Engineering extended single-domain antibody half-life via charge modification and multimerization

Abstract

Provided herein are single-domain antibodies and methods of making and using the single-domain antibodies with increased β phase half-life. The single-domain antibodies have surface charge modification, multimerization domains, and optionally an FcRn domain.

Description

769449-UIUC-065PC ENGINEERING EXTENDED SINGLE-DOMAIN ANTIBODY HALF-LIFE VIA CHARGE MODIFICATION AND MULTIMERIZATION PRIORITY

[0001] This application claims the benefit of U.S. Ser. No.63 / 702,139, filed October 1, 2024, which is incorporated herein by reference in its entirety. BACKGROUND

[0002] Biologics, and protein-based drugs in particular, have emerged over the last several decades as a unique class of medicine, with successes rivaling those of more traditional small molecule drugs. Antibody-based biologics, including monoclonal antibodies, antibody-drug conjugates, and antibody fragments, have proven especially powerful, both as therapeutics1and as diagnostic imaging agents2. In particular, single-domain antibodies (sdAbs), whose format is based on heavy-chain only antibodies originally identified in camelid and shark species3, have grown in popularity as cancer diagnostics in part due to their ease of expression and chemical conjugation, as well as their high tissue penetration4,5. They also have potential as therapeutics: they retain the binding affinity and antigen specificity of a full-length antibody, are readily amenable to engineering, and can be expressed at high titer in microbial expression systems6-8. However, this class of molecule has relatively poor pharmacokinetic properties for both diagnostic and therapeutic applications, due largely to rapid excretion via kidney filtration9-11. In diagnostic settings, this leads to accumulation of sdAbs in the kidneys and high off-target signals that confound image interpretation12,13. As therapeutics, rapid plasma clearance limits the ability of the circulating drug to accumulate at the disease target site. Efforts to increase sdAb half- life have traditionally involved: conjugation to polyethylene glycol (PEG) polymers14, which requires post-expression processing; fusion to large domains like the IgG Fc15, which limits tissue penetration and microbial expression; or fusion to albumin-binding domains16, which requires dissociation and separation from a highly abundant serum protein in order to access the target site. Thus, decreasing renal filtration rate may be a valuable strategy to enhance the utility of sdAbs as both diagnostics and therapeutics, but requires a unique engineering approach to accommodate the specialized physiology of renal tissue. Page 1 of 45 81158138.v1769449-UIUC-065PC SUMMARY

[0003] Provided herein is a single-domain antibody comprising one or more mutations of one or more positively charged amino acids to one or more negatively charged amino acids; and a multimerization domain. In an aspect, one or more positively charged amino acids are not present in a paratope region of the single-domain antibody and are not in the hydrophobic core of the single-domain antibody. The paratope region of the single-domain antibody can be determined by X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, or peptide array- based epitope mapping. The single-domain antibody can further comprise one or more neonatal Fc receptor (FcRn) binding polypeptides comprising an amino acid sequence set forth in QRFX1TGHFGGLX2PX3NG (SEQ ID NO:1). The single-domain antibody can further comprise one or more neonatal Fc receptor (FcRn) binding polypeptides comprising an amino acid sequence set forth in SEQ ID NO:2, 3, 4, 5, or 32. The positively charged amino acids can be lysine, arginine, and histidine and the negatively charged amino acids can be aspartic acid and glutamic acid. There can be 1, 2, 3, 4, or 5 mutations of one or more positively charged amino acids to one or more negatively charged amino acids. One or more of the one or more mutations can be in a framework region of the single-domain antibody. One or more of the one or more mutations can be in a complementarity-determining region (CDR) region of the single-domain antibody. The multimerization domain can be coiled-coil dimerization domain GCN4, GCN4-pII, GCN4-pLI, ATF6, CREB1, C / EBPα, Fos, Jun, influenza hemagglutinin, or HIVgp41. The single-domain antibody can be a VHH antibody, nanobody, camelid- derived single-domain antibody, shark IgNAR-derived single-domain antibody (VNAR), diabody, or triabody. The hydrodynamic radius of the single-domain antibody can be increased as compared to a control single-domain antibody. The single-domain antibody can be transported across mucosal surfaces.

[0004] In an aspect, the single-domain antibody can be A20.1. One more of the following positively charged amino acids can be changed to a negatively charged amino acid: K43, R56, K65, K76, and K87.

[0005] In an aspect, the single-domain antibody is 2Rs15D. One or more of the following positively charged amino acids can be changed to a negatively charged amino acid: K19, K64, K75, K76, and K86. Page 2 of 45 81158138.v1769449-UIUC-065PC

[0006] An aspect provides a composition comprising any of the single-domain antibodies described herein and a pharmaceutically acceptable carrier.

[0007] An aspect provides a polynucleotide encoding the any of the single-domain antibodies described herein.

[0008] An aspect provides a vector comprising any polynucleotide described herein.

[0009] An aspect provides a host cell comprising any of the vectors described herein.

[0010] An aspect provides a method of increasing the β-phase half-life of a single- domain antibody. The method can comprising identifying selected positively charged amino acids of the single-domain antibody by identifying all positively charged amino acids of the single-domain antibody that are not present in the paratope of the single- domain antibody and are not in the hydrophobic core of the single-domain antibody. One or more of the selected positively charged amino acids can be mutated to one or more negatively charged amino acids to form a charge modified single-domain antibody. A multimerization domain can be added to the charge modified single- domain antibody, thereby forming a single-domain antibody having increased β-phase half-life. The method can further comprise adding one or more neonatal Fc receptor (FcRn) binding polypeptides comprising an amino acid sequence set forth in QRFX1TGHFGGLX2PX3NG (SEQ ID NO:1) to the single-domain antibody. The FcRn binding polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:2, 3, 4, 5, or 32.

[0011] An aspect provides a method of treatment comprising administering any of the compositions described herein to a subject in need thereof. The single-domain antibody can be transported across mucosal surfaces. The β-phase half-life of the single-domain antibody can be increased as compared to a control single-domain antibody.

[0012] Hydrodynamic radius and electrostatic charge can be factors in determining the fractional clearance of a given solute. Solutes with increasing hydrodynamic radius or with increasing negative surface charge demonstrate decreased fractional clearance through the renal filter (Fig 1A). Based on this understanding of the interplay between the tissue microenvironment in the kidneys and the structural details of sdAbs, we combine rational surface charge engineering with the incorporation of multimerization domains, and optionally FcRn domains to promote decreased fractional renal Page 3 of 45 81158138.v1769449-UIUC-065PC clearance of a model sdAb (Fig 1B), and demonstrate that this strategy can translate between unrelated sdAb scaffolds with similar pharmacokinetic outcomes. DESCRIPTION OF THE DRAWINGS

[0013] FIG.1A-1B show: 1A) Physiology and selectivity of glomerular filtration. 1B) Schematic representation of research strategy. Charge engineering of sdAb (top), fusion of sdAb to multimerization domain (middle), and the combination (bottom).

[0014] FIG. 2A-2E show: Charge engineering of the 2Rs15D scaffold. 2A) Crystal structure of the 2Rs15D-HER2 complex (PDB 5MY6). Positively charged residues are highlighted. Red: excluded residues at the 2Rs15D-HER2 interface. Blue: excluded residues in the sdAb interior. Green: residues selected for charge engineering. 2B) HER2 binding ELISA of 2Rs15D and 2Rs15D-K75-76D. 2C) Electrostatic map of 2Rs15D and (-)2Rs15D. Electrostatic maps generated in Pymol.2D) Analytical anion exchange chromatograms of 2Rs15D (Blue) and (-)2Rs15D (Red). Bound / unbound ratios calculated using Unicorn 5.31. 2E) HER2 binding ELISA of 2Rs15D and (- )2Rs15D. All assays were performed in triplicate. Error bars indicate one standard deviation.

[0015] FIG. 3A-3D show: Charge engineering of the A20.1 scaffold. 3A) Crystal structure of the A20.1-Toxin A complex (PDB 4NBX). Green: residues selected for charge engineering.3B) Electrostatic map of A20.1 and (-)A20.1. Electrostatic maps generated in Pymol.3C) Toxin A binding ELISA of A20.1 and (-)A20.1. All assays were performed in triplicate. Error bars indicate one standard deviation. Estimated KDs were calculated by fitting a saturation binding model in GraphPad Prism. 3D) Analytical anion exchange chromatograms of A20.1 (Blue) and (-)A20.1 (Red). Bound / unbound ratios calculated using Unicorn 5.31. In FIG.3A and 3B the amino acid substitutions for K43E, R56E, K65E, K76E, and K87E are shown as K50E, R63E, K72E, K83E, K94E due to the addition of 7 amino acid His tag in the A20.1 construct.

[0016] FIG.4A-4F show: Fusion of GCN4pII to sdAbs induces multimerization.4A) AlphaFold2-predicted structures of 3xA20.1 (red) and 3x2Rs15D (blue) aligned with the unmodified sdAbs (gray).4B) Size-exclusion chromatograms of unmodified sdAbs (black) and GCN4pII-modified sdAbs (blue). Samples were run at 0.5 mL / min in PBS over a Superdex 200 Increase 10 / 300 column.4C) Native PAGE gel comparing the functional molecular weight of sdAbs with and without GCN4pII.4D) Reducing SDS- Page 4 of 45 81158138.v1769449-UIUC-065PC PAGE of sdAbs with and without GCN4pII. 4E) and 4F) Antigen binding ELISA of A20.1 variants to Toxin A (left) and 2Rs15D variants to HER2 (right). All assays were performed in triplicate. Error bars indicate one standard deviation. Estimated KDs were calculated by fitting a saturation binding model in GraphPad Prism.

[0017] FIG.5A-5D show: Charge engineering and multimerization impacts plasma clearance in vivo.5A) Schematic representation of in vivo study design.5B) Plasma concentrations of A20.1 variants in Tg32 mice (n=4 per group) after one 100 μg intraperitoneal injection. Stars indicate statistical significance over unmodified sdAb control by One-Way ANOVA: * 0.01<p≤0.05.5C) Molar urine concentration of A20.1 and variants normalized to peak concentration in Tg32 mice (n=4 per group). 5D) Plasma concentrations of 2Rs15D variants in Tg32 mice (n=4 per group) after one 100 μg intraperitoneal injection. Stars indicate statistical significance over unmodified sdAb control by One-Way ANOVA: ** 0.001<p≤0.01; * 0.01<p≤0.05. 5E) Molar urine concentration of 2Rs15D and variants normalized to peak concentration in Tg32 mice (n=4 per group).

[0018] FIG. 6 shows: Expression of charge-modified 2Rs15D in E. coli. Protein expression was evaluated by western blot of whole-cell lysates of uninduced (U, no IPTG added) or induced (I, 0.1 mM IPTG) BL21 E. coli expressing a charge-modified 2Rs15D variants. Membranes were probed with camelid anti-VHH-HRP and developed with SuperSignal WestDura substrate. DETAILED DESCRIPTION

[0019] Single-Domain Antibodies

[0020] Provided herein are single-domain antibodies (sdAbs) comprising one or more mutations of one or more positively charged, surface exposed amino acids to one or more negatively charged amino acids and a multimerization domain. In certain aspects, the single-domain antibodies can comprise a FcRn binding region. A single- domain antibody can be, for example, a VHH antibody, a nanobody, a camelid-derived single-domain antibody, a shark IgNAR-derived single-domain antibody (VNAR), a diabody, or a triabody.

[0021] A sdAb comprises a variable region that includes three complementarity- determining regions (CDRs) or hypervariable regions and four highly conserved Page 5 of 45 81158138.v1769449-UIUC-065PC framework (FR) regions. The FRs adopt a β-sheet configuration, connected by the three CDRs, which form loops connecting, and in some cases, forming part of the β- sheet structure.

[0022] Single-domain antibodies can “specifically bind” to an antigen, which means that the single-domain antibodies can form a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1×10−7M or less (e.g., a smaller KD denotes a tighter binding). Methods for determining whether two molecules specifically bind include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

[0023] Single-domain antibodies described herein can be humanized. “Humanized” forms of non-human (e.g., camelid) antibodies are chimeric antibodies, which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as camelid having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. In general, a humanized antibody in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.

[0024] Any single-domain antibody can be used in the compositions and methods described herein. In an aspect, a sdAb is useful in the treatment of one or more diseases or disorders can be considered a therapeutic antibody. Therapeutic antibodies are widely used in the treatment of cancer, autoimmunity, and inflammatory diseases or for drug delivery to target antigen. Therapeutic antibodies recognize and bind to an antigen receptor to activate or inhibit a series of biological process, e.g., for blocking cancer cell growth or triggering immune system.

[0025] In an aspect, a single-domain antibody amino acid sequence can be, for example that of Caplacizumab, Envafolimab (KN-035), Ozoralizumab, Ciltacabtagene Page 6 of 45 81158138.v1769449-UIUC-065PC autoleucel (cilta-cel), Sonelokinab (ALX-0761 / M1095), Gefurulimab (ALXN1720), Lunsekimig, cobarilizumab (ALX-0061), ALX-0171 (Sanofi / Ablynx), ALX-0141 (Ablynx), MK-1697 (Merck), LMN-201, anti-ADAMTS-5 VHH, NCT05759793 (CAR- GPRC5D), NCT06880913 (CD19 / CD22 VHH CAR), A20.1, or 2Rs15D. It is contemplated, however, that any sdAb sequence can be used in the compositions and methods disclosed herein.

[0026] Multimerization Domains

[0027] A multimerization domain is a polypeptide capable of forming a dimer, trimer, or tetramer. A multimerization domain can be joined by covalent bonds or not joined by covalent bonds. A dimer, trimer, or tetramer can be a homodimer formed by two, three or four identical multimerization domains. A dimer, trimer, or tetramer can be formed by two, three, or four different multimerization domains. Multimerization domains are ubiquitous in proteins, and their aggregation can be controlled by many different factors including, for example, temperature, ligand binding, and light.

[0028] In an aspect, a multimerization domain can be any known multimerization domain. In an aspect, a multimerization domain motif is a coiled coil, which is a common domain in various transcription factors, viral proteins, and elsewhere that comprises hydrophobic residues in a 7 amino acid (heptad) periodicity. The periodic hydrophobic residues form the core of the hydrophobic dimer interface. The coiled coil motif of the GCN4 transcription factor, e.g., GCN4-pII, GCN4-pLI, and other coiled coils are particularly amenable to mutation to design unique multimerization properties.

[0029] In an aspect, multimerization domains can be coiled coils from other leucine zippers such as TF6, CREB1, C / EBPα, Fos, or Jun, viral fusion proteins influenza hemagglutinin or HIV gp41, or other coiled coil domains ATF6, APC or ProP. Multimerization domains can be thermo-sensitive dimeric coiled coils or more complex multimeric coil structures in recombinant proteins.

[0030] Multimerization domains are not limited to coil-coils. Others include zinc fingers (e.g., C2H2, C4), Helix-Loop-Helix domains (e.g., MyoD, bHLH, c-Myc), Helix-Turn- Helix (e.g., LuxR, TetR, or cI), PDZ domains, SH3 and SH2 domains, Sterile Alpha Motifs, WD40 Repeats, and Death Domains (DD, DED, CARD, PYD). In an aspect, multimerization domains can be membrane multimerization domains, multimerization domains from transcription factors, G protein βγ complexes from heterotrimeric G Page 7 of 45 81158138.v1769449-UIUC-065PC protein complexes, TIM, ADH5, 14-3-3 proteins or their binding partners Bad or Bax, or other protein multimers. Membrane multimerization domains can be glycophorin A, receptor tyrosine kinases, or GPCRs. Multimerization domains can also be nuclear receptors, estrogen receptors, androgen receptors, or glucocorticoid receptors.

[0031] Any multimerization domain can be used in the compositions described herein. See, Testa et al., 2009, “CC+: a relational database of coiled-coil structures,” Nucleic Acid Research, Vol.37, Database issue, D315-D322; Moutevelis & Woolfson, 2009, “A periodic table of coiled-coil protein structures,” Journal of Molecular Biology 385(3): 726-732, Vincent et al., 2013, “LOGICOIL—Multi-state prediction of coiled-coil oligomeric state,” Bioinformatics 29(1):69-76; Armstrong et al., 2011, “SCORER 2.0: An algorithm for distinguishing parallel dimeric and trimeric coiled-coil sequences,” Bioinformatics 27(14):1908-1914; Woolfson, 2005, “The design of coiled-coil structures and assemblies,” Adv. Prot. Chem. 70, 79-112; Mason & Arndt, 2004, “Coiled coil domains: stability, specificity, and biological implications,” Chembiochem 5 (2): 170-176; Yu, 2002, “Coiled-coils: stability, specificity, and drug delivery potential,” Adv. Drug Deliv. Rev.54 (8): 1113-1129; Brown, 2006, “Breaking symmetry in protein dimers: designs and functions,” Protein Sci.15(1): 1-13; Straussman et al., 2007 “Kinking the coiled coil A-negatively charged residues at the coiled-coil interface,” J. Mol. Biol.366, 1232-1242; Armstrong et al., 2009, “Rational design of peptide-based building blocks for nanoscience and synthetic biology,” Faraday Discuss. 143, 305-317; Banwell et al., 2009 “Rational design and application of responsive alpha-helical peptide hydrogels,” Nat. Mater.8: 596-600; Fletcher et al., 2012 “A Basis Set of de Novo Coiled-Coil Peptide Oligomers for Rational Protein Design and Synthetic Biology,” ACS Synthetic Biology 6: 240-250; Bromley et al., 2010 “Assembly Pathway of a Designed alpha-Helical Protein Fiber,” Biophysical Journal 98(8), 1668-1676; Hadley et al., 2008, “Preferred side-chain constellations at antiparallel coiled-coil interfaces,” Proc. Natl. Acad. Sci. U.S.A 105, 530-535, all of which are incorporated by reference herein.

[0032] Charge Engineering of sdAb Scaffold

[0033] Surface charge polarity can have an effect on the fractional clearance of proteins and small molecules through the glomerular filter in the kidneys. Serum albumin, which has a strong net negative charge at physiological pH, has exceptionally low fractional clearance (sieving coefficient = 0.0006) compared to a mutant, neutrally Page 8 of 45 81158138.v1769449-UIUC-065PC charged albumin (sieving coefficient = 0.026). Engineering in the reverse direction, that is, making a natively neutral protein anionic, can at least modestly decrease the fractional clearance of a protein. Positively charged sdAb residues can result in the significant retention of sdAbs within the kidney, which can be problematic in diagnostic applications where a high target to background ratio is desirable. Therefore, an aspect provides for the rational mutation of positively charged, surface-exposed residues on the sdAb scaffold region into negatively charged residues to solve both the problem of rapid, high fractional clearance through the glomerular barrier and the problem of retention in the kidneys.

[0034] Positively charged amino acids are lysine, arginine, and histidine and negatively charged amino acids are aspartic acid and glutamic acid. In an aspect, there are 1, 2, 3, 4, 5, 6, 7, or more mutations of one or more positively charged amino acids to one or more negatively charged amino acids. One or more of the one or more mutations can be in a framework region of the single-domain antibody. One or more of the one or more mutations can be in a complementarity-determining region (CDR) region of the single-domain antibody.

[0035] In an aspect, a single-domain antibody comprises one or more mutations of one or more surface-exposed, positively charged amino acids to one or more negatively charged amino acids. In an aspect, the one or more positively charged amino acids are not present in the paratope of the single-domain antibody and are not in the hydrophobic core of the single-domain antibody. A paratope comprises the amino acids of the sdAb that physically contact the antigen. The hydrophobic core is the central region of the protein where non-polar, hydrophobic amino acid residues are shielded from the aqueous environment. These hydrophobic residues forms a nucleus that contributes to the overall three-dimensional structure and stability of the sdAb.

[0036] The paratope of the single-domain antibody can be determined by X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, cryo-electron microscopy, or peptide array-based epitope mapping.

[0037] The hydrophobic core of an antibody can be determined from the amino acid sequence of the antibody using hydropathy scales (e.g., Kyte–Doolittle plots) to locate stretches of hydrophobic residues. Often, the hydrophobic core residues are present in the conserved framework residues rather than the variable CDR loops. Comparing Page 9 of 45 81158138.v1769449-UIUC-065PC the antibody sequence to known germline templates (e.g., IMGT database) can identify highlight conserved hydrophobic positions. The hydrophobic core can also be identified using known antibody structures. The 3D structure of the antibody can be obtained or modeled using, e.g., PDB entries or AlphaFold. Amino acids that have solvent accessible surface area, SASA < ~20% are likely in a hydrophobic core. Computational prediction tools can model molecular dynamics and energy minimization to residues are in the interior hydrophobic network. Programs like DSSP, NACCESS, and PyMOL / APBS can calculate solvent accessibility, while programs such as Rosetta, FoldX, and Schrödinger BioLuminate can identify core stabilizing residues vs surface-exposed ones. Other methods to identify a hydrophobic core include X-ray crystallography, Cryo-EM, and NMR which can provide direct visualization of hydrophobic packing.

[0038] The main conserved hydrophobic amino acids, which stabilize the β-sandwich fold of a sdAb, using IMGT numbering, are as follows:

[0039] Framework Region 1 (FR1): Val2, Leu4, Leu11, and Ile12, which pack the N- terminal β-strands together and Val37.

[0040] Framework Region 2 (FR2) Trp47, which is buried in the β-sandwich, and Leu45 and Val50.

[0041] Framework Region 3 (FR3): Ile66, Val67, and Leu71, which contribute to the inner sheet; Ile78, Val79, Leu82, which are buried in the central β-sandwich; and Tyr91 or Phe91, which is part of the hydrophobic packing.

[0042] Framework Region 4 (FR4): Trp103 or Phe103, which are aromatic buried residues; Leu106 and Val107, which stabilize the C-terminal end of the β-sandwich.

[0043] Conserved positively charged amino acids (Lys, Arg, His) in sdAbs, using IMGT numbering, include the following:

[0044] FR1: Lys19 or Arg19, which contribute to solubility; Arg24.

[0045] FR2: Arg45 and Glu49.

[0046] FR3: Arg75, His81 or Lys81; Arg94.

[0047] FR4: Lys103 or Arg103, Lys108.

[0048] In an aspect, a sdAb having one or more mutations of one or more surface exposed positively charged amino acids to one or more negatively charged amino acids and a multimerization domain can have an increased hydrodynamic radius as compared to a control single-domain antibody (e.g., a sdAb that does not have one or Page 10 of 45 81158138.v1769449-UIUC-065PC more mutations of one or more positively charged amino acids to one or more negatively charged amino acids; and does not have a multimerization domain). A hydrodynamic radius (Rh) is the radius of a hypothetical sphere that diffuses through a fluid at the same rate as the actual molecule or particle being studied. It is a measure of a molecule's apparent size in solution, accounting for its shape, mass, and interaction with the surrounding solvent molecules, rather than just its physical dimensions. The hydrodynamic radius is calculated from the molecule's diffusion coefficient using the Stokes-Einstein equation. In an aspect, the hydrodynamic radius is increased by about 5, 10, 20, 30, 40, 50% or more as compared to a control. In an aspect the Rh of a sdAb having one or more mutations of one or more surface exposed positively charged amino acids to one or more negatively charged amino acids and a multimerization domain is increased as compared to a control without dramatically increasing the size of the sdAb monomer (i.e., the sdAb monomer not increased or is increased by only about 20, 15, 10, 5, 3, 1% or less).

[0049] Neonatal Fc receptor (FcRn) Binding Polypeptides

[0050] As used herein, the term “FcRn binding polypeptide” refers to a polypeptide which can, in part or whole, bind to, interact with or otherwise mimic the engagement of a protein (e.g., IgG or albumin) with FcRn. In some aspects, the FcRn binding polypeptide can include a portion of an Fc region that mediates binding to FcRn. FcRn binding polypeptides can be derived from albumin. In some aspects, a FcRn binding polypeptide is a synthetic Fc mimic that binds to the same or similar epitope of FcRn as does the IgG Fc domain. A synthetic Fc mimic can be derived from, for example, a phage display library. In some aspects, the FcRn binding polypeptides are not naturally occurring.

[0051] A single-domain antibody as described herein can further comprise one or more neonatal Fc receptor (FcRn) binding polypeptides comprising an amino acid sequence set forth in SEQ ID NO:1, 2, 3, 4, 5, or 32. An FcRn binding polypeptide can comprise an amino acid sequence as set forth in SEQ ID NO:2, 3, 4, or 5.

[0052] In some aspects, FcRn binding polypeptides described herein comprise an amino acid sequence set forth in SEQ ID NO:1 (QRFX1TGHFGGLX2PX3NG). Optionally, the polypeptide is not naturally occurring. X1, X2, X3, can be any naturally or non-naturally occurring amino acid. For example, X1 can be the amino acid denoted by the single letter code C or V. X2 can be the amino acid denoted by the single letter Page 11 of 45 81158138.v1769449-UIUC-065PC code Y or H. X3 can be the amino acid denoted by the single letter code C or A. Exemplary FcRn binding polypeptides include the amino acid sequence as set forth in QRFCTGHFGGLYPCNG (SEQ ID NO:2); QRFCTGHFGGLHPCNG (SEQ ID NO:3); QRFVTGHFGGLYPANG (SEQ ID NO:4); QRFVTGHFGGLHPANG (SEQ ID NO:5); or YVPKEFNAETFTFH (SEQ ID NO:32). FcRn binding polypeptides can include one or more additional amino acids at the N terminus or C terminus of SEQ ID NO:1, 2, 3, 4, 5, or 32. One or more amino acids can be removed from the N terminus or the C terminus of SEQ ID NO:1, 2, 3, 4, 5, or 32. FcRn binding polypeptides of the disclosure can have from about 60% identity to 100% identity to the sequence of SEQ ID NO:1, 2, 3, 4, 5, or 32 for example from about 60%-70%, 70%-80%, 80%-90%, 90%-100% identity to SEQ ID NO:2, 3, 4, 5, or 32.

[0053] In an aspect, a polypeptide comprising SEQ ID NO:1-5 and 32 is smaller than about 50, 40, 30, 20, 19, 18, 17, 16, 15, or 14 amino acids in length. In an aspect, a polypeptide has one, two, three, or four amino acid substitutions, deletions, or additions as compared to SEQ ID NO:1-5 over the 16 amino acids of SEQ ID NO:1- 5 or over the 14 amino acids of SEQ ID NO:32.

[0054] Ranges of desired degrees of sequence identity are approximately 80% to 100% and integer values in between. Percent identities between a disclosed sequence and a claimed sequence can be at least 80%, at least 83%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%. In general, an exact match indicates 100% identity over the length of the reference sequence (e.g., SEQ ID NO:1-5 or 32).

[0055] Polypeptides and polynucleotides that are sufficiently similar to polypeptides and polynucleotides described herein can be used herein. Polypeptides and polynucleotides that are about 90, 91, 92, 93, 9495, 96, 97, 98, 9999.5% or more identical to polypeptides and polynucleotides described herein can also be used herein.

[0056] Polypeptides of the disclosure can include one, two, three or more FcRn polypeptides. When more than one FcRn binding polypeptide is present it can be the same FcRn polypeptide, or it can be a different FcRn polypeptide.

[0057] FcRn binding polypeptides can be derived from albumin. In some aspects, the FcRn binding polypeptide can be a region or portion of albumin. In an aspect, an FcRn binding polypeptide derived from albumin can be YVPKEFNAETFTFH (SEQ ID Page 12 of 45 81158138.v1769449-UIUC-065PC NO:32). Two or more of polypeptides comprising SEQ ID NO:32 can be used. SEQ ID NO:32 can be used in conjunction with one or more of SEQ ID NO:1, 2, 3, 4, and / or 5 in any orientation.

[0058] In an aspect, sdAbs comprising the FcRn polypeptide can be transported across mucosal surfaces such as the gut epithelium, the lining of the respiratory tract, the lining of the urogenital tract, the surface of the eye, the oral cavity, nose, and ears.

[0059] Polypeptides

[0060] Provided herein are polypeptides that are useful in the present disclosure. “Polypeptide” as used herein refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptides refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides can contain amino acids other than the 20 gene-encoded amino acids. Polypeptides can include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques that are well known in the art.

[0061] Polypeptides described herein can be derived from other proteins or polypeptide (herein referred to as a starting polypeptide). They can have one or more mutations relative to the starting polypeptide, e.g., one or more amino acid residues which have been substituted with another amino acid residue or which has one or more amino acid residue insertions or deletions. The polypeptide can comprise an amino acid sequence which is not naturally occurring. Such variations necessarily have less than 100% sequence identity or similarity with the starting polypeptide. In one aspect, the variant will have an amino acid sequence from about 60% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide. In another aspect, the variant will have an amino acid sequence from about 75% to less than 100%), from about 80% to less than 100%, from about 85% to less than 100%, from about 90% to less than 100%), from about 95% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide.

[0062] Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or to varying degrees at several sites Page 13 of 45 81158138.v1769449-UIUC-065PC in a given polypeptide. The polypeptide can include one or more types of modifications. Polypeptides can be branched as a result of ubiquitination, and they can be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides can result from post translation natural processes or can be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0063] Polypeptides described herein can include one or more linkers. A linker refers to a moiety that links or connects together one or more portions or regions of a polypeptide or one or more polypeptides. The linker can be a peptide linker that includes from about 1-100 amino acids (e.g., about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids). The type of linker can vary depending on the crystal structure of the antibody heavy chain and antibody light chain. The linkers can be derived from naturally occurring sequences of amino acids. Alternatively, the linkers can be artificially designed peptide linker. In some aspects, the linker can be composed of flexible residues such as, Glycine (G) and Serine (S), so that the adjacent polypeptide domains are free to move relative to one another. Non limiting examples of linkers include (Gly4Ser)3 linkers, e.g., GGGGSGGGGSGGGGS. (SEQ ID NO:30) or a single repeat of GGGGS linker (SEQ ID NO:31).

[0064] In an aspect, a polypeptide can comprise two or three portions from -NH3 terminus to -COOH terminus: (i) sdAb polypeptide, multimerization domain polypeptide; (ii) multimerization domain polypeptide, sdAb polypeptide; (iii) sdAb polypeptide, multimerization domain polypeptide, FcRn polypeptide; Page 14 of 45 81158138.v1769449-UIUC-065PC (iv) sdAb polypeptide, FcRn polypeptide, multimerization domain polypeptide; (v) multimerization domain polypeptide, sdAb polypeptide, FcRn polypeptide; (vi) multimerization domain polypeptide, FcRn polypeptide, sdAb polypeptide; (vii) FcRn polypeptide, sdAb polypeptide, multimerization domain polypeptide; or (viii) FcRn polypeptide, multimerization domain polypeptide, sdAb polypeptide.

[0065] In an aspect, one more linkers can be present at the amino terminus, the carboxy terminus, or between one or more of the sdAb polypeptide, the multimerization domain polypeptide, and / or the FcRn polypeptide. In an aspect, more than one copy of the sdAb polypeptide, multimerization domain polypeptide, and / or FcRn polypeptide can be present in a polypeptide. These polypeptides can be fusion proteins of all of the components.

[0066] The terms "sequence identity" or "percent identity" are used interchangeably herein. To determine the percent identity of two polypeptide molecules or two polynucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first polypeptide or polynucleotide for optimal alignment with a second polypeptide or polynucleotide sequence). The amino acids or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical positions / total number of positions (i.e., overlapping positions) x 100).

[0067] In some aspects the length of a comparison sequence aligned for comparison purposes is at least 80% of the length of the reference sequence (e.g. SEQ ID NO:1), and in some aspects is at least 85%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, 99% or 100% of the length of the reference sequence. In an aspect, the two sequences are the same length. Therefore, wherein a length of a comparison sequence is Page 15 of 45 81158138.v1769449-UIUC-065PC required to be at least 100% of the length of a reference sequence, and a reference sequence is 10 amino acids in length and a comparison sequence is 100 amino acids in length, and wherein the 10 amino acids of the reference sequence contiguously align with 10 of the 100 amino acids of the comparison sequence, the sequence identity of the comparison sequence is only 10%.

[0068] Polynucleotides

[0069] The present disclosure provides polynucleotides comprising a nucleic acid sequence that encodes any of the polypeptides as described herein, and host cells into which the nucleic acids that are used are introduced to replicate the polypeptide-encoding nucleic acids and / or to express the polypeptides. In some aspects, the host cell is eukaryotic, for example, a human cell, or a prokaryotic cell, for example, a bacterial cell.

[0070] Polynucleotides described herein can encode a one or more single- domain antibodies, multimerization domains, and / or FcRn polypeptides described herein.

[0071] Polynucleotides can be single-stranded or double-stranded. In some aspects, the polynucleotide is DNA. In particular aspects, the polynucleotide is cDNA. In some aspects, the polynucleotide is RNA. In some aspects, the polynucleotide is included within a nucleic acid construct. In some modalities, the construct is a replicable vector. In some aspects, the vector is selected from a plasmid, a viral vector, a phagemid, a yeast chromosomal vector and a non- episomal mammal vector.

[0072] In some aspects, a polynucleotide is operationally linked to one or more regulatory nucleotide sequences in an expression construct.

[0073] Unless otherwise indicated, the term polynucleotide, nucleic acid molecule, or gene includes reference to the specified sequence as well as the complementary sequence thereof. Polynucleotides can be present as a single-stranded or double- stranded and linear or covalently circularly closed molecule. As used herein, a polynucleotide can include both naturally occurring and non-naturally occurring nucleotides.

[0074] Polynucleotides can be obtained from nucleic acid molecules present in, for example, a mammalian cell. Polynucleotides can also be synthesized in the laboratory, for example, using an automatic synthesizer. Polynucleotides can be Page 16 of 45 81158138.v1769449-UIUC-065PC isolated. An isolated polynucleotide can be a naturally occurring polynucleotide that is not immediately contiguous with one or both of the 5’ and 3’ flanking genomic sequences that it is naturally associated with. An isolated polynucleotide can be, for example, a recombinant DNA molecule of any length, provided that the nucleic acid molecules naturally found immediately flanking the recombinant DNA molecule in a naturally occurring genome is removed or absent. Isolated polynucleotides also include non-naturally occurring nucleic acid molecules. “Isolated polynucleotides” can be (i) amplified in vitro, for example via polymerase chain reaction (PCR), (ii) produced recombinantly by cloning, (iii) purified, for example, by cleavage and separation by gel electrophoresis, (iv) synthesized, for example, by chemical synthesis, or (vi) extracted from a sample.

[0075] Polynucleotides can encode full-length polypeptides, polypeptide fragments, and variant or fusion polypeptides. Polynucleotides can comprise coding sequences for naturally occurring polypeptides or can encode altered sequences that do not occur in nature. Polynucleotides can be purified free of other components, such as proteins, lipids and other polynucleotides. For example, the polynucleotide can be 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% purified. A polynucleotide existing among hundreds to millions of other polynucleotide molecules within, for example, cDNA or genomic libraries, or gel slices containing a genomic DNA restriction digest are not to be considered a purified polynucleotide.

[0076] Vectors

[0077] Provided herein are vectors comprising one or more polynucleotides described herein. Such vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon-based vectors, or any other vector suitable for introduction of the polynucleotide of the disclosure into a given organism or genetic background by any means. For example, polynucleotides encoding sdAbs, multimerization domains, and / or FcRNs of the disclosure can be inserted into vectors. The nucleotide segments encoding the sdAbs, multimerization domains, and / or FcRNs can be operably linked to control sequences in the vector(s) that ensure the expression of the polypeptides. Such control sequences include signal sequences, promoters (e.g., naturally associated or heterologous promoters), enhancer elements, and transcription termination sequences, and are chosen to be compatible with the host cell chosen to express the sdAbs. Once the vector has been incorporated into the appropriate host Page 17 of 45 81158138.v1769449-UIUC-065PC cell, the host cell is maintained under conditions suitable for high level expression of the proteins encoded by the incorporated polynucleotides.

[0078] Suitable vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, vectors contain selection markers such as ampicillin-resistance, hygromycin-resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance to permit detection of those cells transformed with the desired DNA sequences. Suitable vectors, promoter, and enhancer elements are commercially available for generating subject recombinant constructs.

[0079] The polypeptides described herein can be produced by recombinant methods. For example, a polynucleotide sequence encoding a polypeptide can be inserted into a suitable expression vector for recombinant expression. An affinity tag sequence (e.g., a His(6) tag) can optionally be attached or included within the starting polypeptide sequence to facilitate downstream purification. The DNA segments encoding sdAbs can be operably linked to control sequences in the expression vector(s) that ensure the expression of polypeptides. Expression control sequences include, but are not limited to, promoters (e.g., naturally-associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. The expression control sequences can be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Alternatively, the expression control sequences can be prokaryotic promoter systems. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the polypeptide.

[0080] Expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers (e.g., ampicillin-resistance, hygromycin-resistance, tetracycline resistance or neomycin resistance) to permit detection of those cells transformed with the desired DNA sequences.

[0081] Escherichia coli can be useful for cloning the polynucleotides (e.g., DNA sequences). Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Other microbes, such as yeast, are also useful for expression. Page 18 of 45 81158138.v1769449-UIUC-065PC Saccharomyces and Pichia are exemplary yeast hosts, with suitable vectors having expression control sequences (e.g., promoters), an origin of replication, termination sequences and the like as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for methanol, maltose, and galactose utilization.

[0082] In addition to microorganisms, mammalian tissue culture can also be used to express and produce the polypeptides described herein (e.g., polynucleotides encoding sdAbs described herein or fragments thereof).

[0083] Expression vectors for mammalian cells can be used. Non-limiting examples of mammalian cells include, CHO cell lines, various Cos cell lines, HeLa cells, 293 cells, myeloma cell lines, and / or transformed B-cells. Expression vectors can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Expression control sequences can be promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like.

[0084] The vectors containing polynucleotide sequences of interest (e.g., sdAb, multimerization domain, FcRn and / or expression control sequences) can be transferred into the host cell by methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics or viral-based transfection can be used for other cellular hosts. (See generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989). Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally, Sambrook et al., supra). Polypeptides can be expressed using a single vector or two vectors. In one aspect, signal sequences can be used to facilitate expression of polypeptides described herein. Host Cells

[0085] An aspect provides host cells comprising one or more vectors as described herein. The term “host cell” refers to a cell into which a vector has been introduced. It Page 19 of 45 81158138.v1769449-UIUC-065PC is understood that the term host cell is intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Such host cells can be eukaryotic cells, prokaryotic cells, plant cells, or archaeal cells. Escherichia coli, bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species are examples of prokaryotic host cells. Other microbes, such as yeast, are also useful for expression. Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells. Exemplary eukaryotic cells may be of mammalian, insect, avian, or other animal origins.

[0086] Compositions

[0087] An aspect provides one or more single-domain antibodies as described herein and a pharmaceutically acceptable carrier. A therapeutic or pharmaceutical composition can include at least one single-domain antibodies described herein in a pharmaceutically acceptable carrier. A “pharmaceutically acceptable carrier” refers to at least one component of a pharmaceutical preparation that is normally used for administration of active ingredients. As such, a carrier can contain any pharmaceutical excipient used in the art and any form of vehicle for administration.

[0088] Provided herein are methods that comprise administering one or more single- domain antibodies to a patient, wherein the one or more single-domain antibodies are contained within a pharmaceutical composition. A pharmaceutical composition can be formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like. Formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil- in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J. Pharm. Sci. Technol.52:238-311.

[0089] The dose of one or more single-domain antibodies administered to a patient can vary depending upon the age and the size of the patient, symptoms, conditions, route of administration, and the like. The dose is typically calculated according to body Page 20 of 45 81158138.v1769449-UIUC-065PC weight or body surface area. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. Effective dosages and schedules for administering pharmaceutical compositions of one or more single-domain antibodies can be determined empirically. For example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly.

[0090] Various delivery systems can be used to administer the pharmaceutical compositions, e.g., encapsulation in liposomes, microparticles, microcapsules, and recombinant cells capable of expressing the one or more single-domain antibodies. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intra-tracheal, epidural, and oral routes. The compositions can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other biologically active agents.

[0091] A pharmaceutical composition can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device (e.g., an autoinjector pen) can be used to deliver a pharmaceutical composition. A pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition. Examples include, but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany).

[0092] For direct administration to the sinuses pharmaceutical compositions can be administered using, e.g., a microcatheter (e.g., an endoscope and microcatheter), an aerosolizer, a powder dispenser, a nebulizer or an inhaler. The methods include administration of one or more single-domain antibodies to a subject in need thereof, Page 21 of 45 81158138.v1769449-UIUC-065PC in an aerosolized formulation. Aerosolized antibodies can be prepared as described in, for example, U.S. Pat. No. 8,178,098, incorporated herein by reference in its entirety.

[0093] In an aspect, pharmaceutical compositions can be delivered in a controlled release system. In an aspect, a pump can be used. In another aspect, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet another aspect, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol.2, pp.115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0094] Injectable preparations can include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. The injectable preparations can be prepared, e.g., by dissolving, suspending or emulsifying the one or more single-domain antibodies or their salts in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc. An oily medium, e.g., sesame oil, soybean oil, etc., can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection can typically be filled in an appropriate ampoule.

[0095] Pharmaceutical compositions for oral or parenteral use can be prepared in dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0096] Methods of Increasing the Half-Life of a Single-Domain Antibody

[0097] Provided herein are methods of increasing the β-phase half-life of a single- domain antibody. The methods can comprise identifying selected positively charged amino acids by identifying all positively charged amino acids of the single-domain antibody that are not present in a paratope of the single-domain antibody and are not Page 22 of 45 81158138.v1769449-UIUC-065PC in the hydrophobic core of the single-domain antibody. In an aspect the selected positively charged amino acids are surface exposed (i.e., accessible to the surrounding solvent). One or more of the selected positively charged amino acids can be mutated to one or more negatively charged amino acids to form a charge modified single-domain antibody. A multimerization domain can be fused or otherwise added to polypeptide encoding the charge modified single-domain antibody. Optionally, one or more neonatal Fc receptor (FcRn) binding polypeptides comprising an amino acid sequence set forth in SEQ ID NO:1-5 or 32 can also be fused or otherwise added to the single-domain antibody.

[0098] In an aspect, the β-phase half-life of the sdAb is increased by 10, 20, 30, 40, 50, 60, 70, 80, 100, 200, 300, 400, 500, 750, 1,000 % or more as compared to a control single-domain antibody (e.g., a sdAb that does not have one or more mutations of one or more positively charged amino acids to one or more negatively charged amino acids, does not have a multimerization domain, and in certain aspects does not have a FcRn domain). Methods of Treatment

[0099] The present disclosure provides methods of treatment using single-domain antibodies and pharmaceutical compositions of the same. As used herein, the terms “treating”, “treat” or “treatment” include administering polypeptides, antibodies and / or pharmaceutical compositions described herein to thereby reduce or eliminate at least one symptom of a specified disease or condition. As used herein, the term “subject” shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and non-human primates. For example, the subject is a human.

[0100] In an aspect, FcRn engagement can enhance the gut bioavailability and residence time of IV-dosed sdAb via FcRn-mediated blood-to-gut transport. Thus, modification of sdAb with the FcRn-polypeptide described here can dramatically enhance the impact of these therapeutics against GI indications including, but not limited to, inflammatory bowel disease or recalcitrant Clostridioides difficile infection. Furthermore, FcRn engagement can enable gut-to-blood transport and can facilitate alternative routes of sdAb administration including oral dosing, vectored immunoprophylaxis, or in situ production by engineered commensal microbes. Page 23 of 45 81158138.v1769449-UIUC-065PC

[0101] Surface charge modified sdAbs with multimerization domains, and optionally FcRn polypeptides, as described herein, can be useful in the treatment of cancers. Exemplary cancers include cystic and solid tumors, bone and soft tissue tumors, including tumors in anal tissue, bile duct, bladder, blood cells, bowel, brain, breast, carcinoid, cervix, eye, esophagus, head and neck, kidney, larynx, leukemia, liver, lung, lymph nodes, lymphoma, melanoma, mesothelioma, myeloma, ovary, pancreas, penis, prostate, skin, sarcomas, stomach, testes, thyroid, vagina, vulva. Soft tissue tumors include Benign schwannoma Monosomy, Desmoid tumor, lipo- blastoma, lipoma, uterine leiomyoma, clear cell sarcoma, dermatofibrosarcoma, Ewing sarcoma, extraskeletal myxoid chondrosarcoma, liposarcooma myxoid, Alveolar rhabdomyosarcoma and synovial sarcoma. Specific bone tumors include non-ossifying fibroma, unicameral bone cyst, enchondroma, aneurismal bone cyst, osteoblastoma, chondroblastoma, chondromyxofibroma, ossifying fibroma and adamantinoma, Giant cell tumor, fibrous dysplasia, Ewing’s sarcoma eosinophilic granuloma, osteosarcoma, chondroma, chondrosarcoma, malignant fibrous histiocytoma and metastatic carcinoma. Leukemias include acute lymphoblastic, acute myeloblastic, chronic lymphocytic and chronic myeloid.

[0102] Surface charge modified sdAbs with multimerization domains, and optionally FcRn polypeptides, as described herein can be used in the treatment and prevention of human viral infections. Examples of viral infections include infections caused by DNA viruses (e.g., Herpes Viruses such as Herpes Simplex viruses; Epstein-Barn virus; Cytomegalovirus; Pox viruses such as Variola (small pox) virus; Hepadnaviruses (e.g., Hepatitis B virus); Papilloma viruses; Adenoviruses); RNA Viruses (e.g., HIV I, II; HTLV I, II; Poliovirus; Hepatitis A; coronaviruses, such as sudden acute respiratory syndrome (SARS); Orthomyxoviruses (e.g., Influenza viruses); Paramyxoviruses (e.g., Measles virus); Rabies virus: Hepatitis C virus), Flaviviruses, Influenza viruses; caliciviruses; rabies viruses, rinderpest viruses, Arena virus, and the like. Moreover, examples of the types of virus-related diseases include but are not limited to: acquired immunodeficiency; hepatitis; gastroenteritis; hemorrhagic diseases; enteritis; carditis; encephalitis; paralysis; bronchiolitis; upper and lower respiratory disease; respiratory papillomatosis; arthritis; disseminated disease, meningitis, mononucleosis. Page 24 of 45 81158138.v1769449-UIUC-065PC

[0103] Surface charge modified sdAbs with multimerization domains, and optionally FcRn polypeptides, as described herein, are also useful in the treatment of microbial infections including Chlamydia trachomatis, Listeria sp., Helicobacter pylori, Mycobacterium sp., Mycoplasma sp., Bacillus sp., Salmonella sp., and Shigella sp., E. coli, Clostridium sp.

[0104] Dosage

[0105] The amount of one or more single-domain antibodies administered to a subject is, generally, a therapeutically effective amount. As used herein, the phrase “therapeutically effective amount” means an amount of one or more single-domain antibodies that results in a reduction in the incidence of one or more symptoms of a disease or disorder and / or an improvement in one or more symptoms of a disease or disorder. A “therapeutically effective amount” also includes an amount of one or more single-domain antibodies that inhibits, prevents, lessens, or delays the progression of a disease or disorder in a subject.

[0106] A therapeutically effective amount of one or more single-domain antibodies described herein can be from about 0.05 mg to about 700 mg, e.g., about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 3.0 mg, about 5.0 mg, about 7.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 610 mg, about 620 mg, about 630 mg, about 640 mg, about 650 mg, about 660 mg, about 670 mg, about 680 mg, about 690 mg, or about 700 mg of the anti-IL-33 antibody or anti-IL-4R antibody.

[0107] The amount of one or more single-domain antibodies described herein contained within individual doses can be expressed in terms of milligrams of antibody per kilogram of patient body weight (i.e., mg / kg). For example, one or more single- Page 25 of 45 81158138.v1769449-UIUC-065PC domain antibodies described herein can be administered to a patient at a dose of about 0.0001 to about 10 mg / kg of patient body weight. For example, the one or more single-domain antibodies can be administered at a dose of 1 mg / kg, 2 mg / kg, 3 mg / kg, or 4 mg / kg.

[0108] The one or more single-domain antibodies described herein can be administered as a single dose, or every week, every other week, every third week, every fourth week, every three months, every six months, or once a year.

[0109] The compositions and methods are more particularly described below and the Examples set forth herein are intended as illustrative only, as numerous modifications and variations therein will be apparent to those skilled in the art. The terms used in the specification generally have their ordinary meanings in the art, within the context of the compositions and methods described herein, and in the specific context where each term is used. Some terms have been more specifically defined herein to provide additional guidance to the practitioner regarding the description of the compositions and methods.

[0110] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. As used in the description herein and throughout the claims that follow, the meaning of “a”, “an”, and “the” includes plural reference as well as the singular reference unless the context clearly dictates otherwise. The term “about” in association with a numerical value means that the value varies up or down by 5%. For example, for a value of about 100, means 95 to 105 (or any value between 95 and 105).

[0111] All patents, patent applications, and other scientific or technical writings referred to anywhere herein are incorporated by reference herein in their entirety. The aspects illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are specifically or not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising," "consisting essentially of," and "consisting of" can be replaced with either of the other two terms, while retaining their ordinary meanings. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claims. Page 26 of 45 81158138.v1769449-UIUC-065PC Thus, it should be understood that although the present methods and compositions have been specifically disclosed by aspects and optional features, modifications and variations of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of the compositions and methods as defined by the description and the appended claims.

[0112] Any single term, single element, single phrase, group of terms, group of phrases, or group of elements described herein can each be specifically excluded from the claims.

[0113] Whenever a range is given in the specification, for example, a temperature range, a time range, a composition, or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the aspects herein. It will be understood that any elements or steps that are included in the description herein can be excluded from the claimed compositions or methods.

[0114] In addition, where features or aspects of the compositions and methods are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the compositions and methods are also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

[0115] The following are provided for exemplification purposes only and are not intended to limit the scope of the aspects described in broad terms above. EXAMPLES

[0116] Example 1: Plasmid Construction

[0117] Plasmids and primers used to construct modified sdAb expression plasmids are listed in Table 2. C-terminal multimerizing peptide modifications were added to sdAbs by nested PCR. Charge engineered mutants were constructed by site-directed mutagenesis (SDM). Targeted amino acids were mutated in sequence, from N- to C- terminus, with each mutation confirmed by DNA sequencing prior to performing the next SDM reaction. SdAb variants with both charge engineering and peptide Page 27 of 45 81158138.v1769449-UIUC-065PC modifications were charge engineered by sequential SDM first, with peptide modifications added by nested PCR after charge engineering was confirmed by DNA sequencing. Modified sdAbs were either cloned into the BamHI and HindIII sites of the E. coli expression plasmid pET24b+-OmpA20or into the Corynebacterium glutamicum expression plasmid pZ9. Plasmid pZ9 was constructed from pZ8-Ptac21through replacement of the inducible tac promoter with a constitutively active PH36 promoter22by colleague M. Tarabey.

[0118] Residues 2460-2710 of the Clostridioides difficile toxin A were amplified from the C. difficile genome and cloned into the plasmid 2Bc-T (Addgene # 37236) by ligation-independent cloning (LIC) using standard methods to generate 2Bc-T-TxA- A4.

[0119] Table 2 List of primers used to construct plasmids for expression of recombinant proteins. Primer Name Sequence (5’-3’) 2Rs15D_Bam_F TAAGCAGGATCCCAAGTACAGTTGCAGGAATCC SEQ ID NO:6 sdAb_Hind_R TAAGCAAAGCTTTTATTAGTGATGGTGATGGTGATGAGAG SEQ ID NO:7 2Rs15D_K1D_F CAAGCAGGTGGAAGTCTGGATCTTACTTGCGCCGCC SEQ ID NO:8 2Rs15D_K1D_R ATCCAGACTTCCACCTGCTTG SEQ ID NO:9 2Rs15D_K2D_F GGCGACACTTGGCACGATGAGAGCGTAAAAGGTCG SEQ ID NO:10 2Rs15D_K2D_R ATCGTGCCAAGTGTCGCC SEQ ID NO:11 2Rs15D_K3D_F GCACAAGGAGAGCGTAGATGGTCGTTTTACGATTAGTCAG SEQ ID NO:12 2Rs15D_K3D_R ATCTACGCTCTCCTTGTGC SEQ ID NO:13 2Rs15D_K45D_F CGATTAGTCAGGATAATGTAGATGATACATTATACCTTCAGATGAACAG SEQ ID NO:14 2Rs15D_K45D_R ATCATCTACATTATCCTGACTAATCGTAAAAC SEQ ID NO:15 2Rs15D_K6D_F CCTTCAGATGAACAGTCTGGATCCCGAGGACACAGC SEQ ID NO:16 2Rs15D_K6D_R ATCCAGACTGTTCATCTGAAGG SEQ ID NO:17 2Rs15D_GCN4pII_ ACCCCCGCCACCGCTACCGCCCCCGCCGCTGCCGCCACCCCCACTGCTTACCGTTACTTG Inner_R SEQ ID NO:18 TCTTGCTCAGGATCTCCTCTATCTTATCTTCAATTTGTTTCATCCTGCTACCCCCGCCAC GCN4pII_Mid_R SEQ ID NO:19 CAACTTTTTAATACGCGCGATTTCGTTTTCAATGTGGTAGATCTTGCTCAGGATCTCCTC GCN4pII_3_R SEQ ID NO:20 GCN4pII_Out_R TAAGCAAAGCTTTTATTAGCGCTCACCAACCAACTTTTTAATACGCGCG SEQ ID NO:21 A20.1_Bam_F TAAGCAGGATCCCAGGTACAACTGGTCGAGTC SEQ ID NO:22 A20.1_K1E_F GAGGAAAGGGAGTTTGTTGCAG SEQ ID NO:23 A20.1_K1E_R CTGCAACAAACTCCCTTTCCTCACCCGGTGGCTGAC SEQ ID NO:24 A20.1_R5K2E_F ACCACTTATTACGCGGATTCAGTGGAGGGGCGGTTCACTATTAGC SEQ ID NO:25 A20.1_R5K2E_R GAATCCGCGTAATAAGTGGTCTCCCCAGTCGAACTCCCAG SEQ ID NO:26 A20.1_K34E_F GAATACCGTCTATTTACAAATGAATTCGTTGGAGCCTGAGGATACCGCCG SEQ ID NO:27 A20.1_K34E_R GAATTCATTTGTAAATAGACGGTATTCTCCGCGTTGTCGCGG SEQ ID NO:28 A20.1_GCN4pII_I ACCCCCGCCACCGCTACCGCCCCCGCCGCTGCCGCCACCCCCGGATGAAACCGTAACCTG nner_R SEQ ID NO:29

[0120] Example 2: Recombinant protein expression Page 28 of 45 81158138.v1769449-UIUC-065PC

[0121] Residues 2460-2710 of C. difficile toxin A and sdAbs A20.1, (-)A20.1, 3xA20.1, and 3x(-)A20.1 were expressed and purified from E. coli. SdAbs 2Rs15D, (-)2Rs15D, 3x2Rs15D, and 3x(-)2Rs15D were expressed and purified from Corynebacterium glutamicum. The amino acid sequence of 2Rs15D is QVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSRISGD GDTWHKESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYNLETYWGQGT QVTVSS (SEQ ID NO:33).

[0122] For E. coli-expressed proteins, BL21 (DE3) E. coli were transformed with pET24b+-OmpA-SdAb or 2bct-TxA-A4 plasmids. Transformed E. coli were grown in 2xTY (16 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCl, 0.5% v / v glucose, 50 mM NaxHxPO4, 50 mM KxHxPO4, 25 mM (NH4)2SO4) media with 50 μg / mL kanamycin at 37°C to an OD600 of approximately 0.6. Protein expression was induced by addition of isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, followed by incubation at 18°C overnight (~14 hours). Cells were pelleted and lysed by sonication in 1x phosphate buffered saline (PBS) / 1% (v / v) Triton X100. Protein was then purified by NiNTA chromatography (Toxin A) or MonoRab αCamelid-VHH affinity chromatography (sdAbs) (GenScript) from the soluble fraction of the cell lysate. A second purification step using size-exclusion chromatography was performed when necessary. Purity was assessed by SDS-PAGE. All purified proteins were sterile filtered and stored in 1x PBS supplemented with 0.1 mM phenylmethylsulfonyl fluoride (PMSF) protease inhibitor at 4°C.

[0123] For C. glutamicum-expressed proteins, C. glutamicum harboring pZ9-SdAb plasmids were grown in Brain Heart Infusion media (Gibco) with 50 μg / mL kanamycin at 30°C with 200 rpm orbital shaking to an OD600 of approximately 0.6. PMSF was added to a final concentration of 100 μM, and cultures were incubated for 20 hours at 18°C, 200 rpm. Cultures were pelleted by centrifugation at 5,000xg for 20 minutes at 4°C. Proteins were purified by MonoRab αCamelid-VHH affinity chromatography (GenScript) from the supernatant. A second purification step by size-exclusion chromatography was performed when necessary. Purity was assessed by SDS- PAGE. All purified proteins were sterile filtered and stored in 1x PBS supplemented with 0.1 mM phenylmethylsulfonyl fluoride (PMSF) protease inhibitor at 4°C.

[0124] Example 3: Size exclusion chromatography Page 29 of 45 81158138.v1769449-UIUC-065PC

[0125] Multimerization of GCN4pII-modified sdAbs was confirmed by comparison of SEC chromatograms with non-multimerizing sdAbs. 100 mg protein was injected in 250 mL total volume onto a Superdex 200 Increase 10 / 300 column (Cytiva) on an AKTA FPLC. Samples were run through the column in a PBS mobile phase at a flowrate of 0.5 mL / min. Chromatograms were overlayed and retention times were determined using Unicorn 5.31 software. For preparative SEC, samples were run in the same way. Fractions were collected manually and the protein content of each peak was determined by SDS-PAGE.

[0126] Example 4: Ion exchange chromatography

[0127] Isoelectric point alteration of charge-engineered sdAbs was confirmed by comparison of ion exchange (IEX) chromatograms with non-engineered sdAbs. Proteins were buffer exchanged by gravity-drip PD-10 column into 20 mM histidine buffer, pH 6, then 100 μg protein was injected in 250 mL total volume onto a HiTrap Q HP column (Cytiva) on an AKTA FPLC. Samples were run through the column in a 20 mM histidine, pH 6 mobile phase at a flowrate of 1 mL / min. After sample injection, the column was washed with 10 column volumes of running buffer. Bound proteins were eluted from the column with 20 mM histidine, pH 6, 500 mM NaCl. Chromatograms were overlayed, and the fraction of bound protein (fbound) was calculated by using peak integration on Unicorn 5.31 to determine peak area, then using the following equationwhere Aelution is the area under the curve of the elution peak and Atotal is the total area under the curve of all bound and unbound chromatogram peaks.

[0128] Example 5: Binding ELISA

[0129] Purified HER2 (SinoBiological) or C. difficile Toxin A Fragment 4 was coated at 2 μg / mL on 2HB microtiter plates (Immulon) overnight at 4°C in 50 mM sodium carbonate buffer, pH 9. Coated plates were brought to room temperature (RT), then blocked with 3% (w / v) bovine serum albumin (BSA) in PBS + 0.05% (v / v) Tween 20 (PBS-T) for one hour at RT. Prior to measuring binding by ELISA, 2Rs15D and its variants were conjugated with NHS-Biotin (ApexBio) per the manufacturer’s instructions. After blocking, sdAbs were diluted in 3% BSA, added to blocked plates, and incubated at RT for 1-2 hours with gentle shaking. After 3 washes with PBS-T, Page 30 of 45 81158138.v1769449-UIUC-065PC bound sdAb was detected by 1 hour incubation with either a 1:5,000 dilution of HRP- conjugated MonoRab anti-camelid VHH antibody (GenScript) or a 1:10,000 dilution of streptavidin-HRP (Jackson ImmunoResearch) for A20.1 and 2Rs15D, respectively, followed by 5 washes with PBS-T and development with o-phenylenediamene (OPD) (BioBasic) per the manufacturer’s instructions.

[0130] Absorbance was measured at 450 nm (A450), and the A450 of blank wells was subtracted before analysis. All assays were performed in triplicate, and statistical analysis was performed using GraphPad Prism.

[0131] Example 6: Mice – handling and husbandry

[0132] Mice used in this study were purchased initially from The Jackson Laboratory (Bar Harbor, ME). A breeding colony was maintained using standard triad breeding. Mice were housed in pathogen-free conditions at the University of Illinois Urbana- Champaign (UIUC). All mouse breeding and experimental procedures were approved by the UIUC Institutional Animal Care and Use Committee (IACUC). The mouse strain used in this study was B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr / DcrJ (Tg32; stock number 014565). Tg32 mice are homozygous for a knockout of mouse Fcgrt, and express human FCGRT under the control of the native human FCGRT promoter. Mice were weaned from their dams at 21 days post-birth, and all animals utilized in the evaluation of protein half-life were between 8-10 weeks of age at the time of dosing.

[0133] Example 7: Plasma clearance in mice

[0134] For evaluation of sdAb plasma clearance, Tg32 mice (n=4 per group) were injected with 100 mg protein (£ 200 mL total volume) into the right peritoneal cavity using a 1 mL insulin syringe (BD). All samples were filtered through a 0.2 mm polyethersulfone filter prior to injection.

[0135] Blood samples were collected at 0, 1, 3, 6, 8, 12, and 24 hours post-dosing, with additional samples collected at 24-hour intervals after that if signal was still detectable. Samples were obtained by tail vein puncture using a 25-gauge needle.5- 10 mL of whole blood was collected using a heparinized glass capillary tube (Globe Scientific). Blood samples were dispensed into 20-50 mL sterile PBS in a microcentrifuge tube, mixed well, and centrifuged at 3,000 x g for 10 minutes. Diluted plasma was removed by careful pipetting, transferred to a new tube, and stored at - 80°C until analysis. Plasma concentrations were measured by antigen-binding ELISA as described above. A standard curve of purified protein of known concentrations was Page 31 of 45 81158138.v1769449-UIUC-065PC included for each protein tested to enable absorbance-to-concentration conversion. Beta-phase half-lives were calculated by plotting the log10 of sdAb plasma concentration, then fitting with a simple linear regression model in graphpad prism. Half-life was calculated using the following equation.

[0136] Urine samples were collected by briefly placing mice into a plastic Broome style restraint device (ThermoFisher). Upon urination, mice were removed from the device and samples were transferred from the base of the restrainer to a microcentrifuge tube by pipette. The restraint device was thoroughly cleaned between mice. Urine samples were stored at -80°C until analysis. Protein concentrations in urine were determined by ELISA as described above.

[0137] Example 8: Single-Domain Antibody 2Rs15D

[0138] For our initial studies, we selected the sdAb 2Rs15D, which targets human epidermal growth factor receptor 2 (HER2) – an important antigen for the treatment and detection of breast and colon cancers. 2Rs15D was initially derived via immunization of a dromedary camel with a HER2-IgG Fc fusion protein. We first inspected the 2Rs15D-HER2 cocrystal structure (PDB 5MY6), and identified all positively charged amino acid residues not involved in antigen binding.2Rs15D is a unique sdAb in that the majority of the antibody-antigen contacts lie within the scaffold region, as opposed to the hypervariable CDR loops. We therefore excluded Arg43, Arg45, Arg50, and Lys60, as these residues lie within or close to the 2Rs15D-HER2 interface. We also excluded Arg38 and Arg66, as these face toward the interior of the sdAb. The remaining targets for charge engineering were Lys19, Lys64, Lys75, Lys76, and Lys86 (Fig 2A). We first made individual or double Lys to Asp mutants at each position to generate 2Rs15D-K19D, -K64D, -K75-76D, and -K86D. We expressed each of these variants in BL21 E. coli and evaluated expression by western blot (Fig. 6). We prepared a large-scale batch of 2Rs15D-K75-76D, measured HER2 binding by ELISA, and observed only a small reduction in apparent binding affinity, from 1.2 nM to 1.9 nM (Fig 2B).

[0139] Because we hypothesized that a large change in surface charge would be required to observe an effect on plasma clearance, and because the individual Page 32 of 45 81158138.v1769449-UIUC-065PC mutations were generally well-tolerated, we combined all five single-amino acid mutations to generate (-)2Rs15D. The unmodified and 5x mutant sdAbs display dramatically different electrostatic potential across their surfaces (Fig 2C), with theoretical isoelectric points of 7.2 and 4.8, respectively. We expressed and purified (- )2Rs15D from E. coli and did not observe a decrease in yield compared to the unmodified 2Rs15D (~3 mg / L). In order to experimentally validate that the engineering had indeed altered the charge distribution of the sdAbs, we performed analytical ion exchange chromatography by separately running both variants over an anion exchange column and analyzing the fraction of protein bound to the column.2Rs15D, which natively carries a net charge of 0 at physiological pH, had a bound fraction of 0.56 while (-)2Rs15D, with a theoretical net charge of -10 at physiological pH, had a bound fraction of 0.96 (Fig 2D). We next tested the effects of the 5x KD mutations on antigen binding, and observed a small decrease in binding affinity compared to unmodified 2Rs15D, from 1.2 nM to 2 nM. We observed a similar decrease after only two KD mutations (Fig 2B, E). Page 33 of 45 81158138.v1769449-UIUC-065PC

[0140] Example 9: Single-Domain Antibody A20.1

[0141] To demonstrate that this rational charge engineering strategy is applicable across sdAb scaffolds, we applied the same charge modifications to A20.1, a llama- derived sdAb that targets Clostridioides difficile toxin A. Excluding the hypervariable CDR regions, A20.1 and 2Rs15D share 75% sequence identity. The amino acid sequence of A20.1 is QVQLVESGGGLAQAGGSLRLSCAASGRTFSMDPMAWFRQPPGKEREFVAAGSST GRTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAAPYGANWYRDE YDYWGQGTQVTVSS (SEQ ID NO:34). After inspection of the A20.1-Toxin A crystal structure (PDB 4NBX), we identified five negatively charged amino acid residues using the same criteria we used for 2Rs15D (Fig 3A). We then constructed an A20.1 mutant with five charge mutations: K43E, R56E, K65E, K76E, and K87E ((-)A20.1). The mutations reduced the theoretical isoelectric point and net charge of A20.1 from 7.9 and +1 to 4.2 and -9, respectively, and had a similar effect on the theoretical electrostatic distribution on the sdAb surface as for 2Rs15D (Fig 3B). We expressed and purified both A20.1 and (-)A20.1 in BL21 E. coli with only a small effect on protein yield (~8 mg / L vs ~5 mg / L). As with 2Rs15D, the mutations resulted in a small decrease in affinity, from 0.5 nM to 1.3 nM (Fig 3C). We also experimentally validated the impact of the mutations on the surface charge of A20.1 using analytical ion exchange chromatography as described above for 2Rs15D, and observed a similar expected shift in the bound fraction, from 0.51 to 1 (Fig 3D).

[0142] Example 10: Fusion to GCN4pII results in sdAb trimerization

[0143] Hydrodynamic radius can affecting glomerular sieving. We therefore hypothesized that increasing sdAb hydrodynamic radius would be an effective strategy to decrease renal excretion and slow plasma clearance. In developing our engineering strategy, we sought a method that would functionally increase hydrodynamic radius without dramatically increasing the size of the sdAb monomer in order to retain the sdAb’s inherent ability to penetrate tissue and to be expressed at high titer by microbial expression systems – both features enabled by the small size and simplicity of the sdAb. With this in mind, we identified GCN4pII, a 33-amino acid leucine zipper that is a variant of the multimerization domain of the yeast transcription factor GCN4. The amino acid sequence of GCN4pII is RMKQIEDKIEEILSKIYHIENEIARIKKLVGER (SEQ ID NO:35). Fusion of GCN4pII to recombinant proteins can induce formation of Page 34 of 45 81158138.v1769449-UIUC-065PC stable homotrimers. We fused GCN4pII to the C-terminus of A20.1 and 2Rs15D to generate 3xA20.1 and 3x2Rs15D. We then used ColabFold to generate predicted structures of both sdAb-GCN4pII fusions and observed no major structural changes (Fig 4A). We then used size-exclusion chromatography and native polyacrylamide gel electrophoresis (PAGE) to evaluate the effects of GCN4pII fusion on sdAb size. The addition of GCN4pII decreased the SEC retention time by approximately 25%-36% (35 min to 26 min for A20.1 and 36 min to 23 min for 2Rs15D) (Fig 4B). We also observed peaks corresponding to cleaved monomer and GCN4pII domains, confirmed by denaturing SDS-PAGE (Fig 4D). Native PAGE clearly showed the expected ~3x increase in molecular weight corresponding to a trimerized sdAb (Fig 4C). We also tested the effects of multimerization on antigen binding by ELISA and observed an increase in apparent affinity for both 3xA20.1 (0.5 nM to 0.2 nM) and 3x2Rs15D (3 nM to 1.4 nM), likely due to the increased avidity of the trimerized sdAb (Fig 4E).

[0144] We next fused GCN4pII onto (-)A20.1 and (-)2Rs15D to generate the multimerizing, charge-engineered variants 3x(-)A20.1 and 3x(-)2Rs15D. Following the same workflow described for the charge-only and multimerization-only modified sdAbs, we evaluated the effects of these modifications on both antigen binding and protein size. By SEC, the combination of charge-engineering and GCN4pII fusion further decreased retention time to 24 min for A20.1 and 18 min 2Rs15D (data not shown). We hypothesize that increased inter-monomer repulsion caused by the strongly anionic charge-engineered sdAb domains compared to the unmodified versions resulted in an additional functional increase in hydrodynamic radius. Monomer size was not significantly altered, as measured by reducing SDS-PAGE (Fig 4D). Antigen binding was similarly unaffected when comparing 3xA20.1 and 3x(- )A20.1, though we did observe a decrease in 3x(-)2Rs15D from 1.4 to 3.3 nM (Fig 4E). This decrease is expected when considering the affinity decrease observed during initial charge engineering (Fig 2E).

[0145] Example 11: Charge engineering and multimerization impact plasma clearance in mice

[0146] Because there is not an in vitro model that captures the full complexity of plasma clearance and renal filtration, we used a mouse model to evaluate the pharmacokinetics of our engineered sdAbs. We selected the mouse model Tg32 (B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr / DcrJ) which expresses the human isoform of Page 35 of 45 81158138.v1769449-UIUC-065PC the neonatal Fc receptor (FcRn) in place of the mouse isoform. We chose this mouse line to permit comparison of sdAb half-life with full-length human IgG and to enable future studies incorporating FcRn binding peptides we developed previously as antibody fragment fusions.

[0147] To determine if the charge- and multimerization-modified antibody fragments demonstrate enhanced pharmacokinetics, we administered unmodified A20.1, (- )A20.1, 3xA20.1, 3x(-)A20.1, or a saline control to the right peritoneal cavity of 8-10- week old Tg32 mice (n=4 per group), and monitored plasma concentrations of the sdAbs over time by ELISA (Fig 5A). As expected, the unmodified sdAb was rapidly cleared from circulation, with no detectable sdAb at 8h post-injection (Fig 5B, black bars). The charge-engineering alone had no detectable effect on plasma clearance (Fig 5B, pink bars), while the addition of GCN4pII resulted in detectable levels of sdAb as late as 12h post-injection and a ~3-fold increase in β-phase half-life (Fig 5B, cyan bars, Table 1). Compellingly, the combination of charge engineering and GCN4pII fusion had a dramatic effect on clearance: 3x(-)A20.1 was still detectable as far as 72h post-injection, when the study was terminated (Fig 5B, purple bars), and displayed more than a 10-fold increase in half-life (Table 1). To investigate the effects of our half- life extending modifications on renal filtration, we also quantified sdAb concentrations in the urine. Urine concentration of unmodified A20.1, (-)A20.1, and 3xA20.1 all peaked at 3 hours post-injection while 3x(-)A20.1 did not reach peak urine concentration until 6 hours post-injection, indicating that the combination of charge engineering and multimerization delays plasma clearance at least in part due to a delay in renal filtration (Fig 5C). Table 1 β-phase half-lives of modified and unmodified sdAb Construct t1 / 2 (hours) A20.1 1 (-)A20.1 1 3xA20.1 2.8 3x(-)A20.1 10.5 2Rs15D 1 3x2Rs15D 1.3 3x(-)2Rs15D 5.8 Page 36 of 45 81158138.v1769449-UIUC-065PC

[0148] We then repeated these in vivo studies with unmodified 2Rs15D, 3x2Rs15D, and 3x(-)2Rs15D. (-)2Rs15D was not included due to insufficient limit of detection in endpoint assays. Following a similar pattern that observed with A20.1, the unmodified 2Rs15D was rapidly cleared from circulation within 6-8 hours post-injection, 3x2Rs15D was still detectable at 12 hours post-injection, and the double-modified 3x(-)2Rs15D could still be detected at 24 hours post-injection (Fig 5D). The β-phase half-lives of 3x2Rs15D and 3x(-)2Rs15D were extended by 1.3- and 5.8-fold, respectively (Table 1). While there seemed to be a general trend of 2Rs15D and its mutants clearing more rapidly than the A20.1 variants, the relative effects of our engineering on plasma clearance were remarkably similar between the two unrelated sdAbs. Similarly, urine concentrations of unmodified 2Rs15D and 3x2Rs15D peaked 1 hour post-injection, while 3x(-)2Rs15D peaked in the urine at 3 hours post-injection (Fig 5E). Taken together, these data suggest that fusion to a multimerization domain, with or without rational charge engineering, are valid strategies for manipulating the plasma clearance and renal filtration of sdAbs.

[0149] In this work, we have demonstrated that rational charge engineering, combined with fusion to a small multimerization domain, can be used to extend plasma circulation of sdAbs, in part through delaying renal filtration. Further, we showed that these strategies can be translated between unrelated sdAb scaffolds with remarkably similar effects in vivo half-life and near-complete retention of antigen binding affinity. This is, to our knowledge, the first demonstration of rational charge engineering and self-assembly of sdAbs as a means to alter their pharmacokinetics.

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Claims

769449-UIUC-065PC Claims We claim:

1. A single-domain antibody comprising: (a) one or more mutations of one or more positively charged amino acids to one or more negatively charged amino acids; and (b) a multimerization domain.

2. The single-domain antibody of claim 1, wherein the one or more positively charged amino acids are not present in a paratope region of the single-domain antibody and are not in the hydrophobic core of the single-domain antibody.

3. The single-domain antibody of claim 2, wherein the paratope region of the single-domain antibody is determined by X-ray crystallography, hydrogen- deuterium exchange mass spectrometry, or peptide array-based epitope mapping.

4. The single-domain antibody of any one of claims 1-3, wherein the single- domain antibody further comprises one or more neonatal Fc receptor (FcRn) binding polypeptides comprising an amino acid sequence set forth in QRFX1TGHFGGLX2PX3NG (SEQ ID NO:1).

5. The single-domain antibody of any one of claims 1-4, wherein the single- domain antibody further comprises one or more neonatal Fc receptor (FcRn) binding polypeptides comprising an amino acid sequence set forth in SEQ ID NO:2, 3, 4, 5, or 32.

6. The single-domain antibody of any one of claims 1-5, wherein the positively charged amino acids are lysine, arginine, and histidine and the negatively charged amino acids are aspartic acid and glutamic acid. Page 41 of 45 81158138.v1769449-UIUC-065PC 7. The single-domain antibody of any one of claims 1-6, wherein there are 1, 2, 3, 4, or 5 mutations of one or more positively charged amino acids to one or more negatively charged amino acids.

8. The single-domain antibody of any one of claims 1-7, wherein one or more of the one or more mutations are in a framework region of the single-domain antibody.

9. The single-domain antibody of any one of claims 1-7, wherein one or more of the one or more mutations are in a complementarity-determining region (CDR) region of the single-domain antibody.

10. The single-domain antibody of any one of claims 1-9, wherein the multimerization domain is coiled-coil dimerization domain GCN4, GCN4-pII, GCN4-pLI, ATF6, CREB1, C / EBPα, Fos, Jun, influenza hemagglutinin, or HIVgp41.

11. The single-domain antibody of any one of claims 1-10, wherein the single- domain antibody is VHH antibody, nanobody, camelid-derived single-domain antibody, shark IgNAR-derived single-domain antibody (VNAR), diabody, or triabody.

12. The single-domain antibody of any one of claims 1-11, wherein the hydrodynamic radius of the single-domain antibody is increased as compared to a control single-domain antibody.

13. The single-domain antibody of any one of claims 4-12, wherein single-domain antibody can be transported across mucosal surfaces.

14. The single-domain antibody of any one of claims 1-13, wherein the single- domain antibody is A20.

1. Page 42 of 45 81158138.v1769449-UIUC-065PC 15. The single-domain antibody of claim 14, wherein one more of the following positively charged amino acids is changed to a negatively charged amino acid: K43, R56, K65, K76, and K87.

16. The single-domain antibody of any one of claims 1-14, wherein the single- domain antibody is 2Rs15D.

17. The single-domain antibody of claim 16, wherein one more of the following positively charged amino acids is changed to a negatively charged amino acid K19, K64, K75, K76, and K86.

18. A composition comprising the single-domain antibody of any one of claims 1- 17 and a pharmaceutically acceptable carrier.

19. A polynucleotide encoding the single-domain antibody of any one of claims 1- 17.

20. A vector comprising the polynucleotide of claim 19.

21. A host cell comprising the vector of claim 20.

22. A method of increasing the β-phase half-life of a single-domain antibody comprising: (a) identifying selected positively charged amino acids of the single-domain antibody by identifying all positively charged amino acids of the single- domain antibody that are not present in the paratope of the single-domain antibody and are not in the hydrophobic core of the single-domain antibody; (b) mutating one or more of the selected positively charged amino acids to one or more negatively charged amino acids to form a charge modified single- domain antibody; and Page 43 of 45 81158138.v1769449-UIUC-065PC (c) adding a multimerization domain to the charge modified single-domain antibody, thereby forming a single-domain antibody having increased β- phase half-life.

23. The method of claim 22, further comprising adding one or more neonatal Fc receptor (FcRn) binding polypeptides comprising an amino acid sequence set forth in QRFX1TGHFGGLX2PX3NG (SEQ ID NO:1) to the single-domain antibody.

24. The method of claim 22, wherein the FcRn binding polypeptide comprises an amino acid sequence as set forth in SEQ ID NO:2, 3, 4, 5, or 32.

25. A method of treatment comprising administering the composition of claim 18 to a subject in need thereof.

26. The method of claim 25, wherein the single-domain antibody can be transported across mucosal surfaces.

27. The method of claim 25, wherein the β-phase half-life is increased as compared to a control single-domain antibody. Page 44 of 45 81158138.v1

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