WO2024215805A2 - Single-domain antibodies that bind the constant region of immunoglobulins - Google Patents

Abstract

Single-domain antibodies that bind the constant (Fc) region of an immunoglobulin antibody are described. In embodiments, the single-domain antibodies covalently bind the Fc region of IgG. The single-domain antibodies can include a crosslinker for covalent attachment to a primary antibody and can be conjugated to a detectable label for use in techniques such as oligo barcoding, mass spectrometry, various imaging/omics platforms, liquid biopsy, and therapeutics.

Description

SINGLE-DOMAIN ANTIBODIES THAT BIND THE CONSTANT REGION OF IMMUNOGLOBULINS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/495,229 filed April 10, 2023, which is incorporated herein by reference in its entirety as if fully set forth herein.

REFERENCE TO SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the file containing the Sequence Listing is 31 H8471.xml. The file is 110,592 bytes, was created on April 10, 2024, and is being submitted electronically via Patent Center.

FIELD OF THE DISCLOSURE

[0003] The current disclosure describes single-domain antibodies that bind the constant (Fc) region of immunoglobins. The single-domain antibodies can include a crosslinker for covalent attachment to primary antibodies and can be conjugated to detectable labels for use in techniques such as oligo barcoding, mass spectrometry, various imaging/omics platforms, liquid biopsy, and therapeutics.

BACKGROUND OF THE DISCLOSURE

[0004] Immunoglobulins, also known as antibodies, are affinity molecules that specifically bind antigens in cells and tissue. Antibodies are a fundamental tool and have been widely used in molecular and cellular biology, medical research, and clinical diagnosis, enabling the selective, sensitive detection and quantification of proteins and other molecules in techniques, such as immunohistochemistry (IHC), immunofluorescence (IF), and fluorescence-activated cell sorting (FACS) which rely on the antibodies representing standard routine image assays to visualize the abundance of target antigens in tissues or cells under light microscopy. Meanwhile, these applications require the attachment of chemical probes or specific tags to the antibody.

[0005] Detectable labels can be attached to a primary antibody through the use of secondary antibodies. Secondary antibodies can bind primary antibodies and if the secondary antibody is labeled, the location or quantity of primary antibodies are labeled upon secondary antibody binding. Despite these methods being widely used, specificity has long plagued even the more targeted approaches to label antibodies. As such, an efficient, sustainable, and reproducible sitespecific antibody labeling method is needed. SUMMARY OF THE DISCLOSURE

[0006] The current disclosure provides novel single-domain antibodies for covalently labeling primary antibodies. The single-domain antibodies can be conjugated to detectable labels for use in techniques such as oligo barcoding, mass spectrometry, and various imaging platforms, among other uses. For example, the disclosed single-domain antibodies can be used for imaging, diagnostics, and research and can be engineered to deliver therapeutic cargo for anti-cancer or anti-viral treatments.

[0007] In particular embodiments, single-domain antibodies disclosed herein bind the constant (Fc) region of a rabbit immunoglobulin G (IgG) antibody. In particular embodiments, the singledomain antibody includes a sequence having at least 95% or 98% sequence identity to a sequence as set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and/or SEQ ID NO: 7. In particular embodiments, the single-domain antibody includes a complementarity-determining region (CDR) set including a CDR1 including the sequence as set forth in SEQ ID NO: 8, a CDR2 including the sequence as set forth in SEQ ID NO: 9, and a CDR3 including the sequence as set forth in SEQ ID NO: 10. In particular embodiments, the single-domain antibody includes a CDR set including a CDR1 including a sequence having at least 95% or 98% sequence identity to the sequence as set forth in SEQ ID NO: 8, a CDR2 including a sequence having at least 95% or 98% sequence identity to the sequence as set forth in SEQ ID NO: 9, and/or a CDR3 including a sequence having at least 95% or 98% sequence identity to the sequence as set forth in SEQ ID NO: 10. In particular embodiments, a CDR sequence having a percent sequence identity to SEQ ID NO: 8, 9, or 10 includes at least one mutation. In particular embodiments, the at least one mutation includes replacing an amino acid residue with a crosslinker. In particular embodiments, the photocrosslinker is located at position G34, Q42, F55, E58, G60, or G 105 of the sequence as set forth in SEQ ID NO: 5. In particular embodiments, the single-domain antibody is conjugated to a cargo, such as a detectable label or an effector molecule.

BRIEF DESCRIPTION OF THE FIGURES

[0008] Some of the drawings submitted herewith may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0009] FIG. 1. Schematic diagram of the working principle and workflow. This system works through the implementation of photoreactive crosslinkers on a single-domain antibody (also referred to as Nanobody) designed to bind to primary antibody Fc domains. Rabbit primary antibody (labeled IgG) is shortly pre-incubated with the photoactivatable nanobody, followed by a UV irradiation to activate the photoreactive crosslinker on the Nanobodies generating free radical linked to nearby Fc fragment. These quickly form covalent bonds with the Fc domain of the primary antibody, accelerated by the high affinity and specificity afforded by the nanobodies. This system can leverage different types of photoreactive crosslinkers to attain the highest efficiency binding and covalent reaction times.

[0010] FIG. 2. Nanobody expression map. This nanobody is produced through conventional pET bacterial recombinant protein expression system, allowing for an easy-to-use and highly scalable production.

[0011] FIG. 3. Proof of concept photocrosslinking sites in the nanobody (indicated by box and mutation label). Sequence level view of the positions for non-canonical amino acid (NCAA) incorporation as determined via molecular dynamics simulations. Screening via sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS PAGE) analysis from these sites confers the efficacy of the mutated NCAA positions and photocrosslinking proximity. This has been critically validated based on anti-rabbit IgG nanobodies.

[0012] FIG. 4. Coomassie blue G250 stained SDS-PAGE showing purified nanobodies from E.coli cell lysate.

[0013] FIG. 5. Native and denaturing SDS-PAGE showing the kinetic efficiency of the photocrosslinking between the single-domain antibody (IgG-Nanobody*) at various time points after ultraviolet (UV) irradiation. Comparative view of photocrosslinking reactivity over time via both native and denaturing gels. For native gels, the full antibody is kept intact, while the addition of two nanobodies can be clearly seen crosslinked to the intact antibody (150 kDa), resulting in the size shifted 180 kDa complex with two attached nanobodies (15 kDa/Nanobody). Additionally, when viewing the reactivity from a denaturing gel, the targeted component addition is more clearly visualized. The light chain (25 kDa), being a part of the Fab region is not targeted at all by the nanobody, and therefore always retains its size on the gel. Critically, the heavy chain (50 kDa) visibly increases after <5 minutes, as the nanobody specifically and efficiently bound to almost all heavy chain portions of the Fc fragment (65 kDa in complex).

[0014] FIG. 6. Comparative view of the kinetic efficiency of the photocrosslinking between the single-domain antibody (IgG-Nanobody*) and oYo- Link® (Alphathera, Philadelphia, PA) over time after UV irradiation in denaturing SDS-PAGE. Comparative view of photo-crosslinking reactivity and efficiency for the single-domain antibody (IgG-Nanobody*) and the oYo-Link® (Alphathera) product. For both gel regions, the light chain stays consistent, while the heavy chain reacts with either the Nanobody* or the oYo- link® (Alphathera). Critically, while the oYo-Link® requires hours of reaction time to fully cross-link the tag onto the heavy chain, the single-domain antibody (IgG- Nanobody*) has substantially improved reaction efficiency, fully reacting in only 5 minutes (for full time series for oYo-Link® (Alphathera) conjugation, refer to their original publication (Hui et al., 2015, Bioconjug Chem 26(8): 1456-1460).

[0015] FIG. 7. Schematic diagrams of potential applications of this single-domain antibody system. Visual summary of many potential applications for this platform. In particular the predominant use envisioned for this system is in the fields of Oligo barcoding (Cyclic- immunofluorescence (CyclF)/proteomics), fluorophore conjugations for use as either directly sold secondary labels or even as a part of a CycIF labeling system. Other exciting use cases involve other emerging research using probes with elemental isotopes with mass-spectroscopy and even Quantum dots for combined immunofluorescence (IF) and electron microscopy (EM) imaging modalities. Finally, there is a strong use-case as an intermediate product linking primary antibodies of interest to downstream probes such as horseradish peroxidase (HRP) and immunohistochemistry (IHC), as well as incorporation with other self-labeling tags such as Halo® (Promega, Madison, Wl) and Snap® tag (New England Biolabs, Ipswich, MA) modalities.

[0016] FIG. 8. Schematic illustrating two possible models of anti-Rabbit single-domain antibody TP897 interactions with the Rabbit IgG Fc region. Canonical complementarity determining regions (CDR) are labeled. Mutationally screened binding residues for each binding model indicated by triangles (Model 1) and dots (Model 2). (SEQ ID NO: 4)

[0017] FIG. 9. First and second round experimentally validated mutational sites, with each residue matched to corresponding models. Sequence residues for sites selected correspond to numbered positions within SEQ ID NO: 6. Round 1 residues (left) were scored via simulation, while round 2 residues (right) were selected by expanding from round 1 results. p-benzoyl-L-phenylalanine (Bpa) non-canonical amino acid incorporation and photocrosslinking efficiency to Rabbit IgG domains also indicated.

[0018] FIGs. 10A, 10B. (10A) Schematic representation of the strategy used in this example for in vivo incorporation of UAA Bpa into the designed nanobodies. (10B) Non-reducing SDS-PAGE gel showing the crosslink screening of anti-rabbit single-domain antibody TP897 variants incorporated with Bpa and rabbit monoclonal antibody before and after UV irradiation. Sequence residues shown correspond to numbered positions within SEQ ID NO: 6.

[0019] FIGs. 11 A, 11B. (11 A) Representative gel of both E.coli crude cell lysate after Bpa- nanobody (Bpa-NB) expression and highly-specific nickel Nitrilotriacetic acid (Ni-NTA) purification using his-tags run on a SDS gel. (11 B) Photocrosslinking results after 20-minute illuminations for all selected residues of interest run on a native gel. Sequence residues shown correspond to numbered positions within SEQ ID NO: 6. [0020] FIG. 12. Mutational sites on the single-domain antibody incorporating Bpa which showed covalent photocrosslinking to Rabbit IgG after first and second round screening and validations. Sequence residues shown correspond to numbered positions within SEQ ID NO: 6.

[0021] FIG. 13. Photocrosslinking efficiency of single-domain antibody F56X (position F56 of SEQ ID NO: 6) with rabbit monoclonal antibody when exposed to UV for different periods of time run on a reducing SDS-PAGE gel. Numbered moieties of the photocrosslinking product, Rabbit Fc heavy/light chains, and Bpa-NB indicated as 1-4 respectively.

[0022] FIG. 14. Comparison of 355 nm UV illumination power on conjugation efficiency for F56 (position F56 of SEQ ID NO: 6) Bpa-NB with Rabbit IgG. UV illumination using a focused 100mW laser (left). Identically pre-incubated Bpa-NB and Rabbit IgG illuminated with varying laser power for 1 minute.

[0023] FIG. 15. Comparison of mutational sites incorporated with Bpa and Dizpk individually. 355 nM UV illumination was performed using UV lamp.

[0024] FIG. 16. Schematic overview of how to use this technology and its potential applications as a technology platform. In particular predominant use in the fields of Oligo barcoding (Cyclic- IF/proteomics), fluorophore conjugations was envisioned for use as either directly sold secondary labels or even as a part of a cyclic-IF labeling system. Other exciting use cases involve other emerging research using probes with elemental isotopes with mass-spectroscopy and even Quantum dots for combined IF and EM imaging modalities. Finally, there is a strong use-case as an intermediate product linking primary antibodies of interest to downstream probes such as HRP and IHC, as well as incorporation with other self-labeling tags such as Halo® (Promega) and Snap® (New England Biolabs) tag modalities.

[0025] FIG. 17. Comparison of commercial oYo_Link® (AlphaThera LLC, Philadelphia, PA) and the Bpa-NB conjugation efficiency and purity via both denaturing SDS-PAGE (top) and nondenaturing SDS-PAGE gels (bottom). 355 nm UV illumination was performed using UV lamp.

[0026] FIGs. 18A, 18B. (18A) Fluorophore functionalization and photocrosslinking scheme and representative gel of antibody conjugation products. Gel images include the 555-excitation fluorescent gel, Coomassie staining, and overlay respectively. Commercial IgG anti-Tom20 contained BSA within its storage media. (18B) Representative images of IgGs against Tubulin and Tom20 as immunolabeled on U-2 OS cells. Donkey@Rabbit-AF555 commercial secondaries are compared vs 1x and 3x AF-555 conjugated Bpa-NBs as indicated. To visually compare with commercial secondary antibody labelling, 1x and 3x AF-555 conjugated Bpa-NB labelling are displayed at a normalized brightness, in particular the 1x AF-555 had its brightness adjusted to three times higher. All scale bars 10 pm. [0027] FIG. 19. Primer designed for plasmid construction for mutational cloning of single-domain antibody.

[0028] FIG. 20. Cryo-EM analysis of rabbit IgG Fc fragment complexed with nanobodies. 3D reconstructions from the Cryo-EM data with two proposed models docked inside the same map using ChimeraX. The two proposed models shown in ribbon representations are called model 1 and model 2.

[0029] FIG. 21. Comparison of Bpa-NB and the commercial oYo_Link® conjugation efficiency for antibody oligo barcoding. A schematic view (left) shows antibodies mixed with Bpa-NB and oYo_Link® individually attached with DNA oligomers (curved line with dashes), each carrying a Cy3 fluorophore (star). Each mixture undergoes a 5-min 355 nm UV illumination for covalent photocrosslinking. SDS-PAGE gel images (right) include the merged overlay (top), the Cy3- excitation (555nm) fluorescence (middle), and Coomassie staining (bottom), respectively, showing near complete conjugation of Bpa-NB-oligo to antibodies, while not observed with the commercial oYo_Link®.

[0030] FIGs. 22A, 22B. Representative DNA-conjugations with BPA-NB and multi-round DNA- oligomer based CycIF imaging application. (22A) SDS-PAGE gel images show the merged overlay (top), the Cy3 (555nm) fluorescence (middle), and Coomassie staining (bottom). These images show near-complete conjugation of Bpa-NB-Oligos with EpCAM antibody, Tom20 antibody, and CK8 antibody within 5 min 355/365nm UV illumination, individually. Notably, the conjugation of the Tom20 antibody with Bpa-NB-Oligos was also near complete, even in the presence of more concentrated BSA (band between 55kDa and 70kDa) in its storage media. (22B) CycIF imaging of these oligo-barcoded antibodies in MCF7 cells. After initial imaging of DAPI (top left), 3 immunolabeled targets were imaged sequentially. EpCAM (plasma membrane) labeling through its antibody with Bpa-NB-DS1 was revealed after the addition of the fluorescent DNA oligonucleotides (imager strand 1 , IS1 ) to its corresponding complementary target sequence (docking strand 1 , DS1). Following a dehybridization wash to remove IS1 , Tom20 (mitochondria) labeling through its antibody with Bpa-NB-DS2 was displayed after the addition of the fluorescent IS2 to its corresponding complementary DS2. Cytokeratin 8 (Cytoskeleton) labeling through its antibody with Bpa-NB- DS3 was exhibited after introducing the fluorescent IS3 to its corresponding complementary DS3. The merged and enlarged view of the three targets was displayed on the right.

DETAILED DESCRIPTION

[0031] Immunoglobulins, also known as antibodies, are affinity molecules specifically binding antigens in cells and tissue. Antibodies are a fundamental tool and have been widely used in molecular and cellular biology, medical research, and clinical diagnosis, enabling the selective, sensitive detection and quantification of proteins and other molecules in techniques, such as immunohistochemistry (IHC), immunofluorescence (IF), and fluorescence-activated cell sorting (FACS) which rely on the antibodies. These techniques represent standard routine image assays to visualize the abundance of target antigens in tissues or cells. These applications require the attachment of chemical probes or specific tags to the antibody.

[0032] Detectable labels can be attached to primary antibodies through the use of secondary antibodies. Secondary antibodies bind the constant (Fc) region of a primary antibody and if the secondary antibody is labeled, the location or quantity of primary antibodies are labeled upon secondary antibody binding primary antibody. Despite these methods being widely used, specificity has long plagued even the more targeted approaches to label antibodies. As such, an efficient, sustainable, and reproducible site-specific primary antibody labeling method is needed. [0033] The current disclosure provides novel single-domain antibodies for covalently labeling primary antibodies. The single-domain antibodies can be conjugated to detectable labels for use in techniques such as oligo barcoding, mass spectrometry, or various imaging platforms. For example, the disclosed single-domain antibodies can be used for imaging, diagnostics, and research and can also be engineered to deliver therapeutic cargo for anti-cancer and anti-viral treatments.

[0034] Single-domain antibodies disclosed herein bind the constant (Fc) region of a rabbit immunoglobulin G (IgG) antibody. In particular embodiments, the single-domain antibody includes a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and/or SEQ ID NO: 7. In particular embodiments, the single-domain antibody includes a complementarity-determining region (CDR) set including a CDR1 including the sequence as set forth in SEQ ID NO: 8, a CDR2 including the sequence as set forth in SEQ ID NO: 9, and a CDR3 including the sequence as set forth in SEQ ID NO: 10.

[0035] In particular embodiments, the single-domain antibody includes a CDR set including a CDR1 including a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 8, a CDR2 including a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9, and/or a CDR3 including a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 10.

[0036] In particular embodiments, the single-domain antibody includes a CDR set including a CDR1 including a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 8, a CDR2 including a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 9, and/or a CDR3 including a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 10.

[0037] When a CDR sequence has a percent sequence identity to SEQ ID NO: 8, 9, or 10, in particular embodiments the CDR sequence includes at least one mutation (e.g., 1 , 2, 3, 4, or 5 mutations). In particular embodiments, the mutation includes replacement of an amino acid residue with a photocrosslinker. Examples of these sequences include SEQ ID NO: 11 , 12, 13, 14, and 15.

[0038] In particular embodiments, the photocrosslinker is located at position G34, Q42, F55, E58, G60, or G105 of the sequence as set forth in SEQ ID NO: 5. In particular embodiments, the singledomain antibody is conjugated to a cargo, such as a detectable label (e.g., fluorophore or barcode) or an effector molecule (e.g., drug or toxin). If not otherwise described, the position numbering (e.g., position numbering in the figures) is based on SEQ ID NO: 6.

[0039] Single-domain antibodies disclosed herein have advantages over current systems of labeling primary antibodies because they can include a photocrosslinker for covalent attachment to the primary antibody within a few minutes (as opposed to a few (e.g., 2) hours); the fastest covalent labeling efficiency reported, can avoid allosteric hindrances of larger probes, have high affinity for the Fc region of rabbit IgG, and do not hinder the complementarity-determining regions (CDRs) of the primary antibody. In embodiments disclosed herein, the primary antibody includes an Fc region from a rabbit IgG.

[0040] Aspects of the current disclosure are now described in more supporting detail as follows: (i) Conventional Antibodies & Associated Terminology; (ii) Single-Domain Antibodies; (iii) Crosslinkers; (iv) Antibody Conjugates; (v) Methods of Manufacture; (vi) Compositions; (vii) Methods of Use; (viii) Kits; (ix) Exemplary Embodiments; (x) Experimental Example; and (xi) Closing Paragraphs. These headings are provided for organizational purposes only and do not limit the scope or interpretation of the disclosure.

[0041] (i) Conventional Antibodies & Associated Terminology. Unless otherwise indicated, a “conventional antibody” includes a tetramer structure with two full-length heavy chains and two full-length light chains. The amino-terminal portion of each chain includes a variable region that is responsible for antigen recognition and epitope binding. The variable regions exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions (CDRs). The CDRs from the two chains of each pair are aligned by the framework regions, which enables binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions include the domains FR1, CDR1 , FR2, CDR2, FR3, CDR3 and FR4. [0042] The assignment of amino acids to each domain can be in accordance with Kabat numbering (Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,’’ 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme)); Chothia (Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme)), Martin (Abinandan et al., Mol Immunol. 45:3832-3839 (2008), “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains”), Gelfand, Contact (MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (Contact numbering scheme)), IMGT (Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1):55-77 (“IMGT” numbering scheme)), AHo (Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (AHo numbering scheme)), North (North et al., J Mol Biol. 406(2) :228-256 (2011), “A new clustering of antibody CDR loop conformations”), or other numbering schemes.

[0043] Definitive delineation of a CDR and identification of residues including the binding site of an antibody can be accomplished by solving the structure of the antibody and/or solving the structure of the antibody-epitope complex. In particular embodiments, this can be accomplished by methods such as X-ray crystallography and cryoelectron microscopy. Alternatively, CDRs are determined by comparison to known antibodies (linear sequence) and without resorting to solving a crystal structure. To determine residues involved in binding, a co-crystal structure of the Fab (antibody fragment) bound to the target can optionally be determined. Software programs, such as ABodyBuilder can also be used.

[0044] The carboxy-terminal portion of each chain defines a constant region (the Fc region), which is responsible for effector function of the antibody. Examples of effector functions include: C1q binding and complement dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B-cell receptors); and B-cell activation. A portion of an Fc region is a fragment of an Fc region. The fragment can include 10% of an Fc region, 20% of an Fc region, 30% of an Fc region, 40% of an Fc region, 50% of an Fc region, 60% of an Fc region, 70% of an Fc region, 80% of an Fc region, 90% of an Fc region, or 95% of an Fc region. A portion of an Fc region can also include a characterized segment of an Fc region, such as a CH2 region or a CH3 region.

[0045] Within full-length light and heavy chains, the variable and constant regions are joined by a “J” region of amino acids, with the heavy chain also including a “D” region of amino acids. See, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). [0046] Light chains are typically classified as kappa and lambda light chains. Heavy chains are generally classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG causes opsonization and cellular cytotoxicity and crosses the placenta, IgA functions on the mucosal surface, IgM is most effective in complement fixation, and IgE mediates degranulation of mast cells and basophils. The function of IgD is still not well understood. Resting B cells, which are immunocompetent but not yet activated, express IgM and IgD. Once activated and committed to secrete antibodies these B cells can express any of the five isotypes. The heavy chain isotypes of IgG, IgA, IgM, IgD and IgE are respectively designated the y, a, p, 5, and E chains.

[0047] In particular embodiments, an immunoglobulin gamma heavy chain constant region [Oryctolagus cuniculus] includes the sequence: QPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYS LSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMI SRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRG KEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVE WEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSIS RSPGK (SEQ ID NO: 1).

[0048] In particular embodiments, an immunoglobulin kappa light chain constant region [Oryctolagus cuniculus] includes the sequence: SAAAAPTVLLFPPSSDEVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCT YNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFSRKNC (SEQ ID NO: 2).

[0049] In particular embodiments, an immunoglobulin lambda light chain constant region [Oryctolagus cuniculus] includes the sequence: QPAVTPSVILFPPSSEELKDNKATLVCLISDFYPRTVKVNWKADGNSVTQGVDTTQPSKQSNN KYAASSFLHLTANQWKSYQSVTCQVTHEGHTVEKSLAPAECS (SEQ ID NO: 3).

[0050] Antibodies bind epitopes on antigens. The term antigen refers to a molecule or a portion of a molecule capable of being bound by an antibody. An epitope is a region of an antigen that is bound by the variable region of an antibody. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics, and/or specific charge characteristics. When the antigen is a protein or peptide, the epitope includes specific amino acids within that protein or peptide that contact the variable region of an antibody.

[0051] In particular embodiments, an epitope denotes the binding site on the antigen bound by a corresponding variable region of an antibody. The variable region either binds to a linear epitope (e.g., an epitope including a stretch of 5 to 12 consecutive amino acids), or the variable region binds to a three-dimensional structure formed by the spatial arrangement of several short stretches of the protein target. Three-dimensional epitopes recognized by a variable region, e.g., by the epitope recognition site or paratope of an antibody or antibody fragment, can be thought of as three-dimensional surface features of an epitope molecule. These features fit precisely (in)to the corresponding binding site of the variable region and thereby binding between the variable region and its target protein (more generally, antigen) is facilitated. In particular embodiments, an epitope can be considered to have two levels: (i) the “covered patch” which can be thought of as the shadow an antibody variable region would cast on the antigen to which it binds; and (ii) the individual participating side chains and backbone residues that facilitate binding. Binding is then due to the aggregate of ionic interactions, hydrogen bonds, and hydrophobic interactions. For information regarding binding values and methods to measure the same, see the Closing Paragraphs section of this disclosure.

[0052] A monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies including the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be made by a variety of techniques, including the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the immunoglobulin loci.

[0053] A “rabbit antibody” is one which includes an amino acid sequence which corresponds to that of an antibody produced by a rabbit or a rabbit cell or derived from a non-rabbit source that utilizes rabbit antibody repertoires or other rabbit antibody-encoding sequences.

[0054] A “rabbit consensus framework” is a framework that represents the most commonly occurring amino acid residues in a selection of rabbit immunoglobulin V or V_H framework sequences. Generally, the selection of rabbit immunoglobulin V or V sequences is from a subgroup of variable domain sequences. The subgroup of sequences can be a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91- 3242, Bethesda Md. (1991), vols. 1-3. In particular embodiments, for the VL, the subgroup is subgroup kappa I as in Kabat et al. (supra). In particular embodiments, for the V_H, the subgroup is subgroup III as in Kabat et al. (supra).

[0055] (ii) Single-Domain Antibodies. In particular embodiments, single-domain antibodies are the antigen binding fragment of heavy chain only antibodies. Single-domain antibodies are also referred to as VHH antibodies or nanobodies. One of ordinary skill in the art will recognize portions of the discussion related to “conventional rabbit antibodies” that equally apply to single-domain antibodies, and those portions that only apply to conventional rabbit antibodies. Single-domain antibodies are often more stable than scFv and Fab constructs and are more easily introduced into alternative scaffolds as they do not require heavy light chain pairings and long linker sequences.

[0056] In particular embodiments, a single-domain antibody that binds the Fc region of rabbit IgG is single-domain antibody TP897. In particular embodiments, the single-domain antibody includes the sequence:

QVQLVESGGGLVQAGDSLRLSCVASGRSLDGATMRWYRQAPGKEREFVAGIFWDEIGTEYAD TAKGRFTISRDNAKNTIYLQMTNLRSEDTAMYYCNGLVFGGEYWGQGTQVTVSSGG (SEQ ID NO: 4).

[0057] In particular embodiments, the single-domain antibody includes the sequence: AGSQVQLVESGGGLVQAGDSLRLSCVASGRSLDGATMRWYRQAPGKEREFVAGIFWDEIGT EYADTAKGRFTISRDNAKNTIYLQMTNLRSEDTAMYYCNGLVFGGEYWGQGTQVTVSSGG (SEQ ID NO: 5).

[0058] In particular embodiments, the single-domain antibody includes the sequence: MAGSQVQLVESGGGLVQAGDSLRLSCVASGRSLDGATMRWYRQAPGKEREFVAGIFWDEIG TEYADTAKGRFTISRDNAKNTIYLQMTNLRSEDTAMYYCNGLVFGGEYWGQGTQVTVSSGG (SEQ ID NO: 6).

[0059] In particular embodiments, the single-domain antibody includes the sequence:

M H H H H H HAGSQVQLVESGGGLVQAGDSLRLSCVASGRSLDGATM RWYRQAPGKEREFVAG I FWDEIGTEYADTAKGRFTISRDNAKNTIYLQMTNLRSEDTAMYYCNGLVFGGEYWGQGTQVT VSSGG (SEQ ID NO: 7).

[0060] In particular embodiments, the single-domain antibody includes a CDR1 including the sequence GRSLDGAT (SEQ ID NO: 8), a CDR2 including the sequence EFVAGIFWDEIGTEY (SEQ ID NO: 9), and a CDR3 including the sequence LVFGGEY (SEQ ID NO: 10).

[0061] In particular embodiments, a mutation is located at position G34, Q42, F55, E58, G60, or G105 of SEQ ID NO: 5. [0062] In particular embodiments, the single-domain antibody includes a CDR1 having the sequence as set forth in GRSLDXAT (SEQ ID NO: 11), a CDR2 having the sequence as set forth in EFVAGIFWDEIGTEY (SEQ ID NO: 9), and a CDR3 having the sequence as set forth in LVFGGEY (SEQ ID NO: 10), wherein X is a mutation.

[0063] In particular embodiments, the single-domain antibody includes a CDR1 having the sequence as set forth in GRSLDGAT (SEQ ID NO: 8), a CDR2 having the sequence as set forth in EFVAGIXWDEIGTEY (SEQ ID NO: 12), and a CDR3 having the sequence as set forth in LVFGGEY (SEQ ID NO: 10), wherein X is a mutation.

[0064] In particular embodiments, the single-domain antibody includes a CDR1 having the sequence as set forth in GRSLDGAT (SEQ ID NO: 8), a CDR2 having the sequence as set forth in EFVAGIFWDXIGTEY (SEQ ID NO: 13), and a CDR3 having the sequence as set forth in LVFGGEY (SEQ ID NO: 10), wherein X is a mutation.

[0065] In particular embodiments, the single-domain antibody includes a CDR1 having the sequence as set forth in GRSLDGAT (SEQ ID NO: 8), a CDR2 having the sequence as set forth in EFVAGIFWDEIXTEY (SEQ ID NO: 14), and a CDR3 having the sequence as set forth in LVFGGEY (SEQ ID NO: 10), wherein X is a mutation.

[0066] In particular embodiments, the single-domain antibody includes a CDR1 having the sequence as set forth in GRSLDGAT (SEQ ID NO: 8), a CDR2 having the sequence as set forth in EFVAGIFWDEIGTEY (SEQ ID NO: 9), and a CDR3 having the sequence as set forth in LVFXGEY (SEQ ID NO: 15), wherein X is a mutation.

[0067] In particular embodiments, the single-domain antibody includes a CDR1 having the sequence as set forth in GRSLDGAT (SEQ ID NO: 8), a CDR2 having the sequence as set forth in EFVAGIFWDEIGTEY (SEQ ID NO: 9), and a CDR3 having the sequence as set forth in LVFGGEY (SEQ ID NO: 10), wherein the single-domain antibody includes the sequence AGSQVQLVESGGGLVQAGDSLRLSCVASGRSLDGATMRWYRXAPGKEREFVAGIFWDEIGTE YADTAKGRFTISRDNAKNTIYLQMTNLRSEDTAMYYCNGLVFGGEYWGQGTQVTVSSGG (SEQ ID NO: 16), and wherein X is a mutation.

[0068] In each embodiment including a mutation, the mutation can include insertion of a crosslinker. In particular embodiments, the crosslinker replaces an amino acid residue.

[0069] In particular embodiments, the single-domain antibody can include a methionine at the N- terminal end of the single-domain antibody sequence. In particular embodiments, the singledomain antibody can include an affinity tag and/or a linker. In particular embodiments, the affinity tag and/or linker are located at the N-terminal end. In particular embodiments, the affinity tag and/or linker are located at the C-terminal end. Affinity tags are described elsewhere herein. Linkers can include any chemical moiety that is capable of linking. Some linkers serve no purpose other than to link components while many linkers serve an additional purpose. Linkers can be flexible, rigid, or semi-rigid, depending on the desired function of the linker.

[0070] Commonly used flexible linkers include linker sequence with the amino acids glycine and serine (Gly-Ser linkers). In particular embodiments, the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (Gly_xSer_y)n, wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). Particular examples include (Gly4Ser)_n (SEQ ID NO: 30), (Gly₃Ser)n(Gly₄Ser)_n (SEQ ID NO: 31), (Gly₃Ser)n(Gly₂Ser)n (SEQ ID NO: 32), or (Gly₃Ser)n(Gly₄Ser)i (SEQ ID NO: 33). In particular embodiments, the linker is (Gly₄Ser)₄ (SEQ ID NO: 34), (Gly₄Ser)₃ (SEQ ID NO: 35), (Gly₄Ser₎₂ (SEQ ID NO: 36), (Gly₄Ser)i (SEQ ID NO: 37), (Gly₃Ser)₂ (SEQ ID NO: 38), (Gly₃Ser)i (SEQ ID NO: 39), (Gly₂Ser)₂ (SEQ ID NO: 40) or (Gly₂Ser)i, GGSGGGSGGSG (SEQ ID NO: 41), GGSGGGSGSG (SEQ ID NO: 42), or GGSGGGSG (SEQ ID NO: 43).

[0071] (iii) Crosslinkers. Crosslinkers suited for the methods and systems described herein are well known in the art (see, for example, 1994 Pierce Technical Handbook: cross-linking (See Appendix A in WO 04/50711). Crosslinkers include reactive ends to specific functional groups on proteins and antibodies which enable the covalent joining of the two or more molecules. Crosslinkers include homobifunctional crosslinkers and heterobifunctional crosslinkers.

[0072] A homobifunctional crosslinker has the same reactive chemistry at both ends, such as amine-to-amine or sulfhydryl-to-sulfhydryl. They are typically used to form intramolecular crosslinks or to prepare polymers from monomers. The reactive group an amine-to-amine end includes an NHS ester, whereas the sulfhydryl-to-sulfhydryl cross linker includes NHS-maleimide or NHS pyridyldithiol.

[0073] A heterobifunctional crosslinker has different chemistries at each end, working on different reactive groups such as amine-to-sulfhydryl, carboxyl-to-amine, or sulfhydryl-to-carboxyl. They are useful for preparing conjugates between two different biomolecules. An example amin-to sulfhydryl reactive group includes NHS-maleimide or NHS-pyridyldithiol. An example carboxyl-to amine reactive group include carbodiimide/NHS ester. A heterobifunctional crosslinker also includes a photoreactive crosslinker. A photoreactive crosslinker reacts with nucleophiles or form C-H insertion sites after exposure to UV light. Example photoreactive crosslinkers includes reactive groups such as NHS ester/aryl azide or NHS ester/diazirine.

[0074] In particular embodiments, photoreactive non-natural amino acids can be used as covalent crosslinkers. Photoreactive non-natural amino acids (referred to herein as photocrosslinkers) allow for efficient in vivo and in vitro photocrosslinking of polypeptides (e.g., antibodies). Examples of photocrosslinkers include aryl azides, azido-methyl-coumarins, benzophenones, anthraquinones, certain diazo compounds, diazirines, and psoralen derivatives. In particular embodiments, the photocrosslinker includes p-azidophenylalanine (Azi), p-benzoyl-L- phenylalanine (Bpa), 4'-(3-(trifluoromethyl)-3H-diazirine-3-yl)-l-phenylalanine (Tdf), DizPk, ((3-(3- methyl-3H-diazirine-3-yl)propamino) carbonyl-l-lysine (DizPK), 3'-azibutyl-N-carbamoyl-l-lysine (AbK), or p-2-fluoroacetyl-l-phenylalanine (Ff_act). In particular embodiments, the photocrosslinker includes Bpa. The antibodies with the photocrosslinker can be crosslinked at will by excitation of the photoreactive group using an ultraviolet light. Langmuir (2002) 18, 2463-2467 discloses covalent binding of proteins via photocrosslinkers.

[0075] PEGylated crosslinkers are also available which provide enhanced solubility, increased stability, reduced aggregation, and reduced immunogenicity to proteins.

[0076] The crosslinker can be placed at different locations within the single-domain antibody to enable covalent linkage of the single-domain antibody to its primary antibody. The location of the crosslinker should not reduce the ability of the single-domain antibody to bind its primary antibody but should be optimally placed to allow linkage. In particular embodiments, the crosslinker is within or near a CDR. In particular embodiments, the crosslinker is within or near CDR2. In particular embodiments, the crosslinker is at position G34, Q42, F55, E58, G60, or G105 of SEQ ID NO: 5. In particular embodiments, the crosslinker is at position F55 of SEQ ID NO: 5. The numbering of residue mutations will change based on the reference sequence. A person of skill in the art will be able to determine the residue location for mutation for different reference sequences.

[0077] (iv) Antibody Conjugates. Antibody conjugates include a single-domain antibody disclosed herein linked to another molecule (i.e., cargo). Examples of cargo that can be used in antibody conjugates include detectable labels such as fluorescent labels (e.g., fluorescent proteins or fluorophores), enzymatic labels, radioactive isotopes, metal isotopes, particles, barcodes, or other cargo helpful in imaging, detection, diagnosis, or treatment; and effector molecules such as drugs or toxins.

[0078] Fluorescent labels can be particularly useful in cell staining, identification, imaging, and isolation uses. Fluorescent labels can include fluorescent proteins or fluorophores.

[0079] Exemplary fluorescent proteins include blue fluorescent proteins {e.g. eBFP, eBFP2, Azurite, mKalamal , GFPuv, Sapphire, T-sapphire); cyan fluorescent proteins e.g. eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan, mTurquoise); green fluorescent proteins {e.g. GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green (mAzamigreen)), CopGFP, AceGFP, avGFP, ZsGreenl, Oregon Green™(Thermo Fisher Scientific)); luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato); red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRuby, mRFP1 , DsRed-Express, DsRed2, DsRed- Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611 , mRaspberry, mStrawberry, Jred, Texas Red™ (Thermo Fisher Scientific)); far red fluorescent proteins (e.g., mPlum and mNeptune); yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellowl); and tandem conjugates.

[0080] A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Example fluorophores include fluorescein isothiocyanate (FITC), hydroxycoumarin, Aminocoumarin, methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, 3- hydroxyisonicotinaldehyde, Lucifer yellow, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates, PE- Cy7 conjugates, Red 613, PerCP, TruRed, FluorX, BODIPY-FL, G-Dye100, G-Dye200, G- Dye300, G-Dye400, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine, lissamine Rhodamine B, Texas Red, Allophycocyanin (APC), or APC-Cy7. Further examples of fluorophores include CF dye (Biotium, San Francisco, CA), DRAQ and CyTRAK probes (BioStatus, Leicestershire, United Kingdom), BODIPY (Invitrogen, Waltham, MA), EverFluor (Setareh Biotech, Eugene, OR), Alexa Fluor (Invitrogen), Bella Fluor (Setareh Biotech), DyLight Fluor (Thermo Fisher Scientific, Waltham, MA), Atto and Tracy (Sigma Aldrich, St. Louis, MO), FluoProbes (Interchim, France), Abberior Dyes (Abberior, Germany), DY and MegaStokes Dyes (Dyomics, Germany), Sulfo Cy dyes (Cyandye, Hollywood, FL), HiLyte Fluor (AnaSpec, Fremont, CA), Seta, SeTau and Square Dyes (SETA BioMedicals, Urbana, IL), Quasar and Cal Fluor dyes (Biosearch Technologies, England), SureLight Dyes (APC, RPEPerCP, Phycobilisomes) (Columbia Biosciences, Frederick, MD), and Vio Dyes (Miltenyi Biotec, Gaithersburg, MD). In particular embodiments, the fluorophore includes a blue-emitting synthetic fluorophore, greenemitting synthetic fluorophore, yellow-emitting synthetic fluorophore, orange-emitting synthetic fluorophore, or red-emitting synthetic fluorophore.

[0081] Enzymatic labels can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes can include malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI- phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In particular embodiments, the enzymatic label includes horseradish peroxidase.

[0082] Examples of radioactive isotopes (also referred to as elemental isotopes or radioisotopes) that can be conjugated to single-domain antibodies of the present disclosure include iodine-131 , arsenic-72, arsenic-74, iodine-131 , indium-11 1 , yttrium-90, and lutetium-177, as well as alphaemitting radionuclides such as astatine-211 , actinium-225, bismuth-212 or bismuth-213. Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including Zevalin™ (DEC Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the disclosure.

[0083] Additional examples of useful radioisotopes include ²²⁵Ac, ²²⁸Ac, ¹¹¹Ag, ¹²⁴Am, ⁷⁴As, ²¹¹ At, ²⁰⁹At, ¹⁹⁴Au, ¹²⁸Ba, ⁷Be, ²⁰⁶Bi, ²⁴⁵Bk, ²⁴⁶Bk, ⁷⁶Br, ¹¹C, ¹⁴C, ⁴⁷Ca, ²⁵⁴Cf, ²⁴²Cm, ⁵¹Cr, ⁶⁷Cu, ¹⁵³Dy, ¹⁵⁷Dy, ¹⁵⁹Dy, ¹⁶⁵Dy, ¹⁶⁶Dy, ¹⁷¹Er, ²⁵⁰Es, ²⁵⁴Es, ¹⁴⁷Eu, ¹⁵⁷Eu, ⁵²Fe, ⁵⁹Fe, ²⁵¹ Fm, ²⁵²Fm, ²⁵³Fm, ⁶⁶Ga, ⁷²Ga, ¹⁴⁶Gd, ¹⁵³Gd, ⁶⁸Ge, ³H, ¹⁷⁰Hf, ¹⁷¹Hf, ¹⁹³Hg, ¹⁹³mHg, ¹⁶⁰mHo, ¹³⁰l, ¹³¹l, ¹³⁵l, ¹¹⁴mln, ¹⁸⁵lr, ⁴²K, ⁴³K, ⁷⁶Kr, ⁷⁹Kr, ⁸¹mKr, ¹³²La, ²⁶²Lr, ¹⁶⁹Lu, ¹⁷⁴ml_u, ¹⁷⁶mLu, ²⁵⁷Md, ²⁶⁰Md, ²⁸Mg, ⁵²Mn, ⁹⁰Mo, ²⁴Na, ⁹⁵Nb, ¹³⁸Nd, ⁵⁷Ni, ⁶⁶Ni, ²³⁴Np, ¹⁵O, ¹⁸²0s, ¹⁸⁹mOs, ¹⁹¹Os, ³²P, ²⁰¹ Pb, ¹⁰¹ Pd, ¹⁴³Pr, ¹⁹¹ Pt, ²⁴³Pu, ²²⁵Ra, ⁸¹ Rb, ¹⁸⁸Re, ¹⁰⁵Rh, ²¹¹ Rn, ¹⁰³Ru, ³⁵S, ⁴⁴Sc, ⁷²Se, ¹⁵³Sm, ¹²⁵Sn, ⁹¹Sr, ¹⁷³Ta, ¹⁵⁴Tb, ¹²⁷Te, ²²⁷Th, ²³⁴Th, ⁴⁵Ti, ¹⁶⁶Tm, ²³⁰U, ²³⁷U, ²⁴⁰U, ⁴⁸V, ¹⁷⁸W, ¹⁸¹W, ¹⁸⁸W, ¹²⁵Xe, ¹²⁷Xe, ¹³³Xe, ¹³³mXe, ¹³⁵Xe, ⁸⁵mY, ⁸⁶Y, ⁹⁰Y, ⁹³Y, ¹⁶⁹Yb, ¹⁷⁵Yb, ⁶⁵Zn, ⁷¹mZn, ⁸⁶Zr, ⁹⁵Zr, and/or ⁹⁷Zr. Radioisotopes can be used as a type of detectable label called a radiolabel or in radioimmunotherapy.

[0084] Examples of metal isotopes include isotopes of lanthanide, indium, yttrium, palladium, or bismuth. In particular embodiments, the metal isotope includes Y89, Pd102, Rh103, Pd104, Pd105, Pd106, Pd108, Pd1 10, Xe131 , Cs133, Ba138, Ce140, Pr141 , Nd142, Nd143, Nd144, Nd145, Nd146, Sm147, Nd148, Sm149, Nd150, Eu151 , Sm152, Eu153, Sm154, Gd155, Gd156, Gd158, Tb159, Gd160, Dy161 , Dy162, Dy163, Dy164, Ho165, Er166, Er167, Er168, Tm169, Er170, Yb171 , Yb172, Yb173, Yb174, Lu175, Yb176, BCKG190, Ir191 , Ir193, Pt195, Bi209, Ln113, Ln115, or La139.

[0085] Antibody-particle conjugates include an antibody linked to a particle. In particular embodiments, particles include quantum dots, microparticles, nanoparticles, nanoshells, nanobeads, microbeads, or nanodots. Quantum dot a type of fluorescence nanoparticles has been considered highly sensitive substitutes for organic dyes in existing diagnostic assays. They exhibit an extraordinary capability to detect molecule at ultra-low concentration which can be used for very early detection of disease progression. Particles can include, for example, latex beads, polystyrene beads, fluorescent beads, and/or colored beads, and can be made from organic matter and/or inorganic matter. They can be made of any suitable materials that allow for the conjugation of single-domain antibodies to their surface. Examples of suitable materials include: ceramics, glass, polymers, and magnetic materials. Quantum dots are typically made of materials including lead sulfide, lead selenide, cadmium selenide, cadmium sulfide, cadmium telluride, indium arsenide, and indium phosphide. Suitable polymers include polystyrene, poly-(methyl methacrylate), poly-(lactic acid), (poly-(lactic-co-glycolic acid)), polyesters, polyethers, polyolefins, polyalkylene oxides, polyamides, polyurethanes, polysaccharides, celluloses, polyisoprenes, methylstyrene, acrylic polymers, thoria sol, latex, nylon, Teflon cross- linked dextrans (e.g., Sepharose), chitosan, agarose, and cross-linked micelles. Additional examples include carbon graphited, titanium dioxide, and paramagnetic materials. See, e.g., "Microsphere Detection Guide" from Bangs Laboratories, Fishers Ind. In particular embodiments, microparticles can be made of one or more materials. In particular embodiments, microparticles are paramagnetic microparticles.

[0086] Barcodes can also be attached to single-domain antibodies to form antibody-barcode conjugates. The term “barcode” as used herein, refers to any unique nucleic acid sequence that may be used to identify the originating source of a nucleic acid fragment. Such barcodes may be sequences including for example, TTGAGCCT, AGTTGCTT, CCAGTTAG, ACCAACTG, GTATAACA or CAGGAGCC. In particular embodiments, barcodes are 3-100 nucleotides in length. Further details of barcodes that can be used with the systems and methods of the present disclosure are described in United States Patent Application No. 16/992,569 entitled “Systems and Methods for Using the Spatial Distribution of Haplotypes to Determine a Biological Condition,” filed August 13, 2020, and PCT publication 202020176788A1 entitled “Profiling of biological analytes with spatially barcoded oligonucleotide arrays”.

[0087] Other cargo useful for imaging and detection in an antibody conjugate includes chemiluminescent labels, spectral colorimetric labels, affinity tags, and contrast agents.

[0088] Chemiluminescent labels can include lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, or oxalate ester.

[0089] Spectral colorimetric labels can include colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.

[0090] Affinity tags can include, for example, His tag (HHHHHH (SEQ ID NO: 17)), Flag tag (DYKDDDD (SEQ ID NO: 18), Xpress tag (DLYDDDDK (SEQ ID NO: 19)), Avi tag (GLNDIFEAQKIEWHE (SEQ ID NO: 20)), Calmodulin binding peptide (CBP) tag (KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID NO: 21)), Polyglutamate tag (EEEEEE (SEQ ID NO: 22)), HA tag (YPYDVPDYA (SEQ ID NO: 23)), Myc tag (EQKLISEEDL (SEQ ID NO: 24)), Strep tag (WRHPQFGG (SEQ ID NO: 25)), STREP® tag II (WSHPQFEK (SEQ ID NO: 26); IBA Institut fur Bioanalytik, Germany; see, e.g., US 7,981 ,632), Softag 1 (SLAELLNAGLGGS (SEQ ID NO: 27)), Softag 3 (TQDPSRVG (SEQ ID NO: 28)), and V5 tag (GKPIPNPLLGLDST (SEQ ID NO: 29)).

[0091] Contrast agents for magnetic source imaging include paramagnetic or superparamagnetic ions, iron oxide particles, and water-soluble contrast agents. Paramagnetic and superparamagnetic ions can be selected from the group of metals including iron, copper, manganese, chromium, erbium, europium, dysprosium, holmium, and gadolinium.

[0092] In some examples, a single-domain antibody can be linked to an effector molecule such as a drug or toxin.

[0093] Antibody-drug conjugates allow for the targeted delivery of a drug moiety and, in particular embodiments intracellular accumulation therein, where systemic administration of unconjugated drugs may result in unacceptable levels of toxicity to normal cells (Polakis P. (2005) Current Opinion in Pharmacology 5:382-387).

[0094] In particular embodiments, antibody-drug conjugates refer to targeted molecules which combine properties of both antibodies and cytotoxic drugs (e.g., chemotherapeutic drugs) by targeting potent cytotoxic drugs to antigen-expressing cells (Teicher, B. A. (2009) Current Cancer Drug Targets 9:982-1004), thereby enhancing the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter, P. J. and Senter P. D. (2008) The Cancer Jour. 14(3): 154- 169; Chari, R. V. (2008) Acc. Chem. Res. 41:98-107). See also Kamath & Iyer (Pharm Res. 32(11): 3470-3479, 2015), which describes considerations for the development of antibody-drug conjugates.

[0095] The drug moiety (D) of an antibody-drug conjugate may include any compound, moiety or group that has a cytotoxic or cytostatic effect. Drug moieties may impart their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding or intercalation, and inhibition of RNA polymerase, protein synthesis, and/or topoisomerase. Exemplary drugs include actinomycin D, anthracycline, auristatin, calicheamicin, camptothecin, CC1065, colchicin, cytochalasin B, daunorubicin, 1 -dehydrotestosterone, dihydroxy anthracinedione, dolastatin, doxorubicin, duocarmycin, elinafide, emetine, ethidium bromide, etoposide, gramicidin D, glucocorticoids, lidocaine, maytansinoid (including monomethyl auristatin E [MMAE]; vedotin), mithramycin, mitomycin, mitoxantrone, nemorubicin, PNU-159682, procaine, propranolol, puromycin, pyrrolobenzodiazepine (PBD), taxane, taxol, tenoposide, tetracaine, trichothecene, vinblastine, vinca alkaloid, vincristine, and stereoisomers, isosteres, analogs, and derivatives thereof that have cytotoxic activity.

[0096] The drug may be obtained from essentially any source; it may be synthetic or a natural product isolated from a selected source, e.g., a plant, bacterial, insect, mammalian or fungal source. The drug may also be a synthetically modified natural product or an analogue of a natural product.

[0097] In particular embodiments, the antibody-drug conjugates include an antibody conjugated, i.e., covalently attached, to the drug moiety. In particular embodiments, the single-domain antibody is covalently attached to the drug moiety through a linker. A linker can include any chemical moiety that is capable of linking an antibody, antibody fragment (e.g., antigen binding fragments) or functional equivalent to another moiety, such as a drug moiety. Linkers can be susceptible to cleavage (cleavable linker), such as, acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active. Alternatively, linkers can be substantially resistant to cleavage (e.g., stable linker or noncleavable linker). In some aspects, the linker is a procharged linker, a hydrophilic linker, or a dicarboxylic acid-based linker. The antibody-drug conjugate selectively delivers an effective dose of a drug to cells (e.g., cancer cells) whereby greater selectivity, i.e. a lower efficacious dose, may be achieved while increasing the therapeutic index (“therapeutic window”).

[0098] To prepare antibody-drug conjugates, linker-cytotoxin conjugates can be made by conventional methods analogous to those described by Doronina et al. (Bioconjugate Chem. 17: 114-124, 2006). Antibody-drug conjugates with multiple (e.g., four) drugs per antibody can be prepared by partial reduction of the antibody with an excess of a reducing reagent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) at 37°C for 30 min, then the buffer can be exchanged by elution through SEPHADEX G-25 resin with 1 mM DTPA in Dulbecco’s phosphate-buffered saline (DPBS). The eluent can be diluted with further DPBS, and the thiol concentration of the antibody can be measured using 5,5'-dithiobis(2-nitrobenzoic acid) [Ellman's reagent]. An excess, for example 5-fold, of the linker-cytotoxin conjugate can be added at 4°C. for 1 hr, and the conjugation reaction can be quenched by addition of a substantial excess, for example 20-fold, of cysteine. The resulting ADC mixture can be purified on SEPHADEX G-25 equilibrated in PBS to remove unreacted linker-cytotoxin conjugate, desalted if desired, and purified by size-exclusion chromatography. The resulting ADC can then be sterile filtered, for example, through a 0.2 pm filter, and can be lyophilized if desired for storage. Methods used to produce immunotoxins can similarly be used to prepare antibody-drug conjugates.

[0099] Immunotoxins include a single-domain antibody disclosed herein conjugated to one or more toxins or cytotoxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof). A toxin can be any agent that is detrimental to cells. Frequently used plant toxins are divided into two classes: (1) holotoxins (or class II ribosome inactivating proteins), such as ricin, abrin, mistletoe lectin, and modeccin, and (2) hemitoxins (class I ribosome inactivating proteins), such as pokeweed antiviral protein (PAP), saporin, Bryodin 1, bouganin, and gelonin. Commonly used bacterial toxins include diphtheria toxin (DT) and Pseudomonas exotoxin (PE). Kreitman, Current Pharmaceutical Biotechnology 2:313-325 (2001). The toxin may be obtained from essentially any source and can be a synthetic or a natural product.

[0100] Immunotoxins with multiple (e.g., four) cytotoxins per binding domain can be prepared by partial reduction of the binding domain with an excess of a reducing reagent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) at 37°C for 30 min, then the buffer can be exchanged by elution through SEPHADEX G-25 resin with 1 mM DTPA (diethylene triamine penta-acetic acid) in Dulbecco’s phosphate-buffered saline (DPBS). The eluent can be diluted with further DPBS, and the thiol concentration of the binding domain can be measured using 5,5'- dithiobis(2-nitrobenzoic acid) [Ellman's reagent]. An excess, for example 5-fold, of the linker- cytotoxin conjugate can be added at 4°C. for 1 hr, and the conjugation reaction can be quenched by addition of a substantial excess, for example 20-fold, of cysteine. The resulting immunotoxin mixture can be purified on SEPHADEX G-25 equilibrated in PBS to remove unreacted linker- cytotoxin conjugate, desalted if desired, and purified by size-exclusion chromatography. The resulting immunotoxin can then be sterile filtered, for example, through a 0.2 pm filter, and can be lyophilized if desired for storage.

[0101] (v) Methods of Manufacture. Various expression vector and host systems can be utilized for the expression of single-domain antibodies described herein. Examples of such systems include microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing a nucleic acid sequence encoding the single-domain antibody described herein, yeast transformed with recombinant yeast expression vectors containing the aforementioned nucleic acid sequence, insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the aforementioned nucleic acid sequence, plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV), tobacco mosaic virus (TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the aforementioned nucleic acid sequence, or animal cell systems infected with recombinant virus expression vectors (e.g., adenovirus, vaccinia virus) including cell lines engineered to contain multiple copies of the aforementioned nucleic acid sequence, either stably amplified (e.g., CHO/dhfr, CHO/glutamine synthetase) or unstably amplified in double-minute chromosomes (e.g., murine cell lines). The host cell for expressing a single-domain antibody of the present disclosure can be any cell (e.g., a eukaryotic cell such as animal cell, plant cell or yeast; or a prokaryotic cell such as Escherichia coli or Bacillus subtilis).

[0102] With respect to vectors which may be used in the method of the present disclosure, when the host cell is E. coli, the vector can include ori that enables large scale amplification in E. coli (e.g., JM109, DH5a, HB101 and XL1-Blue) and selective markers for transformed E. coli cells (e.g., drug resistance genes capable of selecting transformants with drugs such as ampicillin, tetracycline, kanamycin or chloramphenicol). Specific examples of vectors include pET-21 b(+), M13 vector, plIC vector, pBR322, pBluescript and pCR-Script. Further, when subcloning and excision of cDNA are intended, pGEM-T, pDIRECT, pT7 and the like are also enumerated in addition to the above-listed vectors. When the vector is used for the purpose of producing the single-domain antibody or a conjugate thereof, expression vectors are particularly useful. When the single-domain antibody is to be expressed in E. coli, the expression vector can have the above-described feature to enable amplification in E. coli. Furthermore, when the host is E. coli such as JM109, DH5a, HB101 or XL1 -Blue, the expression vector can have a promoter which enables efficient expression in E. coli, e.g., lacZ promoter (Ward et al, Nature (1989) 341, 544- 546; FASEB J. (1992) 6, 2422-2427), araB promoter (Better et al., Science (1988) 240, 1041- 1043) or T7 promoter. Specific examples of such vectosr include, in addition to those listed above, pGEX-5X-1 (Pharmacia), QIAexpress system (Qiagen) pEGFP or pET (e.g., pET-21 b(+)).

[0103] The vector may include a signal sequence for polypeptide secretion. As to the signal sequence for polypeptide secretion, pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379) may be used when the antibody is to be produced in the periplasm of E. coli. Introduction of the vector into host cells may be performed by the calcium chloride method, electroporation, microinjection, etc.

[0104] When the host cell is not E. coli, the vector which may be used in the present disclosure include mammal-derived expression vectors such as pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p. 5322), pEF, pCDM8 and INPEP4 (Biogen-IDEC); insect cell-derived expression vectors such as Bac-to-BAC baculovairus expression system (GIBCO BRL) and pBacPAK8; plant-derived expression vectors such as pMH1 and pMN2; animal virus-derived expression vectors such as pHSV, pMV and pAdexLcw; retrovirus-derived expression vectors such as pZIpneo; yeast-derived expression vectors such as Pichia Expression Kit (Invitrogen), pNV11 and SP-Q01) and B. st/W/7/s-derived expression vectors such as pPL608 and pKTH50.

[0105] When expression of an antibody in an animal cell (such as CHO cell, COS cell or NIH3T3 cell) is intended, the vector can have a promoter necessary for intracellular expression of the antibody; e.g., SV40 promoter (Mulligan et al., Nature (1979) 277, 108), MMLV-LTR promoter, EF1a promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter (Niwa et al., Gene. (1991) 108, 193), mouse globin promoter (mBGP), etc. In particular embodiments, the vector has genes for selecting transformation into cells (e.g., drug resistance genes capable of selection with drugs such as neomycin or G418). Examples of vectors with such features include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13. It is known that mRNA with polyA is stable within cells. Thus, the vector preferably has a polyA signal necessary to add polyA to a gene of interest, e.g., mouse p globin polyA signal, bovine growth hormone polyA signal, SV40 polyA signal, etc.

[0106] The host cell may be expressing the antibody or a fragment thereof either in a transient expression system or in a stable expression system.

[0107] The “transient expression system” means a method in which circular plasmids are taken into cells by the calcium phosphate method, electroporation, lipofection, etc. for the purpose of gene expression. Since circular plasmids are inserted into chromosomes at low efficiency, the gene of interest often remains outside of chromosomes. Therefore, it is difficult to retain the expression of the gene of interest from circular plasmids for a long time.

[0108] The “stable expression system” means a method in which linear plasmids prepared by restriction enzyme treatment or the like are taken into cells by the calcium phosphate method, electroporation, lipofection, etc. for the purpose of gene expression. Since linear plasmids are inserted into chromosomes at higher efficiency than circular plasmids, the gene of interest is maintained on chromosomes at higher efficiency. Therefore, it is possible to retain the expression of this gene for a long time. Further, introduction of drug resistance genes into plasmids enables selection with drugs. Thus, it becomes possible to efficiently select those cells maintaining the gene of interest on their chromosomes.

[0109] Further, for amplification of the copy number of the gene of interest in host cell systems, expression vectors may include the following as selective markers: aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene, etc.

[0110] A vector is useful in retaining a nucleic acids (e.g., DNA) encoding a single-domain antibody of the present disclosure or a conjugate thereof or for expressing the single-domain antibody or conjugate thereof in a host cell.

[0111] The present disclosure also provides a host cell including the vector including nucleic acids encoding the single-domain antibody or conjugates thereof. The host cell may be used as a production system for preparing or expressing the single-domain antibody or conjugates thereof. Examples of in vitro production systems include those using eukaryotic cells and those using prokaryotic cells. [0112] By transforming host cells with a gene of interest and culturing the resultant transformants in vitro, a polypeptide encoded by the gene of interest may be obtained. The culture may be performed according to known methods. For example, as a culture broth for animal cells, DM EM, MEM, RPMI1640 or IMDM may be used. During this culture, a serum supplement such as fetal calf serum (FCS) may be used jointly. Alternatively, the culture may be serum-free culture. In particular embodiments, the pH during the culture is 6-8. Usually, the culture is performed at a temperature of 30-40° C for 15-200 hours. The culture medium may be exchanged, aerated or stirred if necessary.

[0113] As regards in vivo systems for producing single-domain antibodies or conjugates thereof, production systems using an animal or a plant may be given. A gene of interest is introduced into the animal or plant. Then, the animal or plant is allowed to produce a polypeptide of interest in its body, followed by recovery of the polypeptide. The term “host” used in the present disclosure includes such animals or plants.

[0114] After expression in cells, the single-domain antibody can be purified using standard purification methods, such as affinity column, Protein A or G columns, column with target molecules for affinity purification, ion exchange chromatography, hydrophobic interaction columns, size exclusion chromatography, HPLC, FPLC, etc. Other methods of fractionation or protein purification include separation based on physical characteristics such as charge (i.e. , ion exchange chromatography, electrophoresis, isoelectric focusing), polarity (i.e., adsorption chromatography, reverse phase chromatography), size (i.e., dialysis, gel electrophoresis, gel filtration chromatography, ultracentrifugation), specificity (i.e., affinity chromatography, immunopurification), and solubility (i.e., salt precipitation, detergent solubilization). Diafiltration can be performed to remove free reducing agents in a sample.

[0115] Disulfide bond formation and folding of the single-domain antibody and its cargo can occur during expression or after expression or both.

[0116] A host cell can be adapted to express one or more single-domain antibodies or singledomain antibody conjugates described herein. In some cases, the single-domain antibody and the cargo are manufactured separately before they are fused or conjugated to form an antibody conjugate. In other cases, antibody conjugates are manufactured or expressed as a fusion protein. The host cells can be prokaryotic, eukaryotic, or insect cells. In some cases, host cells are capable of modulating the expression of the inserted sequences, or modifying and processing the gene or protein product in the specific fashion desired. For example, expression from certain promoters can be elevated in the presence of certain inducers (e.g., zinc and cadmium ions for metallothionine promoters). In some cases, modifications (e.g., phosphorylation) and processing (e.g., cleavage) of single-domain antibodies or conjugates can be important for the function of the antibody conjugate. Host cells can have characteristic and specific mechanisms for the post- translational processing and modification. In some cases, the host cells that express the singledomain antibody secrete minimal amounts of proteolytic enzymes.

[0117] In particular embodiments, a method of manufacturing a single-domain antibody of the present disclosure includes constructing a plasmid including a sequence encoding the singledomain antibody; transforming a host cell with the plasmid; transforming the host cell with a suppressor plasmid; growing cells; and harvesting the single-domain antibody. In particular embodiments, the host cell is E. coli. In particular embodiments, the vector includes pET-21 b(+). In particular embodiments, the suppressor plasmid includes pEVOL suppressor plasmid which encodes an orthogonal translation system components pair MjTyrRS/tRNATyrCUA for pBpa- specific incorporation.

[0118] (vi) Compositions. Any of the single-domain antibodies described herein, in any exemplary format (e.g., antibody conjugate) can be formulated alone or in combination into compositions for administration to compositions containing the primary antibody or to subjects. Additionally, nucleic acids (e.g., cDNA) encoding the antibodies can also be formulated into compositions for administration. Antibodies and/or nucleic acids encoding antibodies are collectively referred to herein as “active ingredients”.

[0119] Salts and/or pro-drugs of the active ingredients can also be used.

[0120] An acceptable salt includes any salt that retains the activity of the active ingredient and is acceptable for use in the methods described herein.

[0121] Suitable acceptable acid addition salts can be prepared from an inorganic acid or an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids can be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids.

[0122] Suitable acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'- dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N- methylglucamine, lysine, arginine and procaine.

[0123] In particular embodiments, the compositions include active ingredients of at least 0.1% w/v or w/w of the composition; at least 1% w/v or w/w of composition; at least 10% w/v or w/w of composition; at least 20% w/v or w/w of composition; at least 30% w/v or w/w of composition; at least 40% w/v or w/w of composition; at least 50% w/v or w/w of composition; at least 60% w/v or w/w of composition; at least 70% w/v or w/w of composition; at least 80% w/v or w/w of composition; at least 90% w/v or w/w of composition; at least 95% w/v or w/w of composition; or at least 99% w/v or w/w of composition.

[0124] Exemplary generally used acceptable carriers include any and all absorption delaying agents, antioxidants, binders, buffering agents, bulking agents or fillers, chelating agents, coatings, disintegration agents, dispersion media, gels, isotonic agents, lubricants, preservatives, salts, solvents or co-solvents, stabilizers, surfactants, and/or delivery vehicles.

[0125] Exemplary antioxidants include ascorbic acid, methionine, and vitamin E.

[0126] Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and/or trimethylamine salts.

[0127] An exemplary chelating agent is EDTA (ethylene-diamine-tetra-acetic acid).

[0128] Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0129] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0130] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the antibodies or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L- leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, o-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weight based on therapeutic weight.

[0131] The compositions disclosed herein can be formulated for administration by, for example, pipetting. The composition can also be formulated for administration to a subject by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. The compositions disclosed herein can further be formulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral, sublingual, and/or subcutaneous administration. [0132] Compositions can be formulated as aqueous solutions, such as in buffers including Hanks' solution, Ringer's solution, or physiological saline. The aqueous solutions can include formulatory agents such as suspending, stabilizing, and/or dispersing agents. Alternatively, the composition can be in lyophilized and/or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0133] In particular embodiments, compositions include the single-domain antibodies disclosed herein and a buffer. In particular embodiments, the buffer includes phosphate-buffered saline or Tirs/HCl-buffered saline. In particular embodiments, the phosphate-buffered saline includes 0.01M phosphate-buffered saline. In particular embodiments, the buffer further includes bovine serum albumin and/or a preservative.

[0134] Any composition or formulation disclosed herein can advantageously include any other pharmaceutically acceptable carriers which include those that do not produce significantly adverse, allergic, or other untoward reactions that outweigh the benefit of administration. Exemplary pharmaceutically acceptable carriers are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, compositions and formulations can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by U.S. FDA Office of Biological Standards and/or other relevant foreign regulatory agencies.

[0135] (vii) Methods of Use. As indicated, there are numerous uses for the single-domain antibodies disclosed herein. Single-domain antibodies described herein can be used for in vivo, ex vivo, or in vitro labeling of primary antibody. In particular embodiments, detection is for research, diagnostic, and/or prognostic uses. In particular embodiments, methods of detection include administering an effective amount of a single-domain antibody disclosed herein. The single-domain antibody can be associated with or linked to cargo (e.g., a detectable label), and the composition can be suitable for labeling primary antibodies.

[0136] For detection applications, the single-domain antibodies of the presently disclosed subject matter can be labeled with a detectable label. The detectable label can be any label that is capable of producing, either directly or indirectly, a detectable signal. For example, detectable labels are described elsewhere herein and include fluorescent labels, enzymatic labels, radioactive isotopes, metal isotopes, particles, or barcodes.

[0137] Detection and imaging of the antibody is tunable, such that imaging can be performed in under 1, 2, 4, 6, 12, or 18, 24, 36, or 48 hours, or any amount below, above, or between this amount. It has been demonstrated that PEGs/larger molecules increase serum half-life by 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%, or 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times compared to a smaller molecule. This allows for imaging at different time points.

[0138] In particular embodiments, methods of labeling a primary antibody include administering an effective amount of a composition including a single-domain antibody to a composition containing the primary antibody, wherein the single-domain antibody includes a crosslinker and cargo; and activating the crosslinker. In particular embodiments, the primary antibody is a rabbit IgG. In particular embodiments, the single-domain antibody binds the Fc region of a rabbit IgG. In particular embodiments, the crosslinker includes a photocrosslinker. In particular embodiments, the photocrosslinker includes a Bpa. In particular embodiments, the cargo includes a detectable label. In particular embodiments, the detectable label includes a fluorescent labels, enzymatic labels, radioactive isotopes, metal isotopes, particles, or barcodes. In particular embodiments, activating the crosslinker includes exposing the crosslinker to ultraviolet light.

[0139] In particular embodiments, methods of labeling a primary rabbit antibody include administering an effective amount of a composition including an anti-rabbit single-domain antibody to a composition containing the primary rabbit antibody, wherein the anti-rabbit singledomain antibody includes a Bpa photocrosslinker and a detectable label; and activating the crosslinker. In particular embodiments, the detectable label includes a fluorescent labels, enzymatic labels, radioactive isotopes, metal isotopes, particles, or barcodes. In particular embodiments, activating the crosslinker includes exposing the crosslinker to ultraviolet light.

[0140] In particular embodiments, the antibody conjugate disclosed herein can be associated with a primary antibody or it can associate with the primary antibody during the methods Following administration of the antibody conjugate to a sample or subject, and after a time sufficient for binding, the biodistribution of the antibody conjugate can be visualized. The term “time sufficient for binding” refers to a temporal duration that permits binding of the antibody conjugate to a target molecule.

[0141] Ex vivo Imaging and Detection. In particular embodiments, a composition as disclosed herein can be used for ex vivo imaging and/or detection. In particular embodiments, ex vivo methods include (a) contacting a sample with a primary antibody that binds a protein of interest; (b) contacting the sample with a single-domain antibody that binds the primary antibody, wherein the single-domain antibody includes a detectable label; and (c) detecting the detectable label. In particular embodiments, the single-domain antibody further includes a crosslinker. In particular embodiments, the method further includes linking the secondary antibody to the primary antibody via the crosslinker.

[0142] Several methods of ex vivo imaging and detection are known in the art including immunofluorescence, cyclic immunofluorescence, immunohistochemistry, electromagnetic imaging, mass spectrometry, immunoblotting (e.g., Western blotting), and protein profiling.

[0143] Immunofluorescence (IF). Non-invasive imaging methods can also include detection of a fluorescent label (e.g., fluorophore). Examples of fluorescent labels are described elsewhere herein. Fluorescence imaging can be performed ex vivo or in vivo. For in vivo detection of a fluorescent label, an image is created using emission and absorbance spectra that are appropriate for the particular label used. The image can be visualized, for example, by diffuse optical spectroscopy. Additional methods and imaging systems are described in U.S. Pat. Nos. 5,865,754; 6,083,486; and 6,246,901 , among other places.

[0144] One type of fluorescence imaging is called immunofluorescence (IF). IF is an imaging technique using specific antibodies tagged with fluorophores. The specific antibodies bind to cellular antigens (e.g., proteins) and can be visualized under, for example, a fluorescent microscope. In particular embodiments, a single-domain antibody is conjugated to a fluorophore, an affinity tag, or a quantum dot for imaging and/or detection by immunofluorescence.

[0145] Cyclic IF is similar to IF in that specific antibodies conjugated to a detectable label are used to image cellular structures. Cyclic IF, however, involves repeatedly staining and imaging samples with the detectable label. Cyclic IF is a multiplexed immunofluorescence imaging techniques that can still use a conventional epifluorescence microscope. It uses simple reagents and existing antibodies to construct images with up to 30 channels by sequential 4- to 6-channel imaging followed by fluorophore inactivation. Cyclic IF can be performed by staining fixed cells with antibody-detectable label conjugates and imaging in multiple colors, inactivating detectable labels (e.g., fluorophores) using a mild base in the presence of hydrogen peroxide and light, and then performing another round of staining and imaging. Other methods to perform cyclic IF involve indirect immunofluorescence and/or enabling chemical inactivation of genetically encoded fluorescent proteins (Lin et al., Curr Protoc Chem Biol, 2016, 8(4):251-264). In particular embodiments, a single-domain antibody is conjugated to an oligo barcode or a fluorophore for imaging and/or detection by cyclic IF.

[0146] Immunohistochemistry. Disclosed herein are methods of using immunohistochemistry (IHC) utilizing an antibody and the single-domain antibody disclosed herein. IHC detects target molecules through antigen-antibody complexes in a pathological specimen using enzyme-linked antigens or antibodies. The presence of the target molecule can then be detected via an enzyme immunoassay.

[0147] A multitude of benefits are realized with IHC versus traditional immunofluorescence. For example, unlike immunofluorescence, IHC can be used with commonly used formalin-fixed paraffin-embedded tissue specimens. Pathological specimens, including histological tissue sections and/or other biological preparations such as tissue culture cells, are commonly used in diagnostic pathology and can be easily screened via IHC. Further, IHC staining is permanent and preserves cell morphology. A comparison of the cell morphology and antigen proliferation on two different slides can be useful in monitoring the progression of a disease.

[0148] Once an antibody detectable label conjugate has been attached, either directly or indirectly, to the specimen, a substrate, specific for the enzyme, is added to the specimen. When the substrate is added, the enzyme label converts the substrate causing a color change that can be seen with light microscopy. The presence of a color change indicates the presence of the target molecule and allows an observer to determine, assess, and diagnose the disease level and severity.

[0149] Electromagnetic imaging. Electromagnetic imaging refers to the use of electromagnetic fields (from static to microwave and higher frequencies) for providing and imaging an object. The near-infrared (NIR) region of the electromagnetic spectrum is seen as ideal for fluorescence imaging. Because quantum dots are a type of nanoparticle that emit a fluorescence in the NIR region, they are useful in electromagnetic imaging. Electromagnetic (EM) methods cover a broad range of the electromagnetic spectrum and is useful for detecting anomalies or defects in both conducting and dielectric materials by generating two-dimensional (2D) or three-dimensional (3D) image data based on electromagnetic principles. In general, EM imaging uses excitation transducers which couple the EM energy into the test objects, while the receiving sensors measure the response of energy-material interaction. EM imaging depends on different energy types and/or levels and various EM sensors and transducers can be used for a broad range of applications. After image acquisition, the data is passed through filters and algorithms to reconstruct the image. In particular embodiments, a single-domain antibody is conjugated to a quantum dot for imaging and/or detection by electromagnetic imaging.

[0150] Mass spectrometry. In particular embodiments, levels, amounts, or ratios of biomarkers of can be measured by mass spectrometry using the antibodies disclosed herein. Mass spectrometry (MS) refers to an analytical technique to identify compounds by their mass. MS technology generally includes (1) ionizing the compounds to form charged compounds; and (2) detecting the molecular weight of the charged compound and calculating a mass-to-charge ratio (m/z). The compound may be ionized and detected by any suitable means. A “mass spectrometer” generally includes an ionizer and an ion detector. See, e.g., US 6,204,500; 6,107,623; 6,268,144; 6,124,137; Wright et al., Prostate Cancer and Prostatic Diseases. 1999, 2:264-76; and Merchant and Weinberger, Electrophoresis. 2000, 21 :1164-67.

[0151] Samples may be processed or purified to obtain preparations that are particularly suitable for analysis by mass spectrometry. Such purification will usually include chromatography, such as liquid chromatography, and may also often involve an additional purification procedure that is performed prior to chromatography. Various procedures may be used for this purpose depending on the type of sample or the type of chromatography. Examples include filtration, extraction, precipitation, centrifugation, delipidization, dilution, combinations thereof and the like.

[0152] Mass cytometry is a variation on the platform for flow cytometry which uses mass spectrometry techniques to detect metal-conjugated antibodies. Unlike flow cytometry, which uses antibodies linked to fluorescent labels, mass cytometry uses antibodies linked to metals. In particular embodiments, a single-domain antibody is conjugated to a radioactive isotope, metal isotope, or particle for imaging and/or detection using mass cytometry.

[0153] Western blot. Western blotting is a technique used in cell and molecular biology that allows for the identification of specific proteins from mixtures of proteins obtained from cells or tissues. In general, a Western blot is performed by separating the proteins by size, transferring to a solid support, and identifying target proteins using a primary and/or secondary antibody labeled with a detectable label. More specifically, a protein mixture is separated by molecular weight using gel electrophoresis such that the different proteins are separated because different proteins have different molecular weight. Once separated, the separated proteins are transferred to a membrane which can then be incubated in a solution containing an antibody specific for the protein of interest. Any unbound antibody is washed away leaving the bound antibody. The bound antibodies can then be detected. In particular embodiments, the bound antibodies are detected by adding a secondary antibody (e.g., single-domain antibody) conjugated to a detectable label and the detectable label is detected. In particular embodiments, a single-domain antibody is conjugated to an enzyme label for Western blot analysis. In particular embodiments, the enzyme label is horseradish peroxidase.

[0154] Protein profiling or protein expression profiling is a method to identify proteins expressed within a tissue. The expression within the tissue is usually for a specified condition at a particular time and is compared to a reference sample. One method of protein profiling uses two- dimensional gel electrophoresis (2DGE) which uses in-gel proteolysis of selected proteincontaining spots and subsequent mass spectrometry. Protein profiling can measure protein expression spatially and/or temporally as a result of set conditions. In particular embodiments, specific proteins can be identified using antibodies. In particular embodiments, antibodies can be labeled with a barcode such as an oligo barcode.

[0155] The single-domain antibodies disclosed herein can also be used for in vivo imaging or delivery of cargo in vivo. In vivo imaging can be useful for diagnosis (e.g., in pet animals) or for research (e.g., in a laboratory animal). Delivery of cargo in vivo can be useful in treating particular diseases. In particular embodiments, in vivo methods include (a) administering a primary antibody that binds a protein of interest; (b) administering a single-domain antibody that binds the primary antibody, wherein the single-domain antibody includes a cargo. In particular embodiments, the method further includes detecting a detectable label if the cargo includes a detectable label. In particular embodiments, the single-domain antibody further includes a crosslinker. In particular embodiments, the method further includes linking the secondary antibody to the primary antibody via the crosslinker. In particular embodiments, the cargo includes an effector molecule.

[0156] Certain examples include treating subjects. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and/or therapeutic treatments. An "effective amount" is the amount of a composition or formulation necessary to result in a desired physiological change in the subject. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically-significant effect in an animal model or in vitro assay relevant to the assessment of a condition’s development, progression, and/or resolution.

[0157] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of a condition or displays only early signs or symptoms of a condition such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the condition further. Thus, a prophylactic treatment functions as a preventative treatment against a condition. In particular embodiments, prophylactic treatments reduce, delay, or prevent the worsening of a condition.

[0158] A "therapeutic treatment" includes a treatment administered to a subject who displays symptoms or signs of a condition and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the condition. The therapeutic treatment can reduce, control, or eliminate the presence or activity of the condition and/or reduce control or eliminate side effects of the condition.

[0159] Function as an effective amount, prophylactic treatment, or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.

[0160] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and/or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of condition, stage of condition, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.

[0161] Useful doses can range from 0.1 to 5 pg/kg or from 0.5 to 1 pg /kg. In other examples, a dose can include 1 pg /kg, 15 pg /kg, 30 pg /kg, 50 pg/kg, 55 pg/kg, 70 pg/kg, 90 pg/kg, 150 pg/kg, 350 pg/kg, 500 pg/kg, 750 pg/kg, 1000 pg/kg, 0.1 to 5 mg/kg or from 0.5 to 1 mg/kg. In other examples, a dose can include 1 mg/kg, 10 mg/kg, 30 mg/kg, 50 mg/kg, 70 mg/kg, 100 mg/kg, 300 mg/kg, 500 mg/kg, 700 mg/kg, 1000 mg/kg or more.

[0162] Exemplary doses of cell-based compositions can include 10⁴ to 10⁹ cells/kg body weight, or 10³ to 10¹¹ cells/kg body weight. Therapeutically effective amounts to administer can include greater than 10² cells, greater than 10³ cells, greater than 10⁴ cells, greater than 10⁵ cells, greater than 10⁶ cells, greater than 10⁷ cells, greater than 10⁸ cells, greater than 10⁹ cells, greater than 10¹⁰ cells, or greater than 10¹¹ cells.

[0163] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly). In particular embodiments, the treatment protocol may be dictated by a clinical trial protocol or an FDA- approved treatment protocol.

[0164] The compositions described herein can be administered by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. Routes of administration can include intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intralesional, intramuscular, oral, subcutaneous, and/or sublingual administration. Formulations are generally be administered by injection.

[0165] In particular embodiments, a single-domain antibody of the presently disclosed subject matter includes a label that can be detected in vivo. The term “in vivo” as used herein to describe imaging or detection methods, refers to generally non-invasive methods such as fluorescence, scintigraphic methods, magnetic resonance imaging, autoradiographic detection, or radioimmunoguided systems, each described briefly herein below. [0166] Scintigraphic Imaging. Scintigraphic imaging methods include SPECT (Single Photon Emission Computed Tomography). PET (Positron Emission Tomography), gamma camera imaging, and rectilinear scanning. A gamma camera and a rectilinear scanner each represent instruments that detect radioactivity in a single plane. Most SPECT systems are based on the use of one or more gamma cameras that are rotated about the subject of analysis, and thus integrate radioactivity in more than one dimension. PET systems include an array of detectors in a ring that also detect radioactivity in multiple dimensions.

[0167] Imaging instruments suitable for practicing the detection and/or imaging methods of the presently disclosed subject matter, and instruction for using the same, are readily available from commercial sources. For example, a SPECT scanner can be used with a CT scanner, with coregistration of images. As in PET/CT, this allows location of tumors or tissues which may be seen on SPECT scintigraphy but are difficult to precisely locate with regard to other anatomical structures. Both PET and SPECT systems are offered by ADAC of Milpitas, Calif., United States of America, and Siemens of Hoffman Estates, III., United States of America. Related devices for scintigraphic imaging can also be used, such as a radio-imaging device that includes a plurality of sensors with collimating structures having a common source focus.

[0168] When scintigraphic imaging is employed, the detectable label can include a radiolabel as described elsewhere herein. When the labeling moiety is a radionuclide, stabilizers to prevent or minimize radiolytic damage, such as ascorbic acid, gentisic acid, or other appropriate antioxidants, can be added to the composition including the labeled targeting molecule.

[0169] Magnetic Resonance Imaging (MRI). Magnetic resonance image-based techniques create images based on the relative relaxation rates of water protons in unique chemical environments. As used herein, the term “magnetic resonance imaging” refers to magnetic source techniques including conventional magnetic resonance imaging, magnetization transfer imaging (MTI), proton magnetic resonance spectroscopy (MRS), diffusion-weighted imaging (DWI) and functional MR imaging.

[0170] Those skilled in the art of diagnostic labeling recognize that metal ions can be bound by chelating moieties, which in turn can be conjugated to a therapeutic agent in accordance with the methods of the presently disclosed subject matter. For example, gadolinium ions are chelated by diethylenetriaminepentaacetic acid (DTPA). Lanthanide ions are chelated by tetraazacyclododocane compounds. See U.S. Pat. Nos. 5,738,837 and 5,707,605. Alternatively, a contrast agent can be carried in a liposome.

[0171] Images derived used a magnetic source can be acquired using, for example, a superconducting quantum interference device magnetometer (SQUID, available with instruction from Quantum Design of San Diego, Calif., United States of America; see also U.S. Pat. No. 5,738,837).

[0172] Autoradiographic Detection. In the case of a radioisotope (also referred to herein as radiolabel) detection can be accomplished by conventional autoradiography or by using a phosphorimager as is known to one of skill in the art. In particular embodiments, an autoradiographic method employs photostimulable luminescence imaging plates (Fuji Medical Systems of Stamford, Conn., United States of America). Briefly, photostimulable luminescence is the quantity of light emitted from irradiated phosphorous plates following stimulation with a laser during scanning. The luminescent response of the plates is linearly proportional to the activity.

[0173] Radioimmunoguided System (RIGS). Another application of the antibodies disclosed herein is in the radioimmunoguided surgery (RIGS) system. This technique involves the intravenous administration of a radiolabeled antibody prior to surgery. After allowing for tumor uptake and blood clearance of radioactivity, the patient is taken to the operating room where surgical exploration is affected with the aid of a hand-held gamma activity probe, e.g., Neoprobe®1000 (Neoprobe Corporation, Dublin, Ohio). This helps the surgeon identify the tumor metastases and improve the complications of excision. The RIGS system is advantageous because it allows for the detection of tumors not otherwise detectable by visual inspection and/or palpation. See, O'Dwyer et al, Arch. Surg., 121 :1 391-1394 (1986). This technique is described in detail in Hinkle et al, Antibody, Immunoconjugates and Radiopharmaceuticals, 4:(3)339-358 (1991). This technique is useful for cancers including colon cancer, breast cancer, pancreatic cancer, and ovarian cancer.

[0174] The term "diagnosis", as used herein, refers to evaluation of the presence or properties of pathological states or lack thereof. With respect to objects of the present disclosure, in particular embodiments, the diagnosis can be to determine the presence of any protein of interest in which a primary rabbit antibody binds, such as proteins associated with cancer.

[0175] (viii) Kits. Also provided herein are kits including at least one single-domain antibody disclosed herein. Kits may be formed with components to practice, for example, the methods described herein. In particular embodiments, the kit includes a single-domain antibody, an antibody conjugate, or cDNA encoding the single-domain antibody as described herein. The kit may include material(s), which may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and/or other material useful in sample processing, washing, or conducting any other step of the method described herein. The kit may include an ultraviolet light. [0176] In particular embodiments, a kit includes an antibody conjugate and any other materials needed for imaging, diagnosis, or treatment. In particular embodiments, a kit includes a single- domain antibody conjugated to a fluorescent label, enzymatic label, radioactive isotope, metal isotope, particle, barcode, drug, or toxin. In particular embodiments, the single-domain antibody binds the Fc region of an rabbit antibody. In particular embodiments, the single-domain antibody includes a photocrosslinker.

[0177] The kit according to the present disclosure may also include instructions for carrying out the method. Instructions included in the kit of the present disclosure may be affixed to packaging material or may be included as a package insert. While instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site which provides instructions.

[0178] The Exemplary Embodiments and Example below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure. [0179] (ix) Exemplary Embodiments.

1. A single-domain antibody that binds the Fc region of a rabbit antibody, the single-domain antibody including a set of complementarity determining regions (CDRs) including: i) a CDR1 having the sequence as set forth in GRSLDXAT (SEQ ID NO: 11), a CDR2 having the sequence as set forth in EFVAGIFWDEIGTEY (SEQ ID NO: 9), and a CDR3 having the sequence as set forth in LVFGGEY (SEQ ID NO: 10); ii) a CDR1 having the sequence as set forth in GRSLDGAT (SEQ ID NO: 8), a CDR2 having the sequence as set forth in EFVAGIXWDEIGTEY (SEQ ID NO: 12), and a CDR3 having the sequence as set forth in LVFGGEY (SEQ ID NO: 10); iii) A CDR1 having the sequence as set forth in GRSLDGAT (SEQ ID NO: 8), a CDR2 having the sequence as set forth in EFVAGIFWDXIGTEY (SEQ ID NO: 13), and a CDR3 having the sequence as set forth in LVFGGEY (SEQ ID NO: 10); iv) a CDR1 having the sequence as set forth in GRSLDGAT (SEQ ID NO: 8), a CDR2 having the sequence as set forth in EFVAGIFWDEIXTEY (SEQ ID NO: 14), and a CDR3 having the sequence as set forth in LVFGGEY (SEQ ID NO: 10); v) a CDR1 having the sequence as set forth in GRSLDGAT (SEQ ID NO: 8), a CDR2 having the sequence as set forth in EFVAGIFWDEIGTEY (SEQ ID NO: 9), and a CDR3 having the sequence as set forth in LVFXGEY (SEQ ID NO: 15); or vi) a CDR1 having the sequence as set forth in GRSLDGAT (SEQ ID NO: 8), a CDR2 having the sequence as set forth in EFVAGIFWDEIGTEY (SEQ ID NO: 9), and a CDR3 having the sequence as set forth in LVFGGEY (SEQ ID NO: 10), wherein the single-domain antibody of vi) includes the sequence AGSQVQLVESGGGLVQAGDSLRLSCVASGRSLDGATMRWYRXAPGKEREFV AGIFWDEIGTEYADTAKGRFTISRDNAKNTIYLQMTNLRSEDTAMYYCNGLVFG GEYWGQGTQVTVSSGG (SEQ ID NO: 16) wherein X is a mutation. A single-domain antibody that binds the Fc region of a rabbit antibody, the single-domain antibody including the sequence: AGSQVQLVESGGGLVQAGDSLRLSCVASGRSLDGATMRWYRQAPGKEREFVAGIFW DEIGTEYADTAKGRFTISRDNAKNTIYLQMTNLRSEDTAMYYCNGLVFGGEYWGQGTQ VTVSSGG (SEQ ID NO: 5), having a mutation at G34, Q42, F55, E58, G60, or G105. The single-domain antibody of embodiments 1 or 2, wherein the mutation includes a substitution of a residue with a crosslinker. The single-domain antibody of embodiment 3, wherein the crosslinker includes a photocrosslinker. The single-domain antibody of embodiment 4, wherein the photocrosslinker includes p- azidophenylalanine (Azi), p-benzoyl-L-phenylalanine (Bpa), 4-(3-(trifluoromethyl)-3H- diazirine-3-yl)-l-phenylalanine (Tdf), DizPk, ((3-(3-methyl-3H-diazirine-3-yl)propamino) carbonyl-l-lysine (DizPK), 3^Z -azibutyl-N-carbamoyl-l-lysine (AbK), or p-2-fluoroacetyl-l- phenylalanine (Ffact). The single-domain antibody of embodiments 4 or 5, wherein the photocrosslinker includes Bpa. The single-domain antibody of any of embodiments 1-6, wherein the single-domain antibody includes a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 5. The single-domain antibody of any of embodiments 1-7, including a methionine at the N- terminal end of the single-domain antibody. The single-domain antibody of any of embodiments 1-8, including an affinity tag. The single-domain antibody of embodiment 9, wherein the affinity tag is located at the N- terminal end and/or C-terminal end of the single-domain antibody. The single-domain antibody of any of embodiments 1-10, further including a linker. The single-domain antibody of embodiment 11 , wherein the linker includes a Gly-Ser linker. The single-domain antibody of embodiment 12, wherein the Gly-Ser linker includes a sequence as set forth in SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43.. A conjugate including the single-domain antibody of any of embodiments 1-13 linked to cargo. The conjugate of embodiment 14, wherein the cargo includes a detectable label and/or an effector molecule. The conjugate of embodiment 15, wherein the detectable label includes a fluorescent label, enzymatic label, radioactive isotope, metal isotope, particle, barcode, chemiluminescent label, spectral colorimetric label, affinity tag, and/or contrast agent. The conjugate of embodiment 16, wherein the fluorescent label includes a fluorescent protein and/or a fluorophore. The conjugate of embodiment 17, wherein the fluorophore includes a blue-emitting synthetic fluorophore, a green-emitting synthetic fluorophore, a yellow-emitting synthetic fluorophore, an orange-emitting synthetic fluorophore, or a red-emitting synthetic fluorophore. The conjugate of any of embodiments 16-18, wherein the enzymatic label includes malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, betagalactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase, and/or acetylcholinesterase The conjugate of any of embodiments 16-19, wherein the enzymatic label includes horseradish peroxidase. The conjugate of any of embodiments 16-20, wherein the radioactive isotope includes ²²⁵Ac, ²²⁸Ac, ¹¹¹Ag, ¹²⁴Am, ⁷⁴As, ²¹¹At, ²⁰⁹At, ¹⁹⁴Au, ¹²⁸Ba, ⁷Be, ²⁰⁶Bi, ²⁴⁵Bk, ²⁴⁶Bk, ⁷⁶Br, ¹¹C, ¹⁴C, ⁴⁷Ca, ²⁵⁴Cf, ²⁴²Cm, ⁵¹Cr, ⁶⁷Cu, ¹⁵³Dy, ¹⁵⁷Dy, ¹⁵⁹Dy, ¹⁶⁵Dy, ¹⁶⁶Dy, ¹⁷¹ Er, ²⁵⁰Es, ²⁵⁴Es, ¹⁴⁷Eu, ¹⁵⁷Eu, ⁵²Fe, ⁵⁹Fe, ²⁵¹ Fm, ²⁵²Fm, ²⁵³Fm, ⁶⁶Ga, ⁷²Ga, ¹⁴⁶Gd, ¹⁵³Gd, ⁶⁸Ge, ³H, ¹⁷⁰Hf, ¹⁷¹Hf, ¹⁹³Hg, ¹⁹³mHg, ¹⁶⁰mHo, ¹³⁰l, ¹³¹l, ¹³⁵l, ¹¹⁴mln, ¹⁸⁵lr, ⁴²K, ⁴³K, ⁷⁶Kr, ⁷⁹Kr, ⁸¹mKr, ¹³²La, ²⁶²Lr, ¹⁶⁹Lu, ¹⁷⁴ml_u, ¹⁷⁶ml_u, ²⁵⁷Md, ²⁶⁰Md, ²⁸Mg, ⁵²Mn, ⁹⁰Mo, ²⁴Na, ⁹⁵Nb, ¹³⁸Nd, ⁵⁷Ni, ⁶⁶Ni, ²³⁴Np, ¹⁵O, ¹⁸²0s, ¹⁸⁹mOs, ¹⁹¹Os, ³²P, ²⁰¹Pb, ¹⁰¹Pd, ¹⁴³Pr, ¹⁹¹ Pt, ²⁴³Pu, ²²⁵Ra, ⁸¹Rb, ¹⁸⁸Re, ¹⁰⁵Rh, ²¹¹Rn, ¹⁰³Ru, ³⁵S, ⁴⁴Sc, ⁷²Se, ¹⁵³Sm, ¹²⁵Sn, ⁹¹Sr, ¹⁷³Ta, ¹⁵⁴Tb, ¹²⁷Te, ²²⁷Th, ²³⁴Th, ⁴⁵Ti, ¹⁶⁶Tm, ²³⁰U, ²³⁷U, ²⁴⁰U, ⁴⁸V, ¹⁷⁸W, ¹⁸¹W, ¹⁸⁸W, ¹²⁵Xe, ¹²⁷Xe, ¹³³Xe, ¹³³mXe, ¹³⁵Xe, ⁸⁵mY, ⁸⁶Y, ⁹⁰Y, ⁹³Y, ¹⁶⁹Yb, ¹⁷⁵Yb, ⁶⁵Zn, ⁷¹mZn, ⁸⁶Zr, ⁹⁵Zr, and/or ⁹⁷Zr. The conjugate of any of embodiments 16-21 , wherein the metal isotope includes isotopes of lanthanide, indium, yttrium, palladium, and/or bismuth. The conjugate of any of embodiments 16-22, wherein the metal isotope includes Y89, Pd102, Rh103, Pd104, Pd105, Pd106, Pd108, Pd1 10, Xe131 , Cs133, Ba138, Ce140, Pr141 , Nd142, Nd143, Nd144, Nd145, Nd146, Sm147, Nd148, Sm149, Nd150, Eu151 , Sm152, Eu153, Sm154, Gd155, Gd156, Gd158, Tb159, Gd160, Dy161 , Dy162, Dy163, Dy164, Ho165, Er166, Er167, Er168, Tm169, Er170, Yb171 , Yb172, Yb173, Yb174, Lu175, Yb176, BCKG190, Ir191 , Ir193, Pt195, Bi209, Ln113, Ln115, and/or La139. The conjugate of any of embodiments 16-23, wherein the particle includes a quantum dot, a microparticle, a nanoparticle, a nanoshell, a nanobead, a microbead, and/or a nanodot. The conjugate of any of embodiments 16-24, wherein the particle includes a quantum dot. The conjugate of any of embodiments 14-25, wherein the cargo includes 2 or more detectable labels. The conjugate of any of embodiments 14-26, wherein the cargo includes 3 detectable labels. The conjugate of any of embodiments 15-27, wherein the effector molecule includes a drug and/or a toxin. A composition including the single-domain antibody of any of embodiments 1-13 or the conjugate of any of embodiments 14-28 and an acceptable carrier. A kit including the single-domain antibody of any of embodiments 1-13 or the conjugate of any of embodiments 14-28. The kit of embodiment 30, further including an ultraviolet light. A method of labeling a primary antibody including administering the composition of embodiment 29 to a composition including the primary antibody, wherein the composition of embodiment 29 includes a single-domain antibody including a photocrosslinker and cargo; and activating the crosslinker. The method of embodiment 32, wherein the administering includes pipetting. The method of embodiments 32 or 33, wherein the primary antibody includes a rabbit antibody. The method of any of embodiments 32-34, wherein the activating the crosslinker includes exposing the crosslinker to ultraviolet light. 36. The method of embodiment 35, wherein the exposing includes at least 1 min, at least 5 min, at least 10 min, at least 15 min, at least 20 min, at least 25 min, at least 30 min, at least 45 min, at least 1 hour, at least 2 hours, or at least 3 hours.

37. The method of embodiments 35 or 36, wherein the exposing includes at least 5 min.

38. The method of embodiments 35 or 36, wherein the exposing includes at least 1 min.

39. The method of any of embodiments 35-38, wherein the ultraviolet light includes a wavelength of 325 nm to 425 nm.

40. The method of any of embodiments 35-39, wherein the ultraviolet light includes a wavelength of 405 nm.

41. The method of any of embodiments 35-39, wherein the ultraviolet light includes a wavelength of 355 nm.

42. The method of any of embodiments 32-41 , wherein the cargo includes a detectable label and the method further includes detecting the detectable label.

43. The method of any of embodiments 32-42, wherein the cargo includes a therapeutic molecule.

44. A cDNA encoding the single-domain antibody of any of embodiments 1-13.

45. The cDNA of embodiment 44 ligated into a bacterial expression vector.

46. The cDNA of embodiment 45, wherein the bacterial expression vector includes pET- 21b(+).

47. A method of preparing the single-domain antibody of any of embodiments 1-13 including transforming a cell with the cDNA of any of embodiments 44-46.

48. The method of embodiment 47, wherein the cell is an E. coli.

49. The method of embodiment 48, wherein the cell is a BL21(DE3) competent E. coli.

50. The method of any of embodiments 47-49, further including transforming the cell with a suppressor plasmid.

51. The method of embodiment 50, wherein the suppressor plasmid encodes an orthogonal translation system for Bpa-specific incorporation.

52. The method of any of embodiments 47-51 , further including culturing the cell.

53. The method of any of embodiments 47-52, further including harvesting the single-domain antibody.

54. The method of embodiment 53, wherein the harvesting includes lysing the cell and collecting the single-domain antibody.

[0180] (x) Experimental Example.

[0181] Example 1. Developing an efficient and reproducible site-specific rabbit antibody covalent labeling using secondary single-domain antibody (herein referred to as nanobody).

[0182] Abstract. Antibodies and derived components serve a wide variety of uses across multiple areas of scientific research, clinic diagnosis, and other healthcare fields often through functionalization. In order to label and functionalize antibodies, direct or secondary conjugations are typically employed. To ensure unperturbed Fab functions secondary antibodies or Fc specific binders have been developed, although specificity has long plagued even targeted approaches. Therefore, we introduce an efficient, sustainable, reproducible site-specific rabbit antibody covalent labeling was introduced through by combining the anti-rabbit IgG secondary nanobodies and a genetically incorporated photocrosslinker. These infer the stability, high-affinity of species specific and easy to produce IgG binders with the permanence of controlled activation for proximity-driven intermolecular covalent conjugation, with a promise for efficient multiplexity.

[0183] Introduction. Immunoglobulins, also known as antibodies, are affinity molecules specifically targeting antigen in cells and tissue. Antibodies are a fundamental tool and have been widely used in molecular and cellular biology, medical research, and clinical diagnosis, enabling the selective, sensitive detection and quantification of proteins and other molecules in techniques, such as immunohistochemistry (IHC), immunofluorescence (IF), and fluorescence-activated cell sorting (FACS) which rely on the antibodies representing standard routine image assays to visualize the abundance of target antigens in tissues or cells under light microscopy. Meanwhile, these applications require the attachment of chemical probes or specific tags to the antibody.

[0184] Traditionally, non-specific labeling of primary antibodies through amine- or thio-reactive chemistries was widely performed, however more targeted methods have been developed recently to limit off-target impacts to the complementarity-determining regions (CDRs) of antibodies. Evolving research and commercial products have sought to overcome this antibodybased multiplexing limitations by leveraging these affinity reagents which specifically target antibodies. Those site-specific methods utilize either enzymatic reaction-directed chemical reaction that attach secondary functional group in the Fc regions of antibodies, or engineered antibody targeting proteins that could contain affinity to the Fc or Fab regions of antibodies. Although all these methods have been utilized instrumentally in accelerating multiplexed -omics analyses by enabling a large array of biological targets to be labeled and subsequently quantified in parallel, these have come with substantive limitations and side-effects to affinity, conjugation number, buffer incompatibilities, storage incompatibilities, insufficient conjugation, and even aggregations. For example, as a commonly used antibody conjugation method, SiteClick® depends on site-selective modification of the mostly conserved N-linked glycan on the Fc regions of antibodies, however, 15-20% of antibodies contain a second N-linked glycan in their CDRs, resulting in a potential hamper on antigen recognitions. Another newer method named oYo-Link® relies on a relatively small binding fragment derived from Protein G targeting the Fc domain, however, unintended allosteric effects in Fc affinity has been observed through modifications of larger probe moieties (e.g., DNA barcode), further reducing already low affinity and conjugation efficiency. Moreover, to ensure maximum conjugation efficiency, this strategy requires 2 hours UV irradiation which results in incompatibility with fluorescent probes and could also cause photodegradation on antibodies.

[0185] Single-domain antibodies, also known as nanobodies, are the smallest natural antigen binding variable domains of camelid heavy chain antibodies with the size around 15 kDa (3x4 nm). Because of their small size with high modularity, nanobodies have attracted much attention as powerful alternatives for conventional antibodies in antigen targeting and therapeutical application. More recently, Pleiner et al. engineered a comprehensive toolbox of nanobodies with high specificity and affinity against all mouse antibody subclasses and rabbit antibody. These specifically engineered secondary nanobodies were envisioned with high-affinity binding would be particularly suitable to enable proximity-enhanced reaction, making it possible to have a covalent connection.

[0186] In this example, the design and development of site-specific incorporation of a noncanonical amino acid benzoyl-phenylalanine (Bpa) was reported, which contains a photoreactive benzophenone moiety, into the anti-rabbit secondary nanobody. It was hypothesized that the tight binding between nanobodies and antibodies provides in close enough proximity for this genetically encoded photoactivated Bpa generating a free-radical linked to nearby antibodies. To identify the site enabling the photocrosslinking of nanobodies to antibodies, 26 nanobody variants were designed and expressed, each having an amino acid substituted by Bpa. Built on these, several variants were identified having ability to be covalently attached to antibodies, especially one site showing nearly 100% photocrosslinking efficiency in less than 5 minutes. This work is highly valuable for addressing those antibody labeling challenges through current available approaches, and the great modularity of nanobody allows flexible and multiple downstream functionalization and readout for a variety of research and clinical applications.

[0187] Results and discussion. In this example, the computational and experimental validation of a novel set of IgG binders were presented which incorporate photo-crosslinkers for high-efficiency and site-specific labeling of Rabbit primary antibodies.

[0188] Computational modeling. The first step in the rational design of site-specific antibody Fc labeling was to model the interactions of purported anti-rabbit nanobody CDRs with the IgG-Fc domain. As a basis for initial model refinement, flexible docking simulations were performed which revealed 2 potential binding modes. These included an expected CDR-pointed interaction [ref] and an unexpected side orientation an additional 45 degrees rotated within the plane of the Fc domain and nearly 90 degree rotation in the long axis of the NB (FIG. 8). These 2 binding modes were later confirmed through Cryo-EM imaging and single molecule reconstructions (FIG. 20). In order to screen for potential conjugation sites based on proximity, additional simulations were performed to score highest-interacting residues within the 3 CDRs corresponding to each of the models (FIG. 9). Top 15 highest interacting residues sites regardless of model preference would then be used as the baseline for proof of concept site-specific functionalization. Notably, residues in the middle of CDR2 and CDR3 showed close proximity via both models.

[0189] Photo-crosslinking expression and validation. To assess the experimental viability of two models above, unnatural amino acid (UAA) integration was utilized of the photocrosslinker p- Benzoyl-L-phenylalanine (Bpa) through E.coli recombinant expression. In a brief, the site-specific insertion of Bpa was accomplished in vivo using a previously engineered orthogonal amino-acyl tRNA synthetase/tRNA (aaRS/tRNA) pair to integrate Bpa into proteins (FIG. 10A). Utilizing this strategy, 15 different nanobodies incorporated with site-defined Bpa were obtained and purified (FIG. 11A). To test for photocrosslinking activity, these purified Bpa-incorporated nanobodies were incubated with commercial rabbit primary antibodies for 30 minutes, and then exposed to UV light at 355 nm for 30 minutes. Photo-crosslinking performance was determined by coomassie G-250 stain showing degree of size-shift corresponding to either single or double NB cross-linking by a native gel (FIGs. 10B and 11 B). Simulated intermediate proximity residues like G35, Q43, and F56 (positions G34, Q42, and F55 of SEQ ID NO: 5) showed clear evidence of photocrosslinking conjugations, while surprisingly some high-proximity residues including I60 (I59 of SEQ ID NO: 5) and S32 (S31 of SEQ ID NO: 5) showed little to no conjugation had occurred. Notably, the success of F56 (F55 of SEQ ID NO: 5) may be due in part to its relative proximity within both models, although experimental validation was critical to discern this.

[0190] In order to identify potential sites for additional high-efficiency Bpa conjugation, the experimental validation was expanded to residues nearby or within the CDR regions which were initially not screened. An additional 11 sites were selected, representing adjacent residues to computationally modeled high to intermediate-proximity sites as well as an expanded set of residues before CDR2 which ran parallel to the antibody Fc in Model 2 (FIG. 9). Notably residues including G61 (G60 of SEQ ID NO: 5) and G106 (G105 of SEQ ID NO: 5) could also partially react with the rabbit Fc (FIGs. 11 B and 12), yet these still could not outperform F56 (F55 of SEQ ID NO: 5) in terms of complete conjugation efficiency within 20 minutes. [0191] Photo-crosslinking efficiency & specificity. Next, the cross-linking reaction speed and positioning from F56 (F55 of SEQ ID NO: 5) was investigated. Already within screened Bpa incorporation sites, an initial time of 20-30 minutes was used to validate photocrosslinking capabilities, however for F56 (F55 of SEQ ID NO: 5) it had already fully reacted within that time. To identify both the conjugation efficiency as well as the Fc specificity, a UV illumination time series of 20, 10, 5, 2.5, and 1.25 minutes was run on a denaturing gel (FIG. 13). At 0 minute control, both heavy and light chain domains could be seen at 55 and 25 kDa size bands respectively, as well as 5x molar excess of 15 kDa Bpa-NB. Within 5 minutes, a size shift from the heavy chain band at 55 kDa to 70 kDa was nearly completed, indicating near complete NB conjugation onto both sides of the Rabbit Fc domain. Critically, the Rabbit IgG light chain was completely unaffected during photo crosslinking, indicating highly site-specific conjugation. To further optimize this conjugation speed, additional timed conjugations were performed with increasing input UV intensity from multiple sources. Notably, the already fast reaction time was further increased to under 1 minute through use of a 355 nm laser excitation. (FIG. 14). To further assess UV illumination power on conjugation efficiency, increasing laser power was used within 1 minute exposures, this secondary validation showed that 100mW laser power was required to fully-photoscrosslink within 1 minute.

[0192] To explore other photoreactive functional analogs with potentially efficiency, non- canonical amino acid DiZPK was additional tested. DiZPK is an analog of lysine which contains an intrinsically smaller diazirine reactive group than the benzophenone in BPA, which may more readily react with a wider range of amino acids. DiZPK was incorporated at position F56 (F55 of SEQ ID NO: 5) and tested its crosslinking efficiency versus BPA (FIG. 15). Notably, although it demonstrated partial reactivity, it was still relatively inefficient when compared to BPA, likely relating to the increased length of their side chains.

[0193] Workflow and applications. Through the computational design and screening, several sites were identified on the secondary nanobody for an efficient, sustainable, reproducible site-specific antibody covalent labeling through a genetically incorporated photocrosslinker. Here a simple workflow was proposed to exhibit how to utilize the Bpa-NBs for site-specific covalent labeling of Rabbit IgGs (FIG. 16). Starting with primary Rabbit IgGs of choice, a short 5-minute incubation is enough for the Bpa-NBs to initially bind specifically to the IgG Fc domain. Next, a short 5 minute UV illumination is sufficient to photo-crosslink all of the Bpa-NBs onto the Rabbit IgGs. I ntriguingly , due to the highly efficient and site-specific nature of this NB conjugation, it is feasible to preincubate and conjugate multiple Rabbit IgGs against different targets within separate vials. Once fully reacted, these could be highly amenable to improved multiplexity and reduced cross-labeling within multi-omics related fields.

[0194] As a proof of concept of the types of applications for the Bpa-NB, nanobodies conjugated to HRP, DNA, and even fluorophores were tested. Unlike current commercial platforms, like Oyo- Link®, which can require 2 hours conjugation time for photo-crosslinking (FIG. 17), the short reaction time of Bpa-NB allows for direct fluorophore conjugations to easily survive UV illumination. To demonstrate the utility of fast and site-specific labeling fluorophores onto Rabbit IgGs, two nanobodies were utilized which were pre-conjugated to either 1 or 3 AF555 fluorophores. Through cysteine integrations opposite the CDR regions simple and cost-effective maleimide chemistry was used for fluorophore attachment (FIG. 18A). Through imaging unilluminated and photocrosslinked products by both fluorescence and Coomassie staining on a native gel, the size shift increase of NB-conjugations as well as fluorophore functionality postphotocrosslinking were clearly seen. Critically, the site-specific nature of the Bpa-NB binding ensured that even antibodies stored in 55 kDa BSA did not interfere with conjugation. These same types of conjugations could be performed in different batches with different targets and fluorophores, enabling multiplexity irrespective of identical species through pre-conjugations.

[0195] Utilizing these Bpa-NBs pre-conjugated to AF555 fluorophore, cellular labeling of microtubule and mitochondrial proteins were also demonstrated (FIG. 18B). For each target, conventional AF555 labeled secondary antibody as a positive control was also compared. Following the same fixation, permeabilization and blocking prior to antibody labeling, representative images for both targets clearly show their respective well-resolved cellular features when magnified. Notably, these Bpa-NB-AF555 conjugated primary antibodies could label within a single incubation, further improving the throughput of this technology overall.

[0196] Methods. Plasmid construction. The anti-rabbit IgG Fc nanobody TP897 coding region was codon optimized and synthesized at Genscript (GenScript). Synthesized coding sequences were cloned through CloneAmpTM HiFi PCR Premix (TaKaRa Bio, 639298). The resulted fragment was ligated into the linearized bacterial expression vector pET-21 b(+) by fusion enzyme ClonExpress Ultra One Step Cloning Kit (Vazymebiotech, C113-01). The site-directed mutagenesis for generating amber codon was performed on the constructed pET-21 b(+)-TP897 expression vector, with a two-step PCR method using CloneAmpTM HiFi PCR Premix and designed primer pairs. A list of primer sequences used in this example are listed in FIG. 19. The resulted fragments were also fused by ClonExpress Ultra One Step Cloning Kit (Vazymebiotech, C113-01). Chemically NEB® 10-beta competent E. coli cells (NEB, C3019H) were used for plasmid amplification. The plasmids were then purified with QIAprep Spin Miniprep Kit (Qiagen, 27016) following the manufacturer’s instructions. The plasmids were confirmed by DNA sequencing (Genewiz).

[0197] Protein expression and purification. The constructed expression vector containing antirabbit IgG Fc nanobody TP897 variants was transformed into BL21(DE3) competent E. coli cells (NEB, C2527H) together with the pEVOL suppressor plasmid which encodes an orthogonal translation system components pair MjTyrRS/tRNA^TyrcuA for pBpa-specific incorporation (PMID: 12154230). Cells were grown overnight at 37°C on LB agar plates with antibiotic selection 100 ug/ml carbenicillin (Sigmaaldrich, C3416-5G) and 50 ug/ml chloramphenicol (Bio Basic, CB0118-250). A single colony of transformed cells was further inoculated into LB liquid at 37°C until OD₆OO reached 0.6, and then the expression of TP897 variants and MjTyrRS/tRNA^TyrcuA were induced by adding 0.2% (w/v) L(+)-Arabinose (Fisher Scientific, AC104981000), 1 mM IPTG (Fisher Scientific, I2481C50), and 1mM 4-Benzoyl-L-phenylalanine (Fisher Scientific, AAH5208303). After the induction, the cultures were grown at 30°C for another 6 hrs, followed by the cell harvesting using a centrifuge at 10000 x g and 4°C for 20 min. Cell pellets were stored at -20°C until the purification.

[0198] The harvested cell pellets were resuspended in the lysis buffer (50 mM Na2HPC>4), 300mM NaCI, 10mM imidazole, pH 8.0). The resuspended cells were lysed by the sonication following the manufacturer’s instructions. The cellular lysate was centrifuged at 12,000 x g and 4°C for 1 hr. The supernatant was transferred into a Pierce™ centrifuge columns (Fisher Scientific, P189897) containing one volume HisPur™ Ni-NTA resin (Fisher Scientific, 88221) previously equilibrated with lysis buffer. After run through the column, the resin was washed 20 resin volumes of the wash buffer (50 mM Na2HPO4), 300mM NaCI, 20mM imidazole, pH 8.0), followed by the elution of the bound protein with 2 mL of 50 mM Na2HPC>4, 300mM NaCI, 300mM imidazole. To remove imidazole, the collected fractions were applied to a 5 mL HiTrap HP desalting prepacked column (Cytivalifesciences, 17140801) in an AKTA pure FPLC system with the running buffer DPBS (ThermoFisher Scientific, 14190144) at a flow rate of 5 mL/min. The purified proteins were stored at -80°C for further studies.

[0199] Photo-crosslinking, analysis. Purified anti-rabbit nanobody variant was pre-incubated with rabbit primary antibodies at the molar ratio of 1 :10 in DPBS buffer. The mixture was placed on a shaking rocker at RT for 30 min. After incubation, the mixture was irradiated with 365 nm UV light either using either a handheld lamp (UV-BG-40A) at RT or, being placed on an ice bath and irradiated in a commercial nail UV chamber for up to 2hrs. 4X non-reducing Pierce™ LDS sample buffer (ThermoFisher Scientific, 84788) was into the incubated samples. For non-reducing gel, the mixed samples were directly loaded into a Bolt 4 to 12% Bis-Tris SDS-PAGE gel. For reducing gel, the mixed samples were added 0.1M DTT (ThermoFisher Scientific, 20290), and then heated at 94°C for 10 min. After electrophoretic separation, the protein bands were developed by the simplyblue™ safestain (ThermoFisher Scientific, LC6065) following manufacturer’s instructions.

[0200] The images of gels were captured using a photo scanner (Epson, Perfection V19). To assess the efficiency of photocrosslinking, the images were then analyzed using Imaged software. Specifically, the lanes of heavy chain, and crosslinked heavy chain-nanobody were selected for intensity profiles using the rectangle tool. The crosslink efficiency was defined by a percentage of the crosslinked heavy chain-nanobody divided by the total heavy chain and nanobody loaded. The conjugation curve was generated using Graphpad Prism (9.5.0)

[0201] (xi) Closing Paragraphs. The nucleic acid and amino acid sequences provided herein are shown using letter abbreviations for nucleotide bases and amino acid residues, as defined in 37 C.F.R. §1.831-1.835 and set forth in WIPO Standard ST.26 (implemented on July 1, 2022). Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included in embodiments where it would be appropriate.

[0202] Variants of the sequences disclosed and referenced herein are also included. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR™ (Madison, Wisconsin) software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.

[0203] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally divided into conservative substitution families as follows: Group 1 : Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gin), Asp, and Glu; Group 4: Gin and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Vai) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gin, Cys, Ser, and Thr; Group 8 (large aromatic residues): Phenylalanine (Phe), Tryptophan (Trp), and Tyr; Group 9 (nonpolar): Proline (Pro), Ala, Vai, Leu, lie, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Vai, Leu, and lie; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0204] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: lie (+4.5); Vai (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glutamate (-3.5); Gin (-3.5); aspartate (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0205] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.

[0206] As detailed in US 4,554,101 , the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); Ser (+0.3); Asn (+0.2); Gin (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Vai (-1.5); Leu (-1.8); lie (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0207] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. As indicated elsewhere, variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and/or mutations that do not affect the function of an encoded product to a statistically-significant degree. [0208] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.

[0209] “% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, H I- 20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.. Within the context of this disclosure it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. As used herein "default values" will mean any set of values or parameters, which originally load with the software when first initialized.

[0210] Variants also include nucleic acid molecules that hybridize under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence. Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at 50 °C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37°C in a solution including 6XSSPE (20XSSPE=3M NaCI; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 pg/ml salmon sperm blocking DNA; followed by washes at 50 °C with 1XSSPE, 0.1 % SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5XSSC). Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

[0211] "Specifically binds" refers to an association of a binding domain (of, for example, a singledomain antibody ) to its cognate binding molecule with an affinity or K_a (/.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10⁵ M’¹, while not significantly associating with any other molecules or components in a relevant environment sample. Binding domains may be classified as "high affinity" or "low affinity". In particular embodiments, "high affinity" binding domains refer to those binding domains with a K_a of at least 10⁷ M^-1 , at least 10⁸ M^-1 , at least 10⁹ M^-1, at least 10¹⁰ M’¹, at least 10¹¹ M^-1, at least 10¹² M’¹, or at least 10¹³ M’¹. In particular embodiments, "low affinity" binding domains refer to those binding domains with a Ka of up to 10⁷ M’¹, up to 10⁶ M’¹, up to 10⁵ M’¹. Alternatively, affinity may be defined as an equilibrium dissociation constant (K_d) of a particular binding interaction with units of M (e.g., 10'⁵ M to 10'¹³ M). In certain embodiments, a binding domain may have "enhanced affinity," which refers to a selected or engineered binding domains with stronger binding to a cognate binding molecule than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a K_a (equilibrium association constant) for the cognate binding molecule that is higher than the reference binding domain or due to a K_d (dissociation constant) for the cognate binding molecule that is less than that of the reference binding domain, or due to an off- rate (K_Off) for the cognate binding molecule that is less than that of the reference binding domain. A variety of assays are known for detecting binding domains that specifically bind a particular cognate binding molecule as well as determining binding affinities, such as Western blot, ELISA, and BIACORE® analysis (see also, e.g., Scatchard, et al., 1949, Ann. N. Y. Acad. Sci. 57:660; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).

[0212] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2nd Edition (1989); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (1987); the series Methods IN Enzymology (Academic Press, Inc.); M. MacPherson, et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991); MacPherson et al., eds. PCR 2: Practical Approach, (1995); Harlow and Lane, eds. Antibodies, A Laboratory Manual, (1988); and R. I. Freshney, ed. Animal Cell Culture (1987).

[0213] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant decrease in covalently binding to the Fc region of rabbit IgG, as described herein.

[0214] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0215] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0216] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0217] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0218] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0219] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.

[0220] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0221] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and/or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0222] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

CLAIMS What is claimed is:

1. A single-domain antibody having a sequence having at least 95% sequence identity to SEQ ID NO: 6.

2. The single-domain antibody of claim 1 comprising a three-member set of complementarity determining regions (CDRs) comprising: i) a CDR1 having the sequence as set forth in GRSLDXAT (SEQ ID NO: 11), a CDR2 having the sequence as set forth in EFVAGIFWDEIGTEY (SEQ ID NO: 9), and a CDR3 having the sequence as set forth in LVFGGEY (SEQ ID NO: 10); ii) a CDR1 having the sequence as set forth in GRSLDGAT (SEQ ID NO: 8), a CDR2 having the sequence as set forth in EFVAGIXWDEIGTEY (SEQ ID NO: 12), and a CDR3 having the sequence as set forth in LVFGGEY (SEQ ID NO: 10); iii) A CDR1 having the sequence as set forth in GRSLDGAT (SEQ ID NO: 8), a CDR2 having the sequence as set forth in EFVAGIFWDXIGTEY (SEQ ID NO: 13), and a CDR3 having the sequence as set forth in LVFGGEY (SEQ ID NO: 10); iv) a CDR1 having the sequence as set forth in GRSLDGAT (SEQ ID NO: 8), a CDR2 having the sequence as set forth in EFVAGIFWDEIXTEY (SEQ ID NO: 14), and a CDR3 having the sequence as set forth in LVFGGEY (SEQ ID NO: 10); v) a CDR1 having the sequence as set forth in GRSLDGAT (SEQ ID NO: 8), a CDR2 having the sequence as set forth in EFVAGIFWDEIGTEY (SEQ ID NO: 9), and a CDR3 having the sequence as set forth in LVFXGEY (SEQ ID NO: 15); or vi) a CDR1 having the sequence as set forth in GRSLDGAT (SEQ ID NO: 8), a CDR2 having the sequence as set forth in EFVAGIFWDEIGTEY (SEQ ID NO: 9), and a CDR3 having the sequence as set forth in LVFGGEY (SEQ ID NO: 10), wherein the single-domain antibody of vi) comprises the sequence AGSQVQLVESGGGLVQAGDSLRLSCVASGRSLDGATMRWYRXAPGKEREFV AGIFWDEIGTEYADTAKGRFTISRDNAKNTIYLQMTNLRSEDTAMYYCNGLVFG GEYWGQGTQVTVSSGG (SEQ ID NO: 16) wherein X is a mutation.

3. The single-domain antibody of claim 1 comprising the sequence:

AGSQVQLVESGGGLVQAGDSLRLSCVASGRSLDGATMRWYRQAPGKEREFVAGIFW DEIGTEYADTAKGRFTISRDNAKNTIYLQMTNLRSEDTAMYYCNGLVFGGEYWGQGTQ VTVSSGG (SEQ ID NO: 5).

4. The single-domain antibody of claim 3 having a mutation at G34, Q42, F55, E58, G60, or

5. The single-domain antibody of claim 1 , wherein an amino acid residue of the singledomain antibody is replaced with a crosslinker.

6. The single-domain antibody of claim 5, wherein the crosslinker comprises a photocrosslinker.

7. The single-domain antibody of claim 6, wherein the photocrosslinker comprises p- azidophenylalanine (Azi), p-benzoyl-L-phenylalanine (Bpa), 4-(3-(trifluoromethyl)-3H- diazirine-3-yl)-l-phenylalanine (Tdf), DizPk, ((3-(3-methyl-3H-diazirine-3-yl)propamino) carbonyl-l-lysine (DizPK), 3^Z -azibutyl-N-carbamoyl-l-lysine (AbK), or p-2-fluoroacetyl-l- phenylalanine (Ffact).

8. The single-domain antibody of claim 6, wherein the photocrosslinker comprises Bpa.

9. The single-domain antibody of claim 1 , wherein the single-domain antibody comprises a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 5.

10. The single-domain antibody of claim 1 , comprising a methionine at the N-terminal end of the single-domain antibody.

11. The single-domain antibody of claim 1, further comprising an affinity tag.

12. The single-domain antibody of claim 11 , wherein the affinity tag is located at the N-terminal end or C-terminal end of the single-domain antibody.

13. The single-domain antibody of claim 1, further comprising a linker.

14. The single-domain antibody of claim 13, wherein the linker comprises a Gly-Ser linker.

15. The single-domain antibody of claim 14, wherein the Gly-Ser linker comprises a sequence as set forth in SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43..

16. A conjugate comprising the single-domain antibody of claim 1 linked to cargo.

17. The conjugate of claim 16, wherein the cargo comprises a detectable label or an effector molecule.

18. The conjugate of claim 17, wherein the detectable label comprises a fluorescent label, enzymatic label, radioactive isotope, metal isotope, particle, barcode, chemiluminescent label, spectral colorimetric label, affinity tag, or contrast agent.

19. The conjugate of claim 18, wherein the fluorescent label comprises a fluorescent protein or a fluorophore.

20. The conjugate of claim 19, wherein the fluorophore comprises a blue-emitting synthetic fluorophore, a green-emitting synthetic fluorophore, a yellow-emitting synthetic fluorophore, an orange-emitting synthetic fluorophore, or a red-emitting synthetic fluorophore.

21. The conjugate of claim 18, wherein the enzymatic label comprises malate dehydrogenase, staphylococcal nuclease, delta- V-steroid isomerase, yeast alcohol dehydrogenase, alphaglycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase, or acetylcholinesterase

22. The conjugate of claim 18, wherein the enzymatic label comprises horseradish peroxidase.

23. The conjugate of claim 18, wherein the radioactive isotope comprises ²²⁵Ac, ²²⁸Ac, ¹¹¹Ag, 1²⁴Am, ⁷⁴As, ²¹¹At, ²⁰⁹At, ¹⁹⁴Au, ¹²⁸Ba, ⁷Be, ²⁰⁶Bi, ²⁴⁵Bk, ²⁴⁶Bk, ⁷⁶Br, ¹¹C, ¹⁴C, ⁴⁷Ca, ²⁵⁴Cf, 2⁴²Cm, ⁵¹Cr, ⁶⁷Cu, ¹⁵³Dy, ¹⁵⁷Dy, ¹⁵⁹Dy, ¹⁶⁵Dy, ¹⁶⁶Dy, ¹⁷¹Er, ²⁵⁰Es, ²⁵⁴Es, ¹⁴⁷Eu, ¹⁵⁷Eu, ⁵²Fe, 5⁹Fe, ²⁵¹Fm, ²⁵²Fm, ²⁵³Fm, ⁶⁶Ga, ⁷²Ga, ¹⁴⁶Gd, ¹⁵³Gd, ⁶⁸Ge, ³H, ¹⁷⁰Hf, ¹⁷¹ Hf, ¹⁹³Hg, ¹⁹³mHg, 1⁶⁰mHo, ¹³⁰l, ¹³¹ l, ¹³⁵l, ¹¹⁴mln, ¹⁸⁵lr, ⁴²K, ⁴³K, ⁷⁶Kr, ⁷⁹Kr, ⁸¹mKr, ¹³²La, ²⁶²Lr, ^1S9Lu, ¹⁷⁴mLu, 1⁷⁶mLu, ²⁵⁷Md, ²⁶⁰Md, ²⁸Mg, ⁵²Mn, ⁹⁰Mo, ²⁴Na, ⁹⁵Nb, ¹³⁸Nd, ⁵⁷Ni, ⁶⁶Ni, ²³⁴Np, ¹⁵O, ¹⁸²0s, 1⁸⁹mOs, ¹⁹¹Os, ³²P, ²⁰¹Pb, ¹⁰¹ Pd, ¹⁴³Pr, ¹⁹¹ Pt, ²⁴³Pu, ²²⁵Ra, ⁸¹ Rb, ¹⁸⁸Re, ¹⁰⁵Rh, ²¹¹ Rn, ¹⁰³Ru, 3⁵S, ⁴⁴Sc, ⁷²Se, ¹⁵³Sm, ¹²⁵Sn, ⁹¹Sr, ¹⁷³Ta, ¹⁵⁴Tb, ¹²⁷Te, ²²⁷Th, ²³⁴Th, ⁴⁵Ti, ¹⁶⁶Tm, ²³⁰U, ²³⁷U, 2⁴⁰U, ⁴⁸V, ¹⁷⁸W, ¹⁸¹W, ¹⁸⁸W, ¹²⁵Xe, ¹²⁷Xe, ¹³³Xe, ¹³³mXe, ¹³⁵Xe, ⁸⁵mY, ⁸⁶Y, ⁹⁰Y, ⁹³Y, ¹⁶⁹Yb, 1⁷⁵Yb, ⁶⁵Zn, ⁷¹mZn, ⁸⁶Zr, ⁹⁵Zr, or ⁹⁷Zr.

24. The conjugate of claim 18, wherein the metal isotope comprises isotopes of lanthanide, indium, yttrium, palladium, or bismuth.

25. The conjugate of claim 18, wherein the metal isotope comprises Y89, Pd102, Rh103, Pd104, Pd105, Pd106, Pd108, Pd1 10, Xe131 , Cs133, Ba138, Ce140, Pr141 , Nd142, Nd143, Nd144, Nd145, Nd146, Sm147, Nd148, Sm149, Nd150, Eu151 , Sm152, Eu153, Sm154, Gd155, Gd156, Gd158, Tb159, Gd160, Dy161 , Dy162, Dy163, Dy164, Ho165, Er166, Er167, Er168, Tm169, Er170, Yb171 , Yb172, Yb173, Yb174, Lu175, Yb176, BCKG190, Ir191 , Ir193, Pt195, Bi209, Ln113, Ln1 15, or La139.

26. The conjugate of claim 18, wherein the particle comprises a quantum dot, a microparticle, a nanoparticle, a nanoshell, a nanobead, a microbead, or a nanodot.

27. The conjugate of claim 18, wherein the particle comprises a quantum dot.

28. The conjugate of claim 16, wherein the cargo comprises 2 detectable labels.

29. The conjugate of claim 16, wherein the cargo comprises 3 detectable labels.

30. The conjugate of claim 17, wherein the effector molecule comprises a drug or a toxin.

31. A composition comprising the single-domain antibody of claim 1 and an acceptable carrier.

32. A kit comprising the single-domain antibody of claim 1 and an ultraviolet light source.

33. A method of labeling a primary rabbit antibody comprising administering the composition of claim 31 to a composition comprising the primary rabbit antibody, wherein the composition of claim 29 comprises a single-domain antibody of claim 1 comprising a photocrosslinker, wherein the single-domain antibody is linked to cargo; and activating the photocrosslinker.

34. The method of claim 33, wherein the administering comprises pipetting.

35. The method of claim 33, wherein the activating the photocrosslinker comprises exposing the photocrosslinker to ultraviolet light.

36. The method of claim 35, wherein the exposing lasts for at least 1 min, at least 5 min, at least 10 min, at least 15 min, at least 20 min, at least 25 min, at least 30 min, at least 45 min, at least 1 hour, at least 2 hours, or at least 3 hours.

37. The method of claim 35, wherein the exposing lasts for 5 min.

38. The method of claim 35, wherein the exposing lasts for 1 min.

39. The method of claim 35, wherein the ultraviolet light has a wavelength from 325 nm to 425 nm.

40. The method of claim 35, wherein the ultraviolet light has a wavelength of 405 nm.

41. The method of claim 35, wherein the ultraviolet light has a wavelength of 355 nm.

42. The method of claim 33, wherein the cargo comprises a detectable label and the method further comprises detecting the detectable label.

43. The method of claim 33, wherein the cargo comprises a therapeutic molecule.

44. A cDNA encoding the single-domain antibody of claim 1.

45. The cDNA of claim 44 ligated into a bacterial expression vector.

46. The cDNA of claim 45, wherein the bacterial expression vector comprises pET-21b(+).

47. A method of preparing the single-domain antibody of claim 1 comprising transforming a cell with the cDNA of claim 44.

48. The method of claim 47, wherein the cell is an E. coli.

49. The method of claim 48, wherein the cell is a BL21(DE3) competent E. coli.

50. The method of claim 47, further comprising transforming the cell with a suppressor plasmid.

51. The method of claim 50, wherein the suppressor plasmid encodes an orthogonal translation system for Bpa-specific incorporation.

52. The method of claim 47, further comprising culturing the cell.

53. The method of claim 47, further comprising harvesting the prepared single-domain antibody.

54. The method of claim 53, wherein the harvesting comprises lysing the cell and collecting the single-domain antibody.