentation in non-inflammatory settings is known to induce antigen-specific immune tolerance via de novo regulatory T cell (Treg) generation, it was hypothesized, without being bound by a particular theory, that prophylactic vaccination with nAlb-MOG35-55would generate MOG35-55-specfic Tregcells capable of protecting against EAE onset following disease induction. To test this, 8–12-week C57BL / 6 mice were prophylactically treated with 1.15 nmol nAlb-MOG35-55, free MOG35-55, or PBS on Days -7, -5, and -3 prior to disease induction on Day 0. EAE was induced via S.C. injections of an emulsion of MOG35-55and Complete Freund’s adjuvant (CFA), followed by a series of I.P. injections of pertussis toxin (PTX) (FIG.13). Mice were monitored daily during disease onset and scored according to established scoring guidelines in which a lower score reflects a less severe disease state, and a higher score reflects a more severe disease state. Compared to both controls, treatment with nAlb-MOG35-55delayed EAE onset and limited total disease burden through 28 days following model initiation (FIG.14). Additionally, treatment with nAlb-MOG35-55significantly reduced the cumulative clinical EAE score of each mouse over the course of the 28 day study compared to both tested controls, and two of the nAlb-MOG35-55treated mice never developed any EAE symptoms (FIG. 15). These results suggest that treatment with nAlb fused antigens absent any immunostimulatory adjuvant is capable of generating protective antigen-specific immune tolerance. This is possibly due to the likely tolerogenic process of albumin uptake and recycling,Attorney Docket No.093386-0008-WO02 as albumin is a ubiquitously expressed protein towards which an autoimmune response would prove catastrophic.

[0180] Vaccination with nAlb-Antigens adjuvanted with conjugated diABZI or a mixture of nAlb-diABZI generates antigen-specific CD8+T cells with effector memory phenotype. To utilize the nAlb-Antigen platform in the context of an activating vaccine, it was decided to include the STING agonist diABZI in the formulation, which can be conjugated to the C-terminus of nAlb and nAlb-Antigens using a series of sortase and SPAAC reactions. It was hypothesized, without being bound by a particular theory, that spatiotemporal coordination of antigen and adjuvant is required to generate effective vaccine responses, as vaccine antigens should be presented by activated APCs to effect a CD8+T cell response, and ideally only vaccine antigens are presented by activated APCs to mitigate chances of developing an autoimmune disease. To evaluate this hypothesis, mice were vaccinated with 1.25 ^g diABZI and an equivalent 1.47 nmol peptide antigen (OVA251-270) S.C. at the left base-of-tail on Days 0, 7, and 14, prior to sacrifice on Day 21 (FIG.16). Treatment groups included a full conjugate of nAlb-OVA251-270- diABZI, a soluble mixture of nAlb-OVA251-270and nAlb-diABZI (nAlb-OVA251-270 / nAlb-diABZI), a soluble mixture of nAlb-OVA251-270and free diABZI, and a soluble mixture of a synthetic long peptide (SLP) form of the SIINFEKL (SEQ ID NO:110) antigen (SLP SIINFEKL, OVA251-264) and free diABZI (SLP SIINFEKL (SEQ ID NO:110) / diABZI). On Day 14, following two injections of indicated vaccine constructs, blood was collected via submandibular cheek bleed collection and peripheral blood mononuclear cells (PBMCs) were processed and analyzed via flow cytometry. Flow cytometric analysis indicated that vaccination with both nAlb-OVA251-270-diABZI and nAlb- OVA251-270 / nAlb-diABZI significantly increased the number of antigen-specific CD8+T cells in circulation (FIG.17). On Day 21, 7 days following the third injection of indicated vaccine constructs, mice were sacrificed and spleens were collected and processed for flow cytometry. Flow cytometric analysis indicated that following three injections, only treatment with nAlb- OVA251-270 / nAlb-diABZI significantly increased the number of antigen-specific CD8+T cells in the spleen (FIG.18). While this was not necessarily anticipated, loss of antigen-specific CD8+T cells following a third injection in the nAlb-OVA251-270-diABZI group may be due to a more rapid exhaustion of T cells following chronic antigen exposure, as this can happen with enough antigen exposure even in inflammatory conditions. To test this, T cell exhaustion was probed for marker PD-1 and found it to exhibit trending increases in all treatment groups, and a significant increase in the nAlb-OVA251-270 / nAlb-diABZI group (FIG.19). This aligned with the observation herein, as nAlb-OVA251-270 / nAlb-diABZI had the highest number of antigen-specific CD8+T cells, and PD-1 expression is part of a negative feedback loop, with expression increasing as T cellsAttorney Docket No.093386-0008-WO02 become increasingly activated to limit off-target T cell responses. Furthermore, the antigen- specific T cells were probed for effector memory (Tem) and central memory (Tcm) phenotype. Temcells are necessary for the generation of effective antitumoral T cell responses. It was found that vaccination with nAlb-OVA251-270 / nAlb-diABZI most effectively induced a shift from Tcmto Temphenotype (FIG.20). Combined with the findings that nAlb-OVA251-270 / nAlb-diABZI vaccination generated the greatest number of antigen-specific CD8+T cells, it was decided to explore the antitumoral effects of this formulation.

[0181] Prophylactic vaccination with nAlb-OVA251-270 / nAlb-diABZI generates antigen-specific CD8+T cells capable of protecting against B16.F10-OVA melanoma challenge. To evaluate the efficacy of nAlb-OVA251-270 / nAlb-diABZI vaccination in a disease context, 6–8-week C57BL / 6 mice were treated with 50 ^g whole OVA protein or an equivalent 1.171 nmol nAlb-OVA251-270and 1.25 ^g diABZI either as a free compound or conjugated to nAlb via S.C. injection on Days 0, 7, and 14 prior to tumor inoculation. On Days 14 and 21, PBMCs were collected and evaluated via flow cytometry to quantify the number of circulating antigen-specific CD8+T cells following two and three treatments with indicated vaccine constructs. Mice were then inoculated with 5 x 105B16.F10-OVA melanoma cells S.C. in the right flank on Day 22, and tumors were monitored until humane endpoints were met (Fig.21). Flow cytometric analysis indicated at that no formulation exhibited significant increases in the number of circulating antigen-specific CD8+T cells on Day 14 – albeit both nAlb-OVA251-270 / nAlb-diABZI and OVA / diABZI treatment exhibited trending increases – and that only nAlb-OVA251-270 / nAlb-diABZI treatment significantly increased the number of circulating antigen-specific CD8+T cells on Day 21 (Fig.22). Following tumor inoculation, tumors were measured every other day and tumor volumes were calculated according to the formula V = L*W2*0.5, where V is the tumor volume, L is the tumor length, and W is the tumor width. Mice were sacrificed when tumor volume reached 1500 mm3. It was found that nAlb-OVA251-270 / nAlb-diABZI vaccination was capable of slowing tumor growth and extending mouse survival compared to both PBS and nAlb-OVA251-270controls, however not to the equivalent degree of OVA / diABZI vaccination (FIG.23 and FIG.24). This is likely due to the presence of helper epitopes in the full OVA vaccine, capable of inducing CD8+T cell, CD4+T cell, and B cell responses, while the nAlb-OVA251-270peptide vaccine can only induce CD8+T cell responses.

[0182] Therapeutic vaccination with nAlb-OVA251-270 / nAlb-diABZI slows B16.F10-OVA melanoma growth and extends mouse survival. Finally, the efficacy of nAlb-OVA251-270 / nAlb- diABZI vaccination was tested in a therapeutic model of B16.F10-OVA melanoma. This is a more clinically relevant model in which mice already have tumors prior to treatment.6–8-weekAttorney Docket No.093386-0008-WO02 C57BL / 6 mice were inoculated with 5 x 105B16.F10-OVA melanoma cells S.C. on the right flank. Tumors were measured every other day, and treatments began on Day 0 when the average tumor volume reached approximately 50-75 mm3. Mice were administered 1.171 nmol OVA251-270, either fused to nAlb or as a free peptide, and 1.25 ^g diABZI, either fused to nAlb or as a free drug, S.C. on Days 0, 4, and 8. Compared to all tested controls, vaccination with nAlb- OVA251-270 / nAlb-diABZI most significantly inhibited tumor growth (FIG.25) and extended mouse survival (FIG.26). Materials and Methods for Example 2

[0183] Cell Lines and Materials. All chemicals involved in synthesis of target compounds were reagent grade unless stated otherwise. DNase, isopropyl thiogalactoside (IPTG), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. Azido-PEG3-Amine and DBCO- PEG12-NHS Ester were purchased from Broadpharm. Magnesium sulfate, sodium hydroxide, sodium azide, sodium acetate, sodium azide, sodium chloride, sodium bicarbonate, sodium hydroxide, 2xYT media, kanamycin, Nickel NTA resin, and all other organic solvents were purchased from Thermo Fisher Scientific. All DNA block segments involved in cloning protein inserts were purchased from Integrated DNA Technologies (IDT) with standard desalting as means of purification. A 44 pET28-b(+) expression vector, Q5 Hot Start Master Mix 2x, T4 DNA ligase, Golden Gate Master 45 Mix (BsaI-HF v2), DH5Į E. coli, and T7 Shuffle Express were used. E. coli chemically competent cells were purchased from New England Biolabs (NEB). Qiaprep Miniprep Spin kits were purchased from Qiagen. THP1-Dual and A549-Dual cell lines were purchased from InvivoGen. The murine melanoma cell line B16.F10-OVA was cultured in DMEM supplemented with 2 mM L-glutamine, 4.5 g / L glucose, 10% HI-FBS, and 100 U*ml-1penicillin / 100 ^g*mL-1streptomycin. Ovalbumin (OVA) expression was maintained through continuous selection using Geneticin (G418; Gibco) after every cell passage at a concentration of 380 ^g*mL-1. All cell types used in the study were grown in a humidified atmosphere at 37 °C in 5% CO2.

[0184] Cloning of Proteins. Gene cassette was purchased from IDT in the form of a gene block, with cloning restriction sites placed on both flanking regions (BsaI – GGTCTC) (SEQ ID NO:111). In the case of a fusion protein, a sequence was placed between the two domains (XTEN – SGSETPGTSESA) (SEQ ID NO:100). In the case of a direct nanobody-antigen fusion expression, a sequence was placed between the nanobody and antigen (GGGS) (SEQ ID NO:99). For sortase mediated bioconjugation of nanobodies, a C-terminal sequence was incorporated (LPETGGHHHHHHEPEA) (SEQ ID NO:4). The gene fragment was digested withAttorney Docket No.093386-0008-WO02 BsaI-HF v2 in a golden gate master mix (New England Biolabs) and ligated into a pET28-b(+) plasmid. The construct was transformed into chemically competent DH5Į E. coli and plated on LB agar with Kanamycin. The sequence verified nanobody was transformed in pET28b into T7 Shuffle Express (New England Biolabs) with E. coli as the expression strain.

[0185] Sequence Details. Engineered Sortase A (eSrtA) Sequence (No Start Codon). eSrtA – HisTag QAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLNRGVSFAEENESLDDQNISIAGHTFIDR PNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVEVLDEQKGKDKQLTLITCDDYN EETGVWETRKIFVATEVKLE – HHHHHH (SEQ ID NO:82) Formula: C785H1234N220O242S3Molecular Weight: 17721.94 Da İ280 = 14440 M-1cm-1Anti-Albumin (nAlb) Sequence (No Start Codon). nAlb – Ligation Tag MEVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS – LPETGGHHHHHHEPEA (SEQ ID NO:81) Formula: C611H948N176O199S5Molecular Weight: 14103.61 Da İ280 = 17085 M-1cm-1Anti-Albumin-OVA251-270(nAlb-OVA251-270) Sequence (No Start Codon). nAlb-OVA251-270– Ligation Tag MEVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS – GGGSGLEQLESIINFEKLTEWTSSGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:84) Formula: C733H1136N208O244S5Molecular Weight: 16926.63 Da İ280 = 22585 M-1cm-1Anti-Albumin – Anti-PD-L1 (AP) Sequence (No Start Codon). nAlb – XTEN Linker – nPD-L1 – Ligation Tag MEVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS – SGSETPGTSESA – QVQLQESGGGLVHPGGSLRLSCATSGSIFSIISMGWYRQAPGKQRELVALVFRGGSTVYADSV KGRFTISGDIAKSTVYLQMDSLKPEDTAVYYCNAKPIGTAQYWGQGTQVTVSS – LPETGGHHHHHHEPEA (SEQ ID NO:88) Formula: C1200H1872N338O389S9Molecular Weight: 27546.64 Da İ280 = 37150 M-1cm-1Anti-Albumin – Anti-PD-L1 – OVA251-270 (AP-OVA251-270) Sequence (No Start Codon). nAlb – XTEN Linker – nPD-L1 – OVA251-270– Ligation Tag MEVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS –Attorney Docket No.093386-0008-WO02 SGSETPGTSESA – QVQLQESGGGLVHPGGSLRLSCATSGSIFSIISMGWYRQAPGKQRELVALVFRGGSTVYADSV KGRFTISGDIAKSTVYLQMDSLKPEDTAVYYCNAKPIGTAQYWGQGTQVTVSS – GGGSGLEQLESIINFEKLTEWTSSGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:89) Formula: C1322H2060N370O434S9Molecular Weight: 30369.66 Da İ280 = 42650 M-1cm-1Anti-GFP-OVA251-270(nGFP-OVA251-270) Sequence (No Start Codon). nGFP-OVA251-270– Ligation Tag MQVQLQESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSS YEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS – GGGSGLEQLESIINFEKLTEWTSSGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:90) Formula: C758H1152N222O246S6Molecular Weight: 17503.19 Da İ280 = 32555 M-1cm-1Anti-PD-L1-OVA251-270(nPD-L1-OVA251-270) Sequence (No Start Codon). nPD-L1-OVA251-270– Ligation Tag MQVQLQESGGGLVHPGGSLRLSCATSGSIFSIISMGWYRQAPGKQRELVALVFRGGSTVYAD SVKGRFTISGDIAKSTVYLQMDSLKPEDTAVYYCNAKPIGTAQYWGQGTQVTVSS – GGGSGLEQLESIINFEKLTEWTSSGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:91) Formula: C752H1163N211O238S5Molecular Weight: 17128.08 Da İ280 = 25565 M-1cm-1Anti-GFP – Anti-PD-L1 (GP) Sequence (No Start Codon). nGFP – XTEN Linker – nPD-L1 – Ligation Tag MQVQLQESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSS YEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS – SGSETPGTSESA – QVQLQESGGGLVHPGGSLRLSCATSGSIFSIISMGWYRQAPGKQRELVALVFRGGSTVYADSV KGRFTISGDIAKSTVYLQMDSLKPEDTAVYYCNAKPIGTAQYWGQGTQVTVSS – LPETGGHHHHHHEPEA (SEQ ID NO:92) Formula: C1225H1888N352O391S10Molecular Weight: 28123.19 Da İ280 = 47120 M-1cm-1Anti-GFP – Anti-PD-L1 – OVA251-270(GP-OVA251-270) Sequence (No Start Codon). nGFP – XTEN Linker – nPD-L1 – OVA251-270– Ligation Tag MQVQLQESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSS YEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS – SGSETPGTSESA – QVQLQESGGGLVHPGGSLRLSCATSGSIFSIISMGWYRQAPGKQRELVALVFRGGSTVYADSV KGRFTISGDIAKSTVYLQMDSLKPEDTAVYYCNAKPIGTAQYWGQGTQVTVSS – GGGSGLEQLESIINFEKLTEWTSSGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:93) Formula: C1347H2076N384O436S10Molecular Weight: 30946.21 Da İ280 = 52620 M-1cm-1Attorney Docket No.093386-0008-WO02

[0186] Expression and Purification of Proteins.5 ^L of Kanamycin (stocked at 50 mg / mL) was added to a culture tube containing 5 mL 2xYT media and inoculated with a stab of protein (cloned into a NEB T7 Shuffle Express cell line). The culture was incubated at 30 ºC, with shaking at 250 RPM, for 16 hours. Each culture was transferred to a 2 L baffled flask containing 500 mL of 2xYT media and 500 ^L of Kanamycin (25 mg) and shaken at 30 ºC in an Innova 42R (New Brunswick Scientific) incubator for 4.5-5 hours (until the OD600 reached ~0.8). The cultures were then induced with IPTG (2.5 mM final concentration). The induced cultures were shaken overnight (20-24 hours) at 30 ºC. The bacteria were harvested the next day by centrifugation (3900 rpm for 10 min) and the pellet was reconstituted in 1x PBS with Dnase with a tablet of protease inhibitor cocktail (EDTA free). The cells were lysed by sonication on an ice bath in 5 second increments over 10 minutes. The resulting bacterial lysate was centrifuged (11000 rpm for 20 min) to remove cellular debris. The lysate was added to a 50 mL Kontes Flex column (Kimbal Kontes Glassware) containing 3 mL of Nickel NTA histidine binding resin that was preequilibrated with 1x PBS buffer. This column was placed on a rotating shaker at room temperature for 1-2 hrs. After this period, the supernatant was drained from the column using gravity and the column washed with 1x PBS buffer twice. Weakly bound proteins were first washed off of the resin using a low concentration elution buffer (2x 10 mL, 10 mM imidazole, 0.02% NaN3, 1x PBS pH 7.4 at 25 ºC). The bound protein was then eluted from the resin using elution buffer (15 mL, 150 mM imidazole, 0.02% NaN3, 1x PBS pH 7.4 at 25 ºC). The eluate was then concentrated to 0.5 mL in a 15 mL Microcon 10 kDa Centrifugal Filter Unit (Millipore) and subsequently purified by size exclusion chromatography (SEC) via an Akta FPLC (Cytiva), on a Hi-Load 16 / 60 Superdex 200 column using 1x PBS and 0.02% NaN3, pH 7.4 at 4 ºC as the running buffer. Pure fractions were determined by SDS-PAGE, pooled together with buffer exchange to 1x PBS not containing NaN3, and stocked at either -20 ºC or 4 ºC.

[0187] Enzymatic Bioconjugation and Click Chemistry Reactions. Bioconjugation reactions occurred in mild conditions (20 mM HEPES at pH 7.4, 150 mM NaCl, and 10 mM CaCl2) between eSrtA (100 ^M) and a nanobody containing a C-terminal ligation tag (75 ^M) using a primary amine containing functional group (20 mM). Reactions occurred with mixing by a rotary shaker overnight (16 h) and were quenched by the addition of a 1:1 volume of a chelating agent EDTA containing solution (20 mM HEPES at pH 7.4, 300 mM NaCl, and 10 mM EDTA) for one hour. After the reaction was stopped, the solution was concentrated, and buffer exchanged to 1x PBS (without NaCl or MgCl2) three times by centrifugal dialysis. The protein solution was then immobilized to Nickel NTA histidine binding resin over 2 hours, and unbound protein wasAttorney Docket No.093386-0008-WO02 collected by washing the resin with 1x PBS. For nanobodies that contain a histidine in the native sequence, proteins were eluted in mild conditions (10 mM Imidazole in 1x PBS). Collected protein was concentrated and buffer exchanged to 1x PBS by centrifugal dialysis and verified by ESI-MS and SDS-PAGE. Click chemistry reactions proceeded by the addition of 5 eq. (molar) of the complementary handle (e.g., if an azide was placed on the nanobody, the click chemistry reaction would proceed with the addition of 5 eq. of DBCO-containing moiety). After 48 hours of reaction between the protein azide and the DBCO-moiety, the mixture was purified by centrifugal dialysis four times, and verified for purity by UV-VIS, ESI-MS, and SDS-PAGE.

[0188] Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Protein samples were diluted in 1x PBS to 10 ^M before analysis.10 ^L of the protein sample was mixed with 10 ^L of reducing Laemmli buffer. Samples were boiled at 95 ºC for 5 minutes, 170 ^L and 15 ^L of each sample was loaded into a 15-well, 4-15% Tris-glycine precast SDS-PAGE (Biorad) and ran at a constant 150 V with 343 mA for 45 minutes. The gel was then directly stained using Coomassie-B-250 and imaged using a GelDoc EZ Imager (BioRad).

[0189] Electrospray Ionization Liquid Chromatography Mass Spectrometry (ESI-MS). Proteins were buffer exchanged into ammonium acetate (pH 5.5) and concentrated to approximately 100 ^M. ESI-MS data were collected using an Agilent 6210A time-of-flight (TOF) mass spectrometer at a range of 50-20,000 m / z over a period of two minutes. Data were analyzed with Agilent MassHunter IM-MS Acquisition Data software to reveal m / z data, where files were condensed across the two-minute run. These m / z data were deconvoluted using a maximum entropy deconvolution calculation using UniDec to give the deconvoluted mass spectra using background subtraction between a range of 1,000-5,000 m / z and with an export range of 5,000-50,000 Da.

[0190] Synthesis and NMR Verification of DBCO-PEG12-diABZI. First, a STING agonist was generated that features a reactive amine handle, which was synthesized in four steps. Briefly, aryl amination of an aryl chloride 1 with an amine 2 gave a dinitro analog, compound 3. The di- nitro compound 3 was subjected to reduction using sodium dithionite in methanol, generating a di-amine moiety 4. Compound 4 was then treated with isothiocyanate, followed by EDC coupling, to reveal a boc-protected analog, compound 5. Next, the boc-group from compound 5 was deprotected by treating with TFA:DCM. To a stirred solution of amine 6 (100 mg, 0.089 mmol, 1 eq.) in 5 mL DMF was added Hunig’s base (77 ^L, 0.44 mmol, 5 eq.) under argon atmosphere, at room temperature. After stirring for 5 min, a solution of activated NHS ester (98 mg, 0.098 mmol, 1.1 eq.) in DMF (5 mL) was added dropwise and stirred overnight (16 h). The solvent was evaporated to get crude product 7, which was purified by silica gel columnAttorney Docket No.093386-0008-WO02 chromatography using a mixture of methanol / dichloromethane as an eluent (5% to 25% MeOH) to get the desired product as a solid (70 mg, 0.042 mmol, yield 43%). (Rf = 0.5 in 20% MeOH in DCM).1H NMR (400 MHz, DMSO) į 8.01 – 7.93 (m, 2H), 7.88 (t, J = 5.7 Hz, 1H), 7.75 (t, J = 5.7 Hz, 1H), 7.67 – 7.60 (m, 4H), 7.49 – 7.42 (m, 3H), 7.38 – 7.27 (m, 7H), 6.49 (d, J = 7.1 Hz, 2H), 5.88 – 5.79 (m, 2H), 5.01 (d, J = 13.9 Hz, 1H), 4.91 (dd, J = 29.6, 4.2 Hz, 4H), 4.53 – 4.49 (m, 4H), 3.98 (t, J = 6.0 Hz, 2H), 3.72 (s, 3H), 3.60 – 3.57 (m, 2H), 3.54 (t, J = 6.5 Hz, 2H), 3.47 (broads, 46H), 3.30 – 3.26 (m, 2H), 3.14 – 3.05 (m, 4H), 2.26 (t, J = 6.5 Hz, 2H), 2.09 (s, 3H), 2.08 (s, 3H), 2.01 – 1.96 (m, 1H), 1.78 – 1.72 (m, 1H), 1.68 (p, J = 6.5 Hz, 2H), 1.28 – 1.27 (m,6H).13C NMR (151 MHz, DMSO) į 171.57, 171.50, 170.52, 168.06, 167.33, 152.50, 152.45, 152.06, 148.88, 145.50, 145.28, 144.65, 140.37, 140.33, 132.87, 130.54, 130.49, 130.07, 129.37, 128.62, 128.58, 128.44, 128.25, 128.13, 127.24, 125.60, 122.99, 121.86, 120.11, 120.04, 114.67, 109.72, 108.61, 106.00, 105.83, 105.58, 70.21, 70.14, 70.10, 70.00, 69.94, 69.45, 67.26, 56.45, 55.34, 53.85, 46.05, 45.05, 42.12, 38.95, 36.57, 35.61, 30.80, 30.17, 29.13, 18.46, 17.17, 16.58, 13.57, 12.74. HRMS (ESMS) Calculated for C84H111N15O21 [M+Na]+: 1688.7977, found 1688.7982.

[0191] Evaluation of Nanobodies in Tumor Models.6–8-week C57BL / 6 mice (The Jackson Laboratory) were inoculated with B16.F10-OVA tumors via subcutaneous (S.C.) administration of 5 x 105cancer cells, suspended in 100 ^L PBS, into the right flank of the mouse. Treatments began on Day 0 when the average tumor volume reached ~50-75 mm3. These mice were administered three S.C. injections of indicated vaccine constructs and / or three intraperitoneal (I.P.) injections of 100 ^g anti-PD-L1 (aPD-L1) immune checkpoint blockade (ICB) on Days 0, 4, and 8. Tumor volume calculations were calculated using Vtumor= L × W2× 0.5, in which Vtumoris tumor volume, L is tumor length, and W is tumor width. Tumor volume, total murine mass, and murine well-being were recorded for the duration of the study. The endpoint for maximum tumor volume (i.e. survival) during studies was 1500 mm3.

[0192] Statistics. All data were plotted, and statistical analysis performed using Prism 10 (GraphPad) software. Unless indicated in the figures, all data are presented as mean ± SEM. For comparisons between two groups, unpaired two-tailed Student’s t-tests were performed 374 as indicated. For multiple comparisons a one-way ANOVA was performed with post-hoc Tukey’s correction for multiple comparisons. For tumor volume, statistically significance was examined through a two-way ANOVA followed by Tukey’s adjustment for multiple comparisons. A Logrank (Mantel-Cox) test was used to compare Kaplan-Meyer survival data. Example 2Attorney Docket No.093386-0008-WO02 Bivalent Proteins and Conjugates Thereof

[0193] Synthesis of bispecific nanobody-antigen fusions and conjugates thereof. It was hypothesized, without being bound by a particular theory, that the fusion of a peptide antigen to a bispecific nanobody fusion protein containing both an albumin-binding domain – termed nAlb – and a domain targeting the immune checkpoint ligand PD-L1 – termed nPD-L1 – and subsequent conjugation of the stimulator of interferon genes (STING) agonist diABZI to this nAlb-nPD-L1-antigen fusion could generate a potent anticancer vaccine for several reasons:

[0194] 1) Fusion of peptide antigens to nAlb lead to increased antigen delivery to and presentation in the draining inguinal lymph node (iLN) after S.C. injection in the left base-of-tail.

[0195] 2) PD-L1 is highly expressed by many cancer types. As demonstrated herein, even after S.C. injection, much nAlb eventually ends up in circulation with a fairly long circulation half- life. This suggests that an nAlb-nPD-L1 fusion, injected S.C., will eventually enter circulation and enter the highly vascularized tumor microenvironment (TME). diABZI mediated STING activation in tumor cells has been demonstrated to reinvigorate the cancer immunity cycle (CIC), and peptide antigen uptake by tumor cells has the potential to “foreignize” tumor cells by inducing presentation of the exogenous peptide antigen on the tumor cell’s Class I Major Histocompatibility Complex (MHC), lending tumor cells more susceptible to T cell attack.

[0196] 3) PD-L1 is highly expressed by immunosuppressive APCs in the TME. Activating these APCs, in particular the tumor associated macrophages (TAMs), can be critical for the generation of a “hot” inflamed microenvironment as activated TAMs secrete many of the necessary cytokines and chemokines required to initiate potent antitumoral CD8+T cell infiltration into the TME. Additionally, many of these TAMs traffic to lymphoid tissues, where this vaccine can also localize, where they can present peptide antigen to prime CD8+T cells.

[0197] 4) The inherent blockade of the PD-L1 / PD-1 immune checkpoint induced by a PD-L1 targeting nanobody vaccine can mimic the effects of anti-PD-L1 ICB therapy. ICB therapies have been demonstrated to act synergistically with cancer vaccines.

[0198] To create this vaccine, nAlb was recombinantly expressed at the N-terminus of the fusion protein. At the C-terminus of the nAlb sequence, a XTEN linker (SGSETPGTSESA) (SEQ ID NO:100) was introduced, followed by nPD-L1. At the C-terminus of nPD-L1, the OVA251-270peptide antigen was expressed, flanked by two flexible spacers (GGGS) (SEQ ID NO:99). After the C-terminal GGGS spacer, a selective ligation tag (LPETGGHHHHHHEPEA) (SEQ ID NO:4) was cloned to include a site-specific substrate for an enzymatic engineered sortase pentamutant A (eSrtA) mediated ligation of any primary amine containing small molecule to the C-terminal of the nAlb-antigen fusion (Fig.27). It was demonstrated that this approach could beAttorney Docket No.093386-0008-WO02 used to facilitate the ligation of an NH2-PEG3-N3linker to the C-terminus of the nanobody, which was able to modularly ligate other cargos via strain-promoted azide-alkyne cycloaddition (SPAAC) to a dibenzocyclooctyne (DBCO) containing molecule. A variant of the STING agonist diABZI was synthesized that was functionalized with an azide-reactive DBCO group and a PEG12spacer (DBCO-PEG12-diABZI). Conjugation of the NH2-PEG3-N3and / or DBCO-PEG12- diABZI was confirmed via SDS PAGE and / or ESI-MS (FIG.28 and FIG.29).

[0199] nAlb-nPD-L1-OVA251-270-diABZI (AP-OVA251-270-diABZI) inhibits cancer progression and extends mouse survival in a B16.F10-OVA model of melanoma. Female 6–8-week C57BL / 6 mice were inoculated with 5 x 105B16.F10-OVA melanoma cells S.C. in the right flank. Tumors were measured every other day, and treatments began on Day 0 when the average tumor volume reached ~50-75 mm3. Mice were administered 1.25 ^g diABZI via S.C. injection, an equivalent 1.47 nmol nPD-L1 and / or peptide antigen via S.C. injection, and / or 100 ^g aPD-L1 ICB via I.P. injection on Days 0, 4, and 8 (FIG.30). Compared to a PBS control, all treatment groups except for aPD-L1 ICB alone significantly slowed tumor growth and extended survival, however none to the equivalent degree as AP-OVA251-270(FIG.31, FIG.32, and FIG.33).

[0200] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the disclosed technology, may be made without departing from the spirit and scope thereof.

[0201] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[0202] Clause 1. A protein-based composition comprising: a nanobody domain, the nanobody domain comprising an albumin-binding nanobody, wherein the albumin-binding nanobody is capable of specifically binding albumin; and a peptide antigen domain.

[0203] Clause 2. The protein-based composition of clause 1, wherein the albumin-binding nanobody has a binding affinity (Kd) to albumin of less than or equal to 100 nM at a pH of about 7 to about 8.

[0204] Clause 3. The protein-based composition of clause 1 or 2, wherein the albumin- binding nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 81.Attorney Docket No.093386-0008-WO02

[0205] Clause 4. The protein-based composition of any one of clauses 1-3, wherein the nanobody domain comprises a second nanobody, the second nanobody being capable of specifically binding to an immune checkpoint ligand, an antigen presenting cell ligand, or both.

[0206] Clause 5. The protein-based composition of clause 4, wherein the immune checkpoint ligand is CTLA-4, PD-1, PD-L1, B7-H3, B7-H4, HVEM, GITRL, CD80 / 86, CD155, PD-L2, Galectin 9, LAG3, TIM3, VISTA, TIGIT, PD1, MMR, or GITR.

[0207] Clause 6. The protein-based composition of clause 4, wherein the antigen presenting cell ligand is PD-L1, PD-L2, B7-H3, B7-H4, MMR, MHC-II, CD11b, CD11c, C-type lectin receptors, Fc receptors, complement receptors, scavenger receptors, PS receptors, B cell receptor, folate receptor, CD40, CD80, CD86, TLR2, TLR4, or TLR5.

[0208] Clause 7. The protein-based composition of any one of clauses 4-6, wherein the second nanobody comprises an amino acid sequence of SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:106, SEQ ID NO:107, or SEQ ID NO:108.

[0209] Clause 8. The protein-based composition of any one of clauses 1-7, wherein the peptide antigen domain comprises an autoantigen, a foreign antigen, a xenoantigen, an alloantigen, a tumor antigen, or a combination thereof.

[0210] Clause 9. The protein-based composition of any one of clauses 1-8, wherein the peptide antigen domain comprises a tumor antigen.

[0211] Clause 10. The protein-based composition of any one of clauses 1-9, wherein the peptide antigen domain comprises an amino acid sequence of SEQ ID NO:96, SEQ ID NO:97, or SEQ ID NO:98.

[0212] Clause 11. The protein-based composition of any one of clauses 1-10, wherein the nanobody domain is positioned N-terminal to the peptide antigen domain.

[0213] Clause 12. The protein-based composition of any one of clauses 4-11, comprising, in a N-terminus to a C-terminus direction: the albumin-binding nanobody; the second nanobody; and the peptide antigen domain.

[0214] Clause 13. The protein-based composition of any one of clause 4-12 comprising, in a N-terminus to a C-terminus direction: the nanobody domain, the nanobody domain comprising the albumin-binding nanobody, wherein the albumin-binding nanobody is capable of specifically binding albumin, and the second nanobody, wherein the second nanobody is capable of specifically binding to an immune checkpoint ligand; and the peptide antigen domain, the peptide antigen domain comprising a tumor antigen.

[0215] Clause 14. The protein-based composition of any one of clauses 1-13, wherein the nanobody domain is attached at its C-terminal end to the N-terminal end of the peptide antigenAttorney Docket No.093386-0008-WO02 domain through a first linker, the first linker comprising a peptide linker, a sortase moiety, the sortase moiety comprising an amino acid sequence of LPXT (SEQ ID NO:1), wherein X is any amino acid, or a combination thereof.

[0216] Clause 15. The protein-based composition of any one of clauses 4-14, wherein the albumin-binding nanobody is attached at its C-terminal end to the N-terminal end of the second nanobody through a peptide linker.

[0217] Clause 16. The protein-based composition of clause 14 or 15, wherein the peptide linker comprises an amino acid sequence of GGGS (SEQ ID NO:99), SGSETPGTSESA (SEQ ID NO:100), SLVR (SEQ ID NO: 101), SLVRYLL (SEQ ID NO:102), or a combination thereof.

[0218] Clause 17. The protein-based composition of any one of clauses 1-16 comprising an amino acid sequence that is at least 95% identical to any one of SEQ ID NO:84 to SEQ ID NO:87, SEQ ID NO:89 to SEQ ID NO:91, SEQ ID NO:93, or SEQ ID NO:109.

[0219] Clause 18. A conjugate, or a pharmaceutically acceptable salt thereof, comprising: the protein-based composition of any one of clauses 1-17; a drug; a second linker attaching the peptide antigen domain at its C-terminal end to the drug.

[0220] Clause 19. The conjugate of clause 18, or a pharmaceutically acceptable salt thereof, wherein the second linker comprises a sortase moiety, the sortase moiety comprising an amino acid sequence of LPXT (SEQ ID NO:1), wherein X is any amino acid.

[0221] Clause 20. The conjugate of clause 19, or a pharmaceutically acceptable salt thereof, wherein the second linker comprises a polyethylene glycol (PEG) moiety attaching the sortase moiety to the drug.

[0222] Clause 21. The conjugate of clause 20, or a pharmaceutically acceptable salt thereof, wherein the PEG moiety is of formula (a):wherein: n is 2 to 20.

[0223] Clause 22. The conjugate of any one of clauses 18-21, or a pharmaceutically acceptable salt thereof, wherein the drug comprises an immunomodulator, an agonist, an antagonist, an inhibitor, or a hormone.

[0224] Clause 23. The conjugate of any one of clauses 18-22, or a pharmaceutically acceptable salt thereof, wherein the immunomodulator comprises a STING agonist.

[0225] Clause 24. The conjugate of clause 23, or a pharmaceutically acceptable salt thereof, wherein the STING agonist is a dimeric amidobenzimidazole, 2’3’ cGAMP, 2’2’ cGAMP, 3’2’ cGAMP, 3’3’ cGAMP, c-di-GMP, c-di-AMP, ADU-S100, cIAMP 2-5, or ML RR-S2 CDA.Attorney Docket No.093386-0008-WO02

[0226] Clause 25. The conjugate of clause 23, or a pharmaceutically acceptable salt thereof, wherein the immunomodulator is a non-nucleotide STING agonist.

[0227] Clause 26. A pharmaceutical composition comprising: a protein-based composition according to any one of clauses 1-17 or a conjugate according to any one of clauses 18-25; and a pharmaceutically acceptable excipient.

[0228] Clause 27. A method of treating a disease or a disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the protein-based composition according to any one of clauses 1-17, optionally in combination with a pharmaceutically acceptable excipient.

[0229] Clause 28. The method of clause 27, wherein the disease or disorder is multiple sclerosis, Type I diabetes, organ transplantation, celiac disease, rheumatoid arthritis, lupus, inflammatory bowl disease, psoriasis, or graves’ disease.

[0230] Clause 29. A method of treating a disease or a disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the conjugate, or a pharmaceutically acceptable salt thereof, according to any one of clauses 18-25, optionally in combination with a pharmaceutically acceptable excipient.

[0231] Clause 30. The method of clause 27 or 28, wherein the disease or disorder is a cancer, a viral infection, or an infectious disease mediated by T-cell immunity.

[0232] Clause 31. The method of clause 30, wherein the cancer is melanoma, breast cancer, neuroblastoma, renal cell carcinoma, colon cancer, lung cancer, glioma, glioblastoma, or pancreatic cancer.

[0233] Clause 32. A method of modulating an immune system of a subject in need thereof, the method comprising administering to the subject an effective amount of the protein-based composition according to any one of clauses 1-17 or a conjugate according to any one of clauses 18-25, optionally in combination with a pharmaceutically acceptable excipient.

[0234] Clause 33. The method of clause 32, wherein modulating the subject’s immune system comprises increasing an amount of regulatory T cells in the subject specific to the peptide antigen domain, increasing an amount of CD8+ T cells in the subject, increasing the amount of CD4+ Th cells, or a combination thereof.

[0235] Clause 34. The method of any one of clauses 27-33, further comprising identifying, isolating, or both a peptide antigen from the subject and including the peptide antigen within the peptide antigen domain of the protein.Attorney Docket No.093386-0008-WO02 SEQUENCES LPXT (SEQ ID NO:1) LPET (SEQ ID NO:2) LPXTG (SEQ ID NO:3) LPETGGHHHHHHEPEA (SEQ ID NO:4) Further details of SEQ ID NO: 5 to SEQ ID NO: 75, such as CDRs, PI, hydrophobicity, etc., can be found in Shen et al., A resource of high-quality and versatile nanobodies for drug delivery, iScience, Volume 24, Issue 9, 24 September 2021, which is incorporated by reference herein in its entirety. Nanobody ID NO.13 AHVQLVESGGELVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGTEREFVAAISRSGGSTYYA DSVKGRFIISRDNAKNTVWLQMNMLKPEDTSVYYCAAAEGLASGSYDYAPPLKSSWYDYWGQ GTQVTVSEPKTPKGGCGGG (SEQ ID NO:5) Nanobody ID NO.18 AEVQLVESGGGLAQAGGSLRLSCAASGRTFSNECLGWFRQAPGKEREFVATIRSTGHTSYAD AVSGRFTVSRDIAKNTVYLEMSNLKPEDTAVYSCAAGFSDYGCYRTSGINYWGQGTQVTVSEP KTPKGGCGGG (SEQ ID NO:6) Nanobody ID NO.20 AQVQLVESGGGLVQAGGSLRLSCTASGRTFSSYYAMGWFRRAPGKEREFVAAISESGRTTDY ADSVKGRFTISRDTAKNTVYLQMISLKPEDTAVYYCAAAGPQEAFWFPSDYAQRALYDYWGQ GTQVTVSEPKTPKGGCGGG (SEQ ID NO:7) Nanobody ID NO.23 AQVQLVESGGGLVQAGDSLRLSCAASERTFGAHVMGWFRQAPGKEREFVATITSSGRNTRYA DSVKGRFTISSDNAKNTVYLQMISLEPEDTAVYYCAYAYGAGLYNIARQYDYWGQGTQVTVSE PKTPKGGCGGG (SEQ ID NO:8) Nanobody ID NO.27Attorney Docket No.093386-0008-WO02 AHVQLVESGGGLMQAGDSLRLSCAASGLTFSNYAMGWFRQAPGREREFVAALSWSGRNGY YADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAIYYCASAGGGGLYKIATQYDYWGQGTQVTV SEPKTPKGGCGGG (SEQ ID NO:9) Nanobody ID NO.29 AQVQLVESGGGLVQPGGSLRLSCVASGIMFDIYTMRWYRQAPGKQRELVAAITGAGRANYND DSVKGRFTISRDNAKNTVYLQMNRMKPEDTALYECNTEILGGGPNYWGRGTQVTVSEPKTPK GGCGGG (SEQ ID NO:10) Nanobody ID NO.33 ADVQLVESGGGLAQAGGSLRLSCAASGRTFSNSCMGWFRQAPGKEREFVVTIRSTGHTSYAD AVSGRFTVSRDIAKNTVYLEMNSLKPEDTAVYSCGAGVSDYGCYHTSGYKYWGQGTQVTVSE PKTPKGGCGGG (SEQ ID NO:11) Nanobody ID NO.35 AHVQLVESGGGLAQAGGSLRLSCAASGGTFSNSCMGWFRQAPGMEREFVATIRSTGHTTYA DSVEGRFTVSRDIAKNTVYLEMNSLKPEDTAVYSCGAGISDYGCYRTSGYNYWGQGTQVTVS EPKTPKGGCGGG (SEQ ID NO:12) Nanobody ID NO.36 AQVQLVESGGGLVQAGGSLRLSCRASGLPFGPYTMGWFRQTPGQEREFVAAITWSSMNTNY ADSVKGRFTISRDSAKNTVYLQMNTLKPDDTAVYYCAAAPGVGYYRHTFQYDYWGQGTQVTV SEPKTPKGGCGGG (SEQ ID NO:13) Nanobody ID NO.39 AHVQLVESGGGLVQAGGSLRVSCAASGREFSNYGMGWFRQAPGKEREFVATISWNGRITFY ADSVKGRFTISRDNAEKTGYLQMNSLKPEDTALYYCAAETSGWGSKVVPNYDYWGQGTQVTV SEPKTPKGGCGGG (SEQ ID NO:14) Nanobody ID NO.46 AQVQLVESGGGLVQAGGSLRLSCTIPGHTISSYIMGWFRQAPGKEREFVAAINWSGGRTNSAD SVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAVFDRISDSALPEERSRYDYWGQGTQVT VSEPKTPKGGCGGG (SEQ ID NO:15) Nanobody ID NO.47Attorney Docket No.093386-0008-WO02 ADVQLVESGGGLAQAGGSLRLSCAASGRTFSNSCMGWFRQAPGKEREFVATIRSTGHITYAD SVEGRFTVSRDIAKNTVYLEMSNLKPEDTAVYSCGAGVSDYGCYRTSGYNYWGQGTQVTVSE PKTPKGGCGGG (SEQ ID NO:16) Nanobody ID NO.48 AQVQLVESGGGLVQAGASLRLSCAASGGTFSSYIMGWFRQAPGREREFVAAISWSGRSTHYA DSVKGRFAISRDNDRVYLQMDSLKPEDTAVYSCAADPNYTWRDDRYYREEGYTYWGQGTQV TVSEPKTPKGGCGGG (SEQ ID NO:17) Nanobody ID NO.53 AQVQLVESGGGLVQSGGSLRLSCAASGSIGVTNTMGWYRQAPGKQRELAATITNDGNTNYAD SVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAGAGKRIPVSTMGWANDNEYEYWGRGTQ VTVSEPKTPKGGCGGG (SEQ ID NO:18) Nanobody ID NO.57 AQVQLVESGGGLVQAGGSLRVSCAASGSTFSNYGMGWFRQAPGKEREFVAVIAWIGGKTDY SDSVKGRFTIFRDNAKNTVYLQMNSLKPEDTAVYYCAATSYGTISRRSEYEYGYWGQGTQVTV SEPKTPKGGCGGG (SEQ ID NO:19) Nanobody ID NO.59 AQVQLVESGGGLVQAGGSLRLSCAASGRTFSTYHMGWFRQAPGKAREFVAAITENGGITYYA DSVRGRFTISRDDARNTVYLQMGSLKPEDTAVYYCAASSALIGRKYFGNENYSWGQGTQVTV SEPKTPKGGCGGG (SEQ ID NO:20) Nanobody ID NO.60 AQVQLVESGGGLVQAGGSLRLSCAASGDTFSTYGVAWFRQAPGKERELVAITPWMGSSTYYA DSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASSYGSISRRSDYEYGYWGQGTQVTV SEPKTPKGGCGGG (SEQ ID NO:21) Nanobody ID NO.61 AEVQLVESGGGLVQAGGSLRLSCAASGDTFGTYGVAWFRQAPGKEREFVAVTPWMGSNTYY ADSMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASSYGTVSRRSEYEYGYWGQGTQVT VSEPKTPKGGCGGG (SEQ ID NO:22) Nanobody ID NO.64Attorney Docket No.093386-0008-WO02 AQVQLVESGGGLVQAGDSLRLSCAASGRTFETHAMGWFRQAPGKEREFVATITPSGRSTSYG DSVKGRFTISSDNAKNTVYLQMNSLKPDDTAIYYCAFAYGVGLYKLARQYDYWGQGTQVTVSE PKTPKGGCGGG (SEQ ID NO:23) Nanobody ID NO.66 AQVQLVESGGGLVQAGDSLRLSCAASGRTFSTHGTGWFRQAPGKEREFVATITSSGRSTSYA DSTKGRFTISSDNAKNTVYLQMNSLKPEDTAIYYCAYAYGVGLYKFATQYDYWGQGTQVTVSE PKTPKGGCGGG (SEQ ID NO:24) Nanobody ID NO.68 AQVQLVESGGGLVQAGSSLRLSCAASGSTFSSHGMGWFRQAPGKEREFVATVSLSGRTTSY GDSVKDRFTISRDNAKNTVYLHMNSLKLEDTAVYYCAATSGGYYSRYAYDYYYWGQGTQVTV SEPKTPKGGCGGG (SEQ ID NO:25) Nanobody ID NO.69 AQVQLVESGGGLVQAGDSLRLSCAASGRTFETHAMGWFRQAPGKEREFVATITPSGRSTSYA DSVKGRYTISSDNAKNTVYLQMDSLKPEDTAIYYCAFAYGVGLYKIARQYDYWGQGTQVTVSE PKTPKGGCGGG (SEQ ID NO:26) Nanobody ID NO.75 AEVQLVESGGGLVQAGGSLRLSCAASGRTYSPLVMGWFRQAPGKEREFVATITPSGGSLSYA DSVTGRFTVSRDNAKKTVFLQMNSLKPEDTAIYYCAAAPGVGNYRYARQYDYWGQGTQVTVS EPKTPKGGCGGG (SEQ ID NO:27) Nanobody ID NO.76 AQVQLVESGGGLVQAGGSLRLSCTASGRTFTPYTMGWFRQAPGKEREFAASILWSGKNTDYA DSVKGRFAISKDNAKNTVYLQMNKLKPEDTAVYYCATGDGLGFYRSVSQYDYWGQGTQVTVS EPKTPKGGCGGG (SEQ ID NO:28) Nanobody ID NO.77 AQVQLVESGGGLVQAGGSLRLSCTASGRTYEPLVMGWFRQAPGKEREFVATITPSGGSLSYA DSVKGRFTVSRDNAKKTVYLQMNRLQPEDTAVYYCAAAPGVGNYRYTRQYDYWGQGTQVTV SEPKTPKGGCGGG (SEQ ID NO:29) Nanobody ID NO.78Attorney Docket No.093386-0008-WO02 AQVQLVESGGGLVQAGGSLRLSCTASGRTFTPYTIGWFRQAPGKEREFVASILWSGINTDYAD SVKGRFAISRDNAKNAAYLQMSNLKPEDTAVYYCATGGGLGYYRSVSQYDYWGQGTQVTVS EPKTPKGGCGGG (SEQ ID NO:30) Nanobody ID NO.80 AEVQLVESGGGLVQAGGSLRLSCTASGRTFTPYTMGWFRQAPGKEREFVASILWSGNNRDY ADSVKGRFAISRDNAKNTAYLQMTSLKPEDTAVYYCAAGDGLGFYRSVNQYDYWGQGTQVTV SEPKTPKGGCGGG (SEQ ID NO:31) Nanobody ID NO.81 AQVQLVESGGGLVQAGGSLKLSCTASGRTFMPYTMGWFRQVPGKEREFVASVLWSGINTDY AESVKGRFAISKDNAKNTMYLQMNSLKPEDTAVYYCAAGDGLGYYRSVSQYDYWGHGTQVT VSEPKTPKGGCGGG (SEQ ID NO:32) Nanobody ID NO.82 AHVQLVESGGGLVQAGGSLRLSCAASGRTFSTYHMGWFRQAPGKAREFVAAITESGGITYYA DSVKGRFTVSRDNAKNTVDLQMNSLKPEDTAVYYCAAAPGVGAYRHATQYDYWGQGTQVTV SEPKTPKGGCGGG (SEQ ID NO:33) Nanobody ID NO.85 AQVQLVESGGGLVQAGGSLRLSCVASGRTFEPFVMGWFRQAPGKEREFVATISWSGGSLSY ADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCAAAPGVGNYRYTFQYDYWGQGTQVT VSEPKTPKGGCGGG (SEQ ID NO:34) Nanobody ID NO.86 AQVQLVESGGGLVQAGGSLRLSCTASGRTFTPYTMGWFRQTPGKEREFAASILWSGINTDYA DSVKGRFAISKDNAKNTVYLQMNSLKPEDTAVYYCAAAYGLGYYRSVSQYDYWGQGTQVTVS EPKTPKGGCGGG (SEQ ID NO:35) Nanobody ID NO.88 AQVQLVESGGGLVQAGGSLRLSCTASGRTFTPYTMGWFRQAPGKEREFAASILWSGENTDYA DSVKGRFAISRDGAKNTVYLQMNSLKPEDTAVYYCASGYGLGFYRSASQYDYWGQGTQVTVS EPKTPKGGCGGG (SEQ ID NO:36) Nanobody ID NO.89Attorney Docket No.093386-0008-WO02 AQVQLVESGGGLVQAGGSLRLSCTASGRTFTPYTMGWFRQAPGKEREFAASILWSGINTDYA DSVKGRFAISRDNAKNTVYLQMNSLKPEDTGVYYCAAADGLGLYRFVSQYDDWGQGTQVTVS EPKTPKGGCGGG (SEQ ID NO:37) Nanobody ID NO.90 AQVQLVESGGGLVQAGGSLRLSCTASGRTFTPYTMGWFRQAPGKEREFAASILWSGINTDYA DSVKGRFAISRDNAKNTVYLQMNSLKPEDTGVYYCATADGLGLYRFVSQYDYWGQGTQVTVS EPKTPKGGCGGG (SEQ ID NO:38) Nanobody ID NO.91 AQVQLVESGGGLVQAGGSLRLSCAASGRTFSPLVMGWFRQAPGHEREFVATITPSGGSQSYA DSVKGRFAVSRDNAKKTVYLQMNSLKPEDTAVYYCAAAPGVGIYRYTSQYDYWGQGTQVTVS EPKTPKGGCGGG (SEQ ID NO:39) Nanobody ID NO.92 AQVQLVESGGGLVQPGKSLRLSCTTSGLPGSWYTLGWFRQVPGKEREFVASVLWSGINTDYA DSVKGRFAISRNNAKNTMYLQMNSLKPEDTAVYYCAAGYGLGFYRSVSQYDYWGHGTQVTV SEPKTPKGGCGGG (SEQ ID NO:40) Nanobody ID NO.93 AQVQLVESGGGLVQAGGSLRLSCSASGSTFSPFVIGWFRQAPGKEREFVGGVRPSGSQYYS DSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCAAAAGVGNYRHTWQYDYWGQGTQVTV SEPKTPKGGCGGG (SEQ ID NO:41) Nanobody ID NO.96 AHVQLVESGGGLVQAGGSLRLSCAASGHTFGPYTMGWFRQTPGKEREFVAAITWSGTSTNY ADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCAAGSGAGRTSMHTSMTTGARGPRSPS EPKTPKGGCGGG (SEQ ID NO:42) Nanobody ID NO.98 AQVQLVESGGGLVQAGDSLRLSCVASGRTFSTYHMGWFRQAPGKAREFVAAITQSGITYYAD SVKGRFTISRDNAKNTAYLQMGSLQPEDTAVYYCAASPKLIGRIYFGNENYSWGQGTQVTVSE PKTPKGGCGGG (SEQ ID NO:43) Nanobody ID NO.99Attorney Docket No.093386-0008-WO02 AQVQLVESGGGLVQAGGSLRLSCAASGRTFSTYHMGWFRQAPGKAREFVAAITQSGGITYYA DSVKGRFTISRDDAKNTVYLQMGSLEPEDTAVYYCAASPTLIGRVYFGNENYSWGQGTQVTVS EPKTPKGGCGGG (SEQ ID NO:44) Nanobody ID NO.100 AQVQLVESGGGLVQAGGSLRLSCAASGRTFSTYHMGWFRQAPGKAREFVAAITGSGGITYYA DSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASVALIGRVYFGNENYSWGQGTQVTVS EPKTPKGGCGGG (SEQ ID NO:45) Nanobody ID NO.101 AQVQLVESGGGLVQAGGSLRLSCAASGRTFSTYHMGWFRQAPGKEREFVAAITQSGGITYYA DAMKGRFTISRDDAKNTVYLQMGSLKPEDTAVYYCVASPALIGRHYFGNENYSWGQGTQVTV SEPKTPKGGCGGG (SEQ ID NO:46) Nanobody ID NO.102 AHVQLVESGGGLVQAGGSLRLSCAASERTFSTYHMGWFRQAPGKGREFVAAITPSGGVTYYA DNLKGRFTISGDNAKNTVYLQMTNLKPEDTAVYYCVASPALIGRVYFGNENYSWGQGTQVTVS EPKTPKGGCGGG (SEQ ID NO:47) Nanobody ID NO.104 AEVQLVESGGGLAQAGGSLRLSCAASGRTFSNSCMGWFRQAPGKEREFVATIRSTGHASYAD SVEGRFTVSRDIAKNTVYLEMNSLKPDDTAVYICGAGVSDYGCYRTSGYNSWGQGTQVTVSE PKTPKGGCGGG (SEQ ID NO:48) Nanobody ID NO.108 AQVQLVESGGGLAQAGGSLRLSCAASGGTFSNNCMGWFRQAPGMEREFVAIIRSTGHTTYAD SVEGRFTVSRDIAKNTVYLEMNSLKPEDTAVYICAAGASDYGCYRTSGINYWGQGTQVTVSEP KTPKGGCGGG (SEQ ID NO:49) Nanobody ID NO.113 AHVQLVESGGGLVQTGGSLRLSCVASGGIFSNSCMGWFRQAPGMERQFVAIIRSTGHTTYAD SVEGRFTVSRDIAKNTVYLEMNSLKPEDTAVYYCAAGVSDYGCYRTSGINYWGQGTQVTVSE PKTPKGGCGGG (SEQ ID NO:50) Nanobody ID NO.117Attorney Docket No.093386-0008-WO02 AQVQLVESGGGLVQAGGSLRLSCAASGRTFSTYHMGWFHQAPGKAREFVAAITESGGITYYA DSVKGRFTISRDIAKNTVNLEMNSLKPEDTAVYSCAAGISDYGCYRTSGIAYWGQGTQVTVSEP KTPKGGCGGG (SEQ ID NO:51) Nanobody ID NO.118 AQVQLVESGGGLVQAGGSLRLSCAASGFSFSSYGMGWFRQAPGKEREFVAAIGWIGSRTSYA DSVKGRFTISKDNAKNTVYLQMDSLRPEDTAVYTCAATSYLNPDSDYARSDRSYGYRGQGTQ VTVSEPKTPKGGCGGG (SEQ ID NO:52) Nanobody ID NO.120 AEVQLVESGGGLVQAGDSLRLSCVASGRTFSTYHMGWFRQAPGKAREFVAAITQSGITYYAD SVKGRFTISRDNAKNTAYLQMGSLQPEDTAVYYCAASPLLIGRVYFGNEDYSWGPQRISEATT GARGPRSPSEPKTPKGGCGGG (SEQ ID NO:53) Nanobody ID NO.122 AQVQLVESGGGLVQAGGSLRLSCAASGRTFSTYHMGWFRQAPGKAREFVAAITGSGGITYYA DSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASAALIGRVYFGNEITPGARGPRSPSEP KTPKGGCGGG (SEQ ID NO:54) Nanobody ID NO.124 AQVQLVESGGGLVQAGGSLRLSCAASERTFSSYAMGWFRQGPGKEREFVAYIHWSGGRTLV VDSVKGRFTISRDNTKNTMYLQMNSLKPADTAVYYCTADQYASTLLRGTGEYWGQGTQVTVS EPKTPKGGCGGG (SEQ ID NO:55) Nanobody ID NO.125 AQVQLVESGGGLVQAGGSLRLSCAAPGDIFSMYVMGWFRQAPGKEREFVAYNHWSGGRTLY ADSVKGRFTISRDNSKNTMSLQMNSLRPEDTAVYYCTADQYASTLLRAAGEYWGQGTQVTVS EPKTPKGGCGGG (SEQ ID NO:56) Nanobody ID NO.126 AQVQLVESGGGLVQAGGSLRLSCAASGLTFSNYVMGWFRQAPGKEREFVAYIHWSGGRILYA DSVKGRFTISRDNTKNTMYLQMNSLKPDDTAVYYCTADQYATTVLRAAGEYWGQGTQVTVSE PKTPKGGCGGG (SEQ ID NO:57) Nanobody ID NO.129Attorney Docket No.093386-0008-WO02 AQVQLVESGGGLVQAGGSLRLSCVASGRTFSPYTTGWFRQAPGKEREFVAAITWSGRSTNYA ASVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCSAGAGGGIYTIRGQYDYWGQGTQVTVS EPKTPKGGCGGG (SEQ ID NO:58) Nanobody ID NO.132 AHVQLVESGGGLVQAGGSLRLSCAASGGTISNYGMGWLRQGPGKEREFVGSINWNGATTHY ADSVKGRFIISRDNAKNTVYLQMNSLKPEDTGVYYCVAQFSVQPTLRTYDYGGQGTQVTVSEP KTPKGGCGGG (SEQ ID NO:59) Nanobody ID NO.133 AHVQLVESGGGLVQAGGSLRLSCAASGLTFRNYAMGWFRRAPGKERDFVAAISYSGGSTDYA DSVKGRFTISRDNAKNTVFLQMSSLKPEDTAVYYCAASPVVYGSLWFKRESYTYWGQGTQVT VSEPKTPKGGCGGG (SEQ ID NO:60) Nanobody ID NO.135 ADVQLVESGGGLVQAGGSLRLSCRASGLPFGPYTMGWFRQTPGQEREFVAAITWSSMNTNY ADSVKGRFTISRDNAKNTVLLQMNSLKPEDTAVYYCAAAEGLASGSYDYFPPLKSSWYDYWG QGTQVTVSEPKTPKGGCGGG (SEQ ID NO:61) Nanobody ID NO.136 AQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVGCISSSDGSPTYA DSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATHFRSANSCFLYYYDMDYSGKGTQVTV SEPKTPKGGCGGG (SEQ ID NO:62) Nanobody ID NO.138 AQVQLVESGGGLVQAGDSLRLSCAASGRTFVAHAMGWFRQAPGKERTFVAMITSSGLTISYA DPVKGRFTISSDNAKNTVYLQMNSLKPEDTAIYYCAFAYGVGKYEIARQYDYWGQGTQVTVSE PKTPKGGCGGG (SEQ ID NO:63) Nanobody ID NO.139 AQVQLVESGGGAVQAGGSLQLSCRASGRTFSPYVMGWFRQAPGKEREFVGLITWSGGTSYA DSVRGRFTASRDRVKNTVYLQMNSLKPEDTAVYYCAAAYGAGYYVHERQYDYWGQGTQVTV SEPKTPKGGCGGG (SEQ ID NO:64) Nanobody ID NO.140Attorney Docket No.093386-0008-WO02 AQVQLVESGGGLVQAGGSLRLSCTIPGHTISSYIMGWFRQAPGKEREFVAAINWSGGRTNSAD SVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAVFDRLSDSALPEERSRYDYWGQGTQV TVSEPKTPKGGCGGG (SEQ ID NO:65) Nanobody ID NO.141 AEVQLVESGGGLAQAGGSLRLSCAASGGTFSNSCMGWFRQAPGMEREFVAIIRSTGHTTYAD SVEGRFTISRDNAKNTVYLEMNSLKPEDTAVYYCAAERWTGACSGAGLHLRSFTSWGQGTQV TVSEPKTPKGGCGGG (SEQ ID NO:66) Nanobody ID NO.143 AQVQLVESGGGLVQAGGSLSVSCAASGRTFRSYVGWFRQAPGKERTFVAGIRWSAGDTYYA DSMKGRFTISRDSAKNTVYLQMNSLKPEDTAVYYCAAAGPQQAFWFPSDYAQRALYDYWGQ GTQVTVSEPKTPKGGCGGG (SEQ ID NO:67) Nanobody ID NO.144 AHVQLVESGGGLVQAGGSLRLSCAASGRTFIPYTTGWFRQTPGKEREFVATITWSGISTKFAD SVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASGPQEAFWFPSDYAQRALYDYWGQGT QVTVSEPKTPKGGCGGG (SEQ ID NO:68) Nanobody ID NO.145 AQVQLVESGGGLVQAGGSLRLSCRASGLPFGPYTMGWFRQTPGQEREFVAAITWSSMNTNY ADSVKGRFTISRDSAKNTVYLQMNSLKREDTAVYYCAAAGPQEAFWFPSDYAQRALYDYWGQ GTQVTVSEPKTPKGGCGGG (SEQ ID NO:69) Nanobody ID NO.147 AHVQLVESGGGLVQPGGSLRLSCVASGIMFDIYTMRWYRQAPGKQRELVAAITGAGRANYND DSVKGRFTISRDNAKNTVYLQMNRMKPEDTALYECNTEILGGGRNYWGRGTQVTVSEPKTPK GGCGGG (SEQ ID NO:70) Nanobody ID NO.148 AQVQLVESGGGLVQAGDSLRLSCAVSGRAFSNDIFGWFRQAPGLEREFVAAHRWNALYADSV KGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAGAGKRFPVSAMGWANDNEYEYWGRGTQV TVSEPKTPKGGCGGG (SEQ ID NO: 71) Nanobody ID NO.150Attorney Docket No.093386-0008-WO02 AHVQLVESGGGLVQAGGSLRLSCAVSGRTFSNDIIGWFRQAPGKDREFVAAHRYNALYADSV KGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAGAGKRYPVSTMGWANDNEYEYWGRGTQV TVSEPKTPKGGCGGG (SEQ ID NO:72) Nanobody ID NO.151 AQVQLVESGGGLAQAGGSLRLSCAASGGTFSNSCMGWFRQAPGMEREFVATIRSTGHTTYA DSVEGRFTVSRDIAKNTVYLEMNSLKREDTAVYTCAAGVSDYGCYHTSGYNYWGQGTQVTVS EPKTPKGGCGGG (SEQ ID NO:73) Nanobody ID NO.154 AQVQLVESGGGLVQAGGSLRLSCTVPGHTISSYIMGWFRQAPGKEREFVAAINWNGGRTNSA DSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAVFDRISDSFLPEERSTYDYWGQGTQV TVSEPKTPKGGCGGG (SEQ ID NO:74) Nanobody ID NO.158 ADVQLVESGGGLVQAGGSLRLSCAASGGTISNYGMGWLRQGPGKEREFVGSINWNGATTHY ADSVKGRFIISRDNAKNTVYLQMNSLKPEDTGVYYCVAQFSVQPTLQTYDYRGQGTQVTVSEP KTPKGGCGGG (SEQ ID NO:75) aAlbumin-eSrtA Tag (Alb1) EVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYA DSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSLPETGGHHH HHHEPEA (SEQ ID NO:76) aAlbumin-eSrtA Tag (M75) QVQLVESGGGFVQAGGSLRLSCAASGRTFDNYVMAWFRQAPGKEREFVASISGSGSITNYAN SVKDRFTISRDSAKNAIYLQMNSLKPEDTALYYCAAGSRRTYYREPKFYPSWGQGTQVTVSSL PETGGHHHHHHEPEA (SEQ ID NO:77) aAlbumin-mCherry-eSrtA Tag (Alb1) EVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYA DSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSSGSETPGTS ESAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPF AWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFI YKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYAttorney Docket No.093386-0008-WO02 KAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKLPETGGHHHHHHE PEA (SEQ ID NO:78) aAlbumin-mCherry-eSrtA Tag (M75) QVQLVESGGGFVQAGGSLRLSCAASGRTFDNYVMAWFRQAPGKEREFVASISGSGSITNYAN SVKDRFTISRDSAKNAIYLQMNSLKPEDTALYYCAAGSRRTYYREPKFYPSWGQGTQVTVSSS GSETPGTSESAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKV TKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQD SSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGH YDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKLPETG GHHHHHHEPEA (SEQ ID NO:79) Anti-Albumin (nAlb) Sequence (No Start Codon / No Ligation Tag). nAlb MEVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS (SEQ ID NO:80) Anti-Albumin (nAlb) Sequence (No Start Codon). nAlb – Ligation Tag MEVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS – LPETGGHHHHHHEPEA (SEQ ID NO:81) Engineered Sortase A (eSrtA) Sequence (No Start Codon). eSrtA – HisTag QAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLNRGVSFAEENESLDDQNISIAGHTFIDR PNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVEVLDEQKGKDKQLTLITCDDYN EETGVWETRKIFVATEVKLE – HHHHHH (SEQ ID NO:82) Anti-GFP (nGFP) Sequence (No Start Codon). nGFP – Ligation Tag MQVQLQESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSS YEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS – LPETGGHHHHHHEPEA (SEQ ID NO:83) Anti-Albumin-OVA251-270(nAlb-OVA251-270) Sequence (No Start Codon). nAlb-OVA251-270– Ligation TagAttorney Docket No.093386-0008-WO02 MEVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS – GGGSGLEQLESIINFEKLTEWTSSGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:84) Anti-Albumin-MOG35-55(nAlb-MOG35-55) Sequence (No Start Codon). nAlb-MOG35-55– Ligation Tag MEVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS – GGGSMEVGWYRSPFSRVVHLYRNGKGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:85) Anti-GFP-OVA251-270(nGFP-OVA251-270) Sequence (No Start Codon). nGFP-OVA251-270– Ligation Tag MQVQLQESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSS YEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS – GGGSGLEQLESIINFEKLTEWTSSGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:86) Anti-GFP-MOG35-55(nGFP-MOG35-55) Sequence (No Start Codon). nGFP-MOG35-55– Ligation Tag MQVQLQESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSS YEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS – GGGSMEVGWYRSPFSRVVHLYRNGKGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:87) Anti-Albumin – Anti-PD-L1 (AP) Sequence (No Start Codon). nAlb – XTEN Linker – nPD-L1 – Ligation Tag MEVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS – SGSETPGTSESA – QVQLQESGGGLVHPGGSLRLSCATSGSIFSIISMGWYRQAPGKQRELVALVFRGGSTVYADSV KGRFTISGDIAKSTVYLQMDSLKPEDTAVYYCNAKPIGTAQYWGQGTQVTVSS – LPETGGHHHHHHEPEA (SEQ ID NO:88) Anti-Albumin – Anti-PD-L1 – OVA251-270(AP-OVA251-270) Sequence (No Start Codon). nAlb – XTEN Linker – nPD-L1 – OVA251-270 – Ligation Tag MEVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS – SGSETPGTSESA – QVQLQESGGGLVHPGGSLRLSCATSGSIFSIISMGWYRQAPGKQRELVALVFRGGSTVYADSV KGRFTISGDIAKSTVYLQMDSLKPEDTAVYYCNAKPIGTAQYWGQGTQVTVSS – GGGSGLEQLESIINFEKLTEWTSSGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:89) Anti-GFP-OVA251-270(nGFP-OVA251-270) Sequence (No Start Codon). nGFP-OVA251-270– Ligation Tag MQVQLQESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSS YEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS – GGGSGLEQLESIINFEKLTEWTSSGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:90)Attorney Docket No.093386-0008-WO02 Anti-PD-L1-OVA251-270(nPD-L1-OVA251-270) Sequence (No Start Codon). nPD-L1-OVA251-270– Ligation Tag MQVQLQESGGGLVHPGGSLRLSCATSGSIFSIISMGWYRQAPGKQRELVALVFRGGSTVYAD SVKGRFTISGDIAKSTVYLQMDSLKPEDTAVYYCNAKPIGTAQYWGQGTQVTVSS – GGGSGLEQLESIINFEKLTEWTSSGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:91) Anti-GFP – Anti-PD-L1 (GP) Sequence (No Start Codon). nGFP – XTEN Linker – nPD-L1 – Ligation Tag MQVQLQESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSS YEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS – SGSETPGTSESA – QVQLQESGGGLVHPGGSLRLSCATSGSIFSIISMGWYRQAPGKQRELVALVFRGGSTVYADSV KGRFTISGDIAKSTVYLQMDSLKPEDTAVYYCNAKPIGTAQYWGQGTQVTVSS – LPETGGHHHHHHEPEA (SEQ ID NO:92) Anti-GFP – Anti-PD-L1 – OVA251-270(GP-OVA251-270) Sequence (No Start Codon). nGFP – XTEN Linker – nPD-L1 – OVA251-270– Ligation Tag MQVQLQESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSS YEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS – SGSETPGTSESA – QVQLQESGGGLVHPGGSLRLSCATSGSIFSIISMGWYRQAPGKQRELVALVFRGGSTVYADSV KGRFTISGDIAKSTVYLQMDSLKPEDTAVYYCNAKPIGTAQYWGQGTQVTVSS – GGGSGLEQLESIINFEKLTEWTSSGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:93) Anti-PD-L1 (nPD-L1) (no start codon / no ligation tag) QVQLQESGGGLVHPGGSLRLSCATSGSIFSIISMGWYRQAPGKQRELVALVFRGGSTVYADSV KGRFTISGDIAKSTVYLQMDSLKPEDTAVYYCNAKPIGTAQYWGQGTQVTVSS (SEQ ID NO: 94) Anti-B7H3 (no start codon / no ligation tag) QEQLKESGGRLVTPGTPLTLTCTVSGFSPNNYGVSWVRQPPGKGLEWIGMSSTAGATYYAN WAKGRFTISKTSTTVDLEITSPTTEDTATYFCAKGTPSLSYGNIWGPGTLVTVSS (SEQ ID NO: 95) OVA251-270GLEQLESIINFEKLTEWTSS (SEQ ID NO: 96) OVA323-339ISQAVHAAHAEINEAGR (SEQ ID NO: 97)Attorney Docket No.093386-0008-WO02 MOG35-55MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 98) Peptide Linkers GGGS (SEQ ID NO:99) SGSETPGTSESA (SEQ ID NO:100) SLVR (SEQ ID NO: 101) SLVRYLL (SEQ ID NO:102) Synthetic azide-antigens N3-GGGS-GLEQLESIINFEKLTEWTSS (SEQ ID NO:103) N3-GGGS-ISQAVHAAHAEINEAGR (SEQ ID NO:104) N3-GGGS-MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO:105) Anti-Class II Major Histocompatibility Complex (nMHCII) Sequence (No Start Codon / no ligation tag). nMHCII – Ligation Tag MQVQLQESGGGLVQAGDSLRLSCAASGRTFSRGVMGWFRRAPGKEREFVAIFSGSSWSGRS TYYSDSVKGRFTISRDNAKNTVYLQMNGLKPEDTAVYYCAAGYPEAYSAYGRE STYDYWGQGTQVTVSSGG (SEQ ID NO:106) Formula: C708H1059N207O223S5Molecular Weight: 16198.75 Da İ280 = 31525 M-1cm-1Anti-CD11b (nCD11b) Sequence (No Start Codon / no ligation tag). nCD11b – Ligation Tag MQVQLQESGGGLVQAGGSHNLSCTASGITFSSLAMGWFRQTPGKEREFVANIMRSGSSVFYA DSVRGRFTISRDNAKNTAHLQMNSLKPEDTAVYFCAATRGAWPAEYWGQGTQVTVSSGG (SEQ ID NO:107) Formula: C640H973N193O200S6Molecular Weight: 14763.30 Da İ280 = 21095 M-1cm-1Attorney Docket No.093386-0008-WO02 Anti-Macrophage Mannose Receptor (CD206) (nMMR) Sequence (No Start Codon / no ligation tag). nMMR – Ligation Tag MQVQLQESGGGSVQAGGSLRLSCTGSRYTYTMGWFRQAPGKEREGVVAITAFGSPFYADSV KGRFTISRDNANNTIFLQMNSLKPEDSAMYYCAARGSSGTSYKWNEYGSYNYWGQGTQVTVS SGG (SEQ ID NO:108) Formula: C675H1006N196O214S6Molecular Weight: 15482.96 Da İ280 = 30035 M-1cm-1Anti-Albumin – Anti-GFP – OVA251-270(AG-OVA251-270) Sequence (No Start Codon) MEVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSSGSETPGT SESAQVQLQESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGD RSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSSGG GSGLEQLESIINFEKLTEWTSSGGGSLPETGGHHHHHHEPEA (SEQ ID NO:109) SIINFEKL (SEQ ID NO:110) (BsaI – GGTCTC) (SEQ ID NO: 111)

Claims

Attorney Docket No.093386-0008-WO02 CLAIMS What is claimed is:

1. A protein-based composition comprising: a nanobody domain, the nanobody domain comprising an albumin-binding nanobody, wherein the albumin-binding nanobody is capable of specifically binding albumin; and a peptide antigen domain.

2. The protein-based composition of claim 1, wherein the albumin-binding nanobody has a binding affinity (Kd) to albumin of less than or equal to 100 nM at a pH of about 7 to about 8.

3. The protein-based composition of claim 1, wherein the albumin-binding nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NO:5 to SEQ ID NO:

81.

4. The protein-based composition of claim 1, wherein the nanobody domain comprises a second nanobody, the second nanobody being capable of specifically binding to an immune checkpoint ligand, an antigen presenting cell ligand, or both.

5. The protein-based composition of claim 4, wherein the immune checkpoint ligand is CTLA- 4, PD-1, PD-L1, B7-H3, B7-H4, HVEM, GITRL, CD80 / 86, CD155, PD-L2, Galectin 9, LAG3, TIM3, VISTA, TIGIT, PD1, MMR, or GITR.

6. The protein-based composition of claim 4, wherein the antigen presenting cell ligand is PD-L1, PD-L2, B7-H3, B7-H4, MMR, MHC-II, CD11b, CD11c, C-type lectin receptors, Fc receptors, complement receptors, scavenger receptors, PS receptors, B cell receptor, folate receptor, CD40, CD80, CD86, TLR2, TLR4, or TLR5.

7. The protein-based composition of claim 4, wherein the second nanobody comprises an amino acid sequence of SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:106, SEQ ID NO:107, or SEQ ID NO:108.Attorney Docket No.093386-0008-WO02 8. The protein-based composition of claim 1, wherein the peptide antigen domain comprises an autoantigen, a foreign antigen, a xenoantigen, an alloantigen, a tumor antigen, or a combination thereof.

9. The protein-based composition of claim 1, wherein the peptide antigen domain comprises a tumor antigen.

10. The protein-based composition of claim 1, wherein the peptide antigen domain comprises an amino acid sequence of SEQ ID NO:96, SEQ ID NO:97, or SEQ ID NO:

98.

11. The protein-based composition of claim 1, wherein the nanobody domain is positioned N- terminal to the peptide antigen domain.

12. The protein-based composition of claim 4, comprising, in a N-terminus to a C-terminus direction: the albumin-binding nanobody; the second nanobody; and the peptide antigen domain.

13. The protein-based composition of claim 4, comprising, in a N-terminus to a C-terminus direction: the nanobody domain, the nanobody domain comprising the albumin-binding nanobody, wherein the albumin-binding nanobody is capable of specifically binding albumin, and the second nanobody, wherein the second nanobody is capable of specifically binding to an immune checkpoint ligand; and the peptide antigen domain, the peptide antigen domain comprising a tumor antigen.

14. The protein-based composition of claim 1, wherein the nanobody domain is attached at its C-terminal end to the N-terminal end of the peptide antigen domain through a first linker, the first linker comprising a peptide linker, a sortase moiety, the sortase moiety comprising an amino acid sequence of LPXT (SEQ ID NO:1), wherein X is any amino acid, or a combination thereof.Attorney Docket No.093386-0008-WO02 15. The protein-based composition of claim 4, wherein the albumin-binding nanobody is attached at its C-terminal end to the N-terminal end of the second nanobody through a peptide linker.

16. The protein-based composition of claim 15, wherein the peptide linker comprises an amino acid sequence of GGGS (SEQ ID NO:99), SGSETPGTSESA (SEQ ID NO:100), SLVR (SEQ ID NO: 101), SLVRYLL (SEQ ID NO:102), or a combination thereof.

17. The protein-based composition of claim 1, comprising an amino acid sequence that is at least 95% identical to any one of SEQ ID NO:84 to SEQ ID NO:87, SEQ ID NO:89 to SEQ ID NO:91, SEQ ID NO:93, or SEQ ID NO:

109.

18. A conjugate, or a pharmaceutically acceptable salt thereof, comprising: the protein-based composition of claim 1; a drug; a second linker attaching the peptide antigen domain at its C-terminal end to the drug.

19. The conjugate of claim 18, or a pharmaceutically acceptable salt thereof, wherein the second linker comprises a sortase moiety, the sortase moiety comprising an amino acid sequence of LPXT (SEQ ID NO:1), wherein X is any amino acid.

20. The conjugate of claim 19, or a pharmaceutically acceptable salt thereof, wherein the second linker comprises a polyethylene glycol (PEG) moiety attaching the sortase moiety to the drug.

21. The conjugate of claim 20, or a pharmaceutically acceptable salt thereof, wherein the PEG moiety is of formula (a):wherein: n is 2 to 20.Attorney Docket No.093386-0008-WO02 22. The conjugate of claim 18, or a pharmaceutically acceptable salt thereof, wherein the drug comprises an immunomodulator, an agonist, an antagonist, an inhibitor, or a hormone.

23. The conjugate of claim 18, or a pharmaceutically acceptable salt thereof, wherein the immunomodulator comprises a STING agonist.

24. The conjugate of claim 23, or a pharmaceutically acceptable salt thereof, wherein the STING agonist is a dimeric amidobenzimidazole, 2’3’ cGAMP, 2’2’ cGAMP, 3’2’ cGAMP, 3’3’ cGAMP, c-di-GMP, c-di-AMP, ADU-S100, cIAMP 2-5, or ML RR-S2 CDA.

25. The conjugate of claim 23, or a pharmaceutically acceptable salt thereof, wherein the immunomodulator is a non-nucleotide STING agonist.

26. A pharmaceutical composition comprising: a protein-based composition according to claim 1; and a pharmaceutically acceptable excipient.

27. A method of treating a disease or a disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the protein-based composition according to claim 1, optionally in combination with a pharmaceutically acceptable excipient.

28. The method of claim 27, wherein the disease or disorder is multiple sclerosis, Type I diabetes, organ transplantation, celiac disease, rheumatoid arthritis, lupus, inflammatory bowl disease, psoriasis, or graves’ disease.

29. A method of treating a disease or a disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the conjugate, or a pharmaceutically acceptable salt thereof, according to claim 18, optionally in combination with a pharmaceutically acceptable excipient.

30. The method of claim 29, wherein the disease or disorder is a cancer, a viral infection, or an infectious disease mediated by T-cell immunity.Attorney Docket No.093386-0008-WO02 31. The method of claim 30, wherein the cancer is melanoma, breast cancer, neuroblastoma, renal cell carcinoma, colon cancer, lung cancer, glioma, glioblastoma, or pancreatic cancer.

32. A method of modulating an immune system of a subject in need thereof, the method comprising administering to the subject an effective amount of the protein-based composition according to claim 1, optionally in combination with a pharmaceutically acceptable excipient.

33. The method of claim 32, wherein modulating the subject’s immune system comprises increasing an amount of regulatory T cells in the subject specific to the peptide antigen domain, increasing an amount of CD8+ T cells in the subject, increasing the amount of CD4+ Th cells, or a combination thereof.

34. The method of claim 32, further comprising identifying, isolating, or both a peptide antigen from the subject and including the peptide antigen within the peptide antigen domain of the protein.

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