Universal covalent crosslinker for antibody-oligo conjugates

Abstract

The present disclosure provides compositions and methods for conjugating a target polypeptide and a target oligonucleotide. More particularly, the present disclosure provides compositions and methods for making and using a recombinant protein harboring a domain recognizing a nucleic acid and a domain capable of binding and crosslinking with antigen-binding polypeptides.

Description

BI-11236 UNIVERSAL COVALENT CROSSLINKER FOR ANTIBODY-OLIGO CONJUGATES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 672,447,filed July 17, 2024. The entire contents of the above-identified application is hereby fully incorporated by reference. FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to compositions and methods for making and using apeptide capable of covalently crosslinking an antigen-binding polypeptide and a single-stranded DNA (ssDNA). BACKGROUND OF THE DISCLOSURE

[0003] Conjugates that join antibodies (Abs) and DNA have wide applications in biologyresearch and biomedical applications. Traditional bioconjugation methods necessitate modifications of DNA and / or Abs to achieve conjugation, with such modifications thereby requiring additional purification steps and consequently resulting in diminished yields, as well as incurring increased costs. Additionally, these traditional methods often mandate the use of carrier- free Abs, which are also costlier and have shorter shelf-lives than other antibody preparations. Moreover, nonspecific conjugation reactions can also compromise the binding affinity of resulting conjugates. Therefore, a need exists for compositions and methods for covalently conjugating Abs and single-stranded DNA (ssDNA), that exhibit robust levels of protection / stability against common denaturation and reduction reagents, thereby enhancing the utility of such conjugates, and avoiding the previously mentioned limitations. BRIEF SUMMARY OF THE DISCLOSURE

[0004] The current disclosure relates, at least in part, to the discovery of compositions andmethods for making and using a recombinant protein harboring a domain specifically recognizing ssDNA with a predetermined sequence and a domain capable of binding and subsequently crosslinking with the Fc domain of antigen-binding polypeptides. Certain compositions and methods of the disclosure therefore feature a recombinant peptide harboring (i) a first domain capable of specifically recognizing and binding single-stranded DNA (ssDNA) with a predetermined sequence and (ii) a second domain capable of binding and subsequently photo- 122903345.1BN00007.2520 BI-11236 crosslinking with the Fc domain of an antigen-binding polypeptide. The methods of the instant disclosure covalently conjugate Abs and ssDNA, forming conjugates that exhibit robust resistance to common denaturation and reduction reagents, which thereby enhances the stability and utility of such conjugates, while avoiding certain known limitations of other art-recognized processes for joining peptides and DNA. In certain embodiments, the recombinant protein is a peptide linker that can be used to join a target polypeptide and a target oligonucleotide.

[0005] In one aspect, the instant disclosure provides a peptide linker for joining a targetpolypeptide and a target oligonucleotide, the peptide linker including: a first polypeptide domain capable of binding the target polypeptide; a second polypeptide domain including a linking polypeptide sequence; and a third polypeptide domain including a histidine-hydrophobic-histidine (HUH) endonuclease, wherein the third polypeptide domain is capable of binding the target oligonucleotide.

[0006] In another aspect, the disclosure provides an isolated nucleic acid that encodes thepeptide linker.

[0007] In another aspect, the disclosure provides an expression vector having the isolatednucleic acid that encodes the peptide linker.

[0008] In another aspect, the disclosure provides a method of conjugating a target polypeptideand a target oligonucleotide including a histidine-hydrophobic-histidine (HUH) endonuclease recognition sequence, the method involving: (a) contacting the target oligonucleotide having the HUH endonuclease recognition sequence with a peptide linker, said peptide linker having: a first polypeptide domain capable of binding the target polypeptide and having a crosslinkable moiety; a second polypeptide domain having a linking polypeptide sequence; and a third polypeptide domain having a HUH endonuclease, thereby forming a first complex comprising the target oligonucleotide and the peptide linker; and (b) contacting the target polypeptide with the first complex under conditions suitable for crosslinking of the target polypeptide and the first complex, thereby conjugating the target polypeptide and the target oligonucleotide.

[0009] In another aspect, the disclosure provides a method of conjugating a target polypeptideand a target oligonucleotide having a histidine-hydrophobic-histidine (HUH) endonuclease recognition sequence, the method involving: (a) contacting the target polypeptide with a peptide linker, said peptide linker including: a first polypeptide domain capable of binding the target polypeptide and having a crosslinkable moiety; a second polypeptide domain having a linking 2 122903345.1BN00007.2520 BI-11236 polypeptide sequence; and a third polypeptide domain having a HUH endonuclease, under conditions suitable for crosslinking of the target polypeptide and the peptide linker, thereby forming a first complex having the target polypeptide and the peptide linker; and (b) contacting the target oligonucleotide having the HUH endonuclease recognition sequence with the first complex; thereby conjugating the target polypeptide and the target oligonucleotide.

[0010] In another aspect, the disclosure provides a composition including: (i) an antigen-binding polypeptide; (ii) a peptide linker having: a first polypeptide domain covalently attached to the antigen-binding polypeptide, a second polypeptide domain having a linking polypeptide sequence, and a third polypeptide domain having a histidine-hydrophobic-histidine (HUH) endonuclease; and (iii) a target oligonucleotide, wherein the third polypeptide domain of the peptide linker is covalently attached to the target oligonucleotide. Definitions

[0011] As used herein, the term "barcode sequence" is intended to mean a series of nucleotidesin a nucleic acid that can be used to identify the nucleic acid, a characteristic of the nucleic acid (e.g., the identity and optionally the location of a bead to which the nucleic acid is attached), or a manipulation that has been carried out on the nucleic acid. The barcode sequence can be a naturally occurring sequence or a sequence that does not occur naturally in the organism from which the barcoded nucleic acid was obtained. A barcode sequence can be unique to a single nucleic acid species in a population or a barcode sequence can be shared by several different nucleic acid species in a population (e.g., all nucleic acid species attached to a single bead might possess the same barcode sequence, while different beads present a different shared barcode sequence that serves to identify each such different bead). By way of further example, each nucleic acid probe in a population can include different barcode sequences from all other nucleic acid probes in the population. Alternatively, each nucleic acid probe in a population can include different barcode sequences from some or most other nucleic acid probes in a population. For example, each probe in a population can have a barcode that is present for several different probes in the population even though the probes with the common barcode differ from each other at other sequence regions along their length. In particular embodiments, one or more barcode sequences that are used with a biological specimen (e.g., a tissue sample) are not present in the genome, transcriptome or other nucleic acids of the biological specimen. For example, barcode sequences can have less than 80%, 3 122903345.1BN00007.2520 BI-11236 70%, 60%, 50% or 40% sequence identity to the nucleic acid sequences in a particular biological specimen.

[0012] As used herein, the term "spatial tag" is intended to mean a nucleic acid having asequence that is indicative of a location. Typically, the nucleic acid is a synthetic molecule having a sequence that is not found in one or more biological specimen that will be used with the nucleic acid. However, in some embodiments the nucleic acid molecule can be naturally derived or the sequence of the nucleic acid can be naturally occurring, for example, in a biological specimen that is used with the nucleic acid. The location indicated by a spatial tag can be a location in or on a biological specimen, in or on a solid support, or a combination thereof. A barcode sequence can function as a spatial tag. In certain embodiments, the identification of the tag that serves as a spatial tag is only determined after a population of beads (each possessing a distinct barcode sequence) has been arrayed upon a solid support (optionally randomly arrayed upon a solid support) and sequencing of such a bead-associated barcode sequence has been determined in situ upon the solid support.

[0013] By “agent” is meant any small compound (e.g., small molecule), antibody, nucleic acidmolecule, or polypeptide, or fragments thereof.

[0014] The term “antigen” or “Ag” as used herein is defined as a molecule that can be targetedby an antibody or antibody fragment thereof.

[0015] As used herein, a “tumor antigen” means a biological molecule having antigenicity,expression of which is associated with a neoplastic cell. The tumor antigens targeted in the present disclosure include a tumor specific antigen (an antigen which is present only in tumor cells and is not found in other normal cells), and a tumor-associated antigen (an antigen which is also present in other organs and tissues or heterogeneous and allogeneic normal cells, or an antigen which is expressed on the way of development and differentiation).

[0016] The terms "antibodies" and "immunoglobulin" include antibodies or immunoglobulinsof any isotype, fragments of antibodies that retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single- chain antibodies (scAb), single domain antibodies (dAb), single domain heavy chain antibodies, a single domain light chain antibodies, bi-specific antibodies, multi-specific antibodies, and fusion proteins comprising an antigen- binding (also referred to herein as antigen binding) portion of an antibody and a non-antibody protein. The antibodies can be detectably labeled, e.g., with a 4 122903345.1BN00007.2520 BI-11236 radioisotope, an enzyme that generates a detectable product, a fluorescent protein, and the like. The antibodies can be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like. The antibodies can also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like. Also encompassed by the term are Fab', Fv, F(ab')2, and or other antibody fragments that retain specific binding to antigen, and monoclonal antibodies. As used herein, a monoclonal antibody is an antibody produced by a group of identical cells, all of which were produced from a single cell by repetitive cellular replication. That is, the clone of cells only produces a single antibody species. While a monoclonal antibody can be produced using hybridoma production technology, other production methods known to those skilled in the art can also be used (e.g., antibodies derived from antibody phage display libraries). An antibody can be monovalent or bivalent. An antibody can be an Ig monomer, which is a "Y-shaped" molecule that consists of four polypeptide chains: two heavy chains and two light chains connected by disulfide bonds.

[0017] The term "humanized immunoglobulin" as used herein refers to an immunoglobulincomprising portions of immunoglobulins of different origin, wherein at least one portion comprises amino acid sequences of human origin. For example, the humanized antibody can comprise portions derived from an immunoglobulin of nonhuman origin with the requisite specificity, such as a mouse, and from immunoglobulin sequences of human origin (e.g., chimeric immunoglobulin), joined together chemically by conventional techniques (e.g., synthetic) or prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of the chimeric antibody can be expressed to produce a contiguous polypeptide chain). Another example of a humanized immunoglobulin is an immunoglobulin containing one or more immunoglobulin chains comprising a CDR derived from an antibody of nonhuman origin and a framework region derived from a light and / or heavy chain of human origin (e.g., CDR- grafted antibodies with or without framework changes).

[0018] Chimeric or CDR-grafted single chain antibodies are also encompassed by the termhumanized immunoglobulin. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No.0125023B1; Boss et al., U.S. Pat. No.4,816,397; Boss et al., European Patent No.0120694B1; Neuberger, M. S. et al., WO 86 / 01533; Neuberger, M. S. et al., European Patent No. 0194276B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0239400B1 ; Padlan, E. A. et al., European Patent Application No. 0519596A1. See also, Ladner et al., U.S. 5 122903345.1BN00007.2520 BI-11236 Pat. No.4,946,778; Huston, U.S. Pat. No.5,476,786; and Bird, R. E. et al., Science, 242: 423-426 (1988)), regarding single chain antibodies.

[0019] The term "nanobody" (Nb), as used herein, refers to the smallest antigen bindingfragment or single variable domain (VHH) derived from naturally occurring heavy chain antibody and is known to the person skilled in the art. They are derived from heavy chain only antibodies, seen in camelids (Hamers- Casterman et al., 1993; Desmyter et al., 1996). In the family of "camelids" immunoglobulins devoid of light polypeptide chains are found. "Camelids" comprise old world camelids (Camelus bactrianus and Camelus dromedarius) and new world camelids (for example, Llama paccos, Llama glama, Llama guanicoe and Llama vicugna). A single variable domain heavy chain antibody is referred to herein as a nanobody or a VHH antibody.

[0020] "Antibody fragments" comprise a portion of an intact antibody, for example, theantigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); domain antibodies (dAb; Holt et al. (2003) Trends Biotechnol.21:484); single-chain antibody molecules; and multi-specific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.

[0021] "Fv" is the minimum antibody fragment that contains a complete antigen-recognitionand -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non- covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind an antigen, although at a lower affinity than the entire binding site.

[0022] The "Fab" fragment also contains the constant domain of the light chain and the firstconstant domain (CHj) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHj domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in 6 122903345.1BN00007.2520 BI-11236 which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0023] "Single-chain Fv" or "sFv" or "scFv" antibody fragments comprise the VH andVL domains of antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer -Verlag, New York, pp.269-315 (1994).

[0024] As used herein, the term “autologous” is meant to refer to any material derived fromthe same individual to which it is later to be re-introduced into the individual.

[0025] As used herein “endogenous” refers to any material from or produced inside anorganism, cell, tissue or system.

[0026] As used herein, the term “exogenous” refers to any material introduced from orproduced outside an organism, cell, tissue, or system.

[0027] As used herein, the term “amino acid with photocrosslinking activity” refers to anamino acid that can covalently crosslink with an amino acid residue of adjacent proteins under suitable light irradiation conditions. The “amino acid with photocrosslinking activity” may include natural amino acids, unnatural amino acids (UAAs), or non-canonical amino acids (ncAAs). The “photocrosslinking activity” includes, but is not limited to, sensitivity to ultraviolet (UV) light. Non-limiting examples of the “amino acid with photocrosslinking activity” include pBpa, pAzF and the like (e.g., such as pAbK, and other UAA derivates with the phenyl azide, benzophenone, or the diazirin structure).

[0028] The term "fusion protein" as used herein refers to an engineered polypeptide thatcombines sequence elements excerpted from two or more other proteins, optionally from two or more naturally-occurring proteins.

[0029] The terms "transfect," "transfects," "transfecting" and "transfection" as used hereinrefer to the delivery of nucleic acids (usually DNA or RNA) to the cytoplasm or nucleus of cells, e.g., through the use of cationic lipid vehicle(s) and / or by means of electroporation, or other art- recognized means of transfection. 7 122903345.1BN00007.2520 BI-11236

[0030] The term "transduction," as used herein refers to the delivery of nucleic acids (usuallyDNA or RNA) to the cytoplasm or nucleus of cells through the use of viral delivery, e.g., via lentiviral delivery vectors / plasmids, or other art-recognized means of transduction.

[0031] The term "plasmid" as used herein refers to a construction comprised of geneticmaterial designed to direct transformation of a targeted cell. The plasmid consist of a plasmid backbone. A "plasmid backbone" as used herein contains multiple genetic elements positional and sequentially oriented with other necessary genetic elements such that the nucleic acid in a nucleic acid cassette can be transcribed and when necessary translated in the transfected or transduced cells. The term plasmid as used herein can refer to nucleic acid, e.g., DNA derived from a plasmid vector, cosmid, phagemid or bacteriophage, into which one or more fragments of nucleic acid may be inserted or cloned which encode for particular genes.

[0032] As used herein, the term “gene expression” refers to the process of converting geneticinformation encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., via the enzymatic action of an RNA polymerase), and for protein encoding genes, into protein through “translation” of mRNA. Gene expression can be regulated at many stages in the process. “Up-regulation” or “activation” refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while “down-regulation” or “repression” refers to regulation that decrease production. Molecules (e.g., transcription factors) that are involved in up-regulation or down-regulation are often called “activators” and “repressors,” respectively.

[0033] Where “amino acid sequence” is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, “amino acid sequence” and like terms, such as “polypeptide” or “protein” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.

[0034] As used herein, the terms “nucleic acid molecule encoding,” “DNA sequenceencoding,” “DNA encoding,” “RNA sequence encoding,” and “RNA encoding” refer to the order or sequence of deoxyribonucleotides or ribonucleotides along a strand of deoxyribonucleic acid or ribonucleic acid. The order of these deoxyribonucleotides or ribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA or RNA sequence thus codes for the amino acid sequence. 8 122903345.1BN00007.2520 BI-11236

[0035] The terms “in operable combination,” “in operable order,” and “operably linked” asused herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.

[0036] As used herein, the term “regulatory element” refers to a genetic element whichcontrols some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, RNA export elements, internal ribosome entry sites, etc.

[0037] Transcriptional control signals in eukaryotes comprise “promoter” and “enhancer”elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis et al., (1987) Science 236:1237). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells, and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review see, Voss et al., (1986) Trends Biochem. Sci., 11:287; and Maniatis et al., supra). For example, the SV40 early gene enhancer is very active in a wide variety of cell types from many mammalian species and has been widely used for the expression of proteins in mammalian cells (Dijkema et al, (1985) EMBO J. 4:761). Two other examples of promoter / enhancer elements active in a broad range of mammalian cell types are those from the human elongation factor 1α gene (Uetsuki et al., (1989) J. Biol. Chem., 264:5791; Kim et al., (1990) Gene 91:217; and Mizushima and Nagata, (1990) Nuc. Acids. Res., 18:5322) and the long terminal repeats of the Rous sarcoma virus (Gorman et al., (1982) Proc. Natl. Acad. Sci. USA 79:6777) and the human cytomegalovirus (Boshart et al., (1985) Cell 41:521).

[0038] As used herein, the term “promoter / enhancer” denotes a segment of DNA that containssequences capable of providing both promoter and enhancer functions (i.e., the functions provided by a promoter element and an enhancer element, see above for a discussion of these functions). For example, the long terminal repeats of retroviruses contain both promoter and enhancer 9 122903345.1BN00007.2520 BI-11236 functions. The enhancer / promoter may be “endogenous” or “exogenous” or “heterologous.” An “endogenous” enhancer / promoter is one which is naturally linked with a given gene in the genome. An “exogenous” or “heterologous” enhancer / promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques such as cloning and recombination) such that transcription of that gene is directed by the linked enhancer / promoter.

[0039] The term “promoter,” “promoter element,” or “promoter sequence” as used herein,refers to a DNA sequence which when ligated to a nucleotide sequence of interest is capable of controlling the transcription of the nucleotide sequence of interest into mRNA. A promoter is typically, though not necessarily, located 5′ (i.e., upstream) of a nucleotide sequence of interest whose transcription into mRNA it controls, and provides a site for specific binding by RNA polymerase and other transcription factors for initiation of transcription.

[0040] Promoters may be constitutive or regulatable. The term “constitutive” when made inreference to a promoter means that the promoter is capable of directing transcription of an operably linked nucleic acid sequence in the absence of a stimulus (e.g., heat shock, chemicals, etc.). In contrast, a “regulatable” promoter is one which is capable of directing a level of transcription of an operably linked nucleic acid sequence in the presence of a stimulus (e.g., heat shock, chemicals, etc.) which is different from the level of transcription of the operably linked nucleic acid sequence in the absence of the stimulus. Certain promoters are also known in the art to impart tissue- specificity and / or temporal / developmental specificity to expression of a nucleic acid sequence under control of such a promoter.

[0041] As used herein the term, the term “in vitro” refers to an artificial environment and toprocesses or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell cultures. The term “in vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.

[0042] As used herein, the term “host cell” refers to any eukaryotic cell (e.g., mammalian cells,avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.

[0043] As used herein, the term "next-generation sequencing" or "NGS" can refer tosequencing technologies that have the capacity to sequence polynucleotides at speeds that were unprecedented using conventional sequencing methods (e.g., standard Sanger or Maxam-Gilbert 10 122903345.1BN00007.2520 BI-11236 sequencing methods). These unprecedented speeds are achieved by performing and reading out thousands to millions of sequencing reactions in parallel. NGS sequencing platforms include, but are not limited to, the following: Massively Parallel Signature Sequencing (Lynx Therapeutics); 454 pyro-sequencing (454 Life Sciences / Roche Diagnostics); solid-phase, reversible dye- terminator sequencing (Solexa / Illumina); SOLiD technology (Applied Biosystems); Ion semiconductor sequencing (ion Torrent); and DNA nanoball sequencing (Complete Genomics). Descriptions of certain NGS platforms can be found in the following: Shendure, et al., "Next- generation DNA sequencing," Nature, 2008, vol.26, No.10, 135-1145; Mardis, "The impact of next-generation sequencing technology on genetics," Trends in Genetics, 2007, vol.24, No.3, pp. 133-141 ; Su, et al., "Next-generation sequencing and its applications in molecular diagnostics" Expert Rev Mol Diagn, 2011 , 11 (3):333-43; and Zhang et al., "The impact of next-generation sequencing on genomics", J Genet Genomics, 201, 38(3): 95-109.

[0044] Unless specifically stated or obvious from context, as used herein, the term “about” isunderstood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

[0045] In certain embodiments, the term "approximately" or "about" refers to a range of valuesthat fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0046] Unless otherwise clear from context, all numerical values provided herein are modifiedby the term “about.”

[0047] By “control” or “reference” is meant a standard of comparison. Methods to select andtest control samples are within the ability of those in the art. Determination of statistical significance is within the ability of those skilled in the art, e.g., the number of standard deviations from the mean that constitute a positive result.

[0048] As used herein, the term "each," when used in reference to a collection of items, isintended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise. 11 122903345.1BN00007.2520 BI-11236

[0049] As used herein, the term "subject" includes humans and mammals (e.g., mice, rats, pigs,cats, dogs, and horses). In many embodiments, subjects are mammals, particularly primates, especially humans. In some embodiments, subjects are livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. In some embodiments (e.g., particularly in research contexts) subject mammals will be, for example, rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine such as inbred pigs and the like.

[0050] Unless specifically stated or obvious from context, as used herein, the term "or" isunderstood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[0051] Ranges can be expressed herein as from “about” one particular value, and / or to “about”another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it is understood that the particular value forms another aspect. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. It is also understood that throughout the application, data are provided in a number of different formats and that this data represent endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0052] Ranges provided herein are understood to be shorthand for all of the values within therange. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With 12 122903345.1BN00007.2520 BI-11236 respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0053] The transitional term “comprising,” which is synonymous with “including,”“containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, non-recited elements or method steps. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed embodiments presented in the disclosure.

[0054] The embodiments set forth below and recited in the claims can be understood in viewof the above definitions.

[0055] Other features and advantages of the disclosure will be apparent from the followingdescription of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The following detailed description, given by way of example, but not intended to limitthe disclosure solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0057] FIGs. 1A-1D show examples of conventional antibody-nucleic acid conjugationmethods involving Thiol-Maleimide / Amine-NHS (FIG.1A), Azide-DBCO (FIG.1B), OYO-link (FIG. 1C), and ribosome displayed nanobody libraries (FIG. 1D). Image for FIGs. 1A-1C is 13 122903345.1BN00007.2520 BI-11236 reproduced from Shen et al. ChemBioChem, 2023, e202300077, while the image for FIG.1D is reproduced from Chen, X. et al. Nat. Commun, 12, 5506 (2021).

[0058] FIG.2 shows an exemplary peptide linker for antibody-oligo conjugation of the presentdisclosure. The peptide linker includes two functional domains: (i) a HUH endonuclease domain; and (ii) an IgG Fc-block binding motif harboring a photo-crosslinkable unnatural amino acid. Both domains are joined by a linking polypeptide sequence, N-GGSGGS-C (SEQ ID NO: 6) as currently shown.

[0059] FIGs. 3A-3C demonstrate the functioning of the HUH endonuclease domain, whichavoids the need to use chemically modified oligonucleotides ("oligos"). FIG.3A shows that the HUH endonuclease breaks and covalently links to specific ssDNA sequences (here, a sequence of 5'-AAGTATTACCAG-3' (SEQ ID NO: 7) is recognized and cleaved, with the ACCAG sequence covalently attached to the HUH endonuclease), and possesses a high efficiency (15 minutes, 37°C). FIG. 3B shows a list of HUH-tag types and associated recognition sequences (recognition sequences, in order from top to bottom are: 5'-AAGTATT|ACCAGAAA-3' (SEQ ID NO: 8); 5'- TGCTTCCGTACTACG|ACCCCCCA-3' (SEQ ID NO: 9); 5'- TTTGCGTGGGGTGT|GGTGCTTT-3' (SEQ ID NO: 10); 5'- CCAGTTTCTCGAAGAGAAACCGGTAAGTGCA|CCCTCCC-3' (SEQ ID NO: 11); and 5'- ACGCGAACGGAACGTTCGCATAAGTGCG|CCCTTACGGGATTTAAC-3' (SEQ ID NO: 12)). The vertical lines indicate the nick site for the corresponding HUH endonucleases. The sequence on the right side of the vertical line will be covalently linked to a HUH endonuclease, while the sequence on the left side of the vertical line will be cleaved off. FIG. 3C shows the percent of covalent adducts (covalent fusions with target oligos carrying an associated HUH endonuclease recognition sequence) that were observed when using various HUH endonucleases (DCV, mMobA, RepB, TraI, and NES) known in the art, as compared to the efficiency of SNAP- tag binding as a positive control. Certain images included in FIGs. 3A-3C are reproduced from Lovendahl et al., J Am Chem Soc, 139: 7030-7035 (2017).

[0060] FIG. 4 demonstrates expression and purification of the peptide linker disclosed herein,which was purified using an NEBTMIMPACT kit. Fusion protein construction utilized the pTXB1 system, and it was demonstrated herein that the fusion protein could be separated from cell lysates and then cleaved from the Mxe intein-CBD component. In the top box (corresponding to the rectangular box on the gel image), the precursor 56 kDa form of the fusion peptide can be seen on 14 122903345.1BN00007.2520 BI-11236 the column pre-elution (lane 7). Cleavage of the full-length fusion protein was then induced on- column by adding DTT, which generated two cleavage products, the first product having the intein-CBD region which still bound the column (middle box, Intein-CBD panel, 28 kDa) and the second product being the eluted Protein G B1 domain-G5 linker-HUH-containing peptide linker (lower rectangular box on the gel image, 18.55 kDa) that was used in the experiments presented herein. The initial construct after on-column cleavage still possessed a large portion of uncleaved precursor bound to the post-DTT resin (rectangle, lane 14), which prompted attempts to further optimize constructs for better cleavage and higher yield, which were subsequently made and validated infra.

[0061] FIG. 5 shows results of testing the linker (peptide linker of the disclosure) forconjugation activity. In left and middle highlighted rectangles (lanes 4-7), gel retardation band shifts were observed where directed covalent binding events occurred, and the reduction in and even absence of the original 18.55 kDa form in lanes exposed to targeted ssDNAs demonstrated the successful and highly efficient conjugation between target ssDNA oligos and the peptide linker. The paired lanes at right demonstrated that covalent conjugation of the peptide linker to Abs was successful and only happened when UV light was used to induce crosslinking. In all the lanes with antibody, DTT was added to the samples separated using SDS-PAGE, except for lanes 12 and 13. Lanes 8 and 9 show that with the reducing agent, IgG antibody (150 kDa) broke down into light chain (bottom bands, 25 kDa) and heavy chain (top bands, 50 kDa), but exposure to the 365 nm UV light did not alter the IgG bands. Lanes 10-13 demonstrate conjugation between the linker of the disclosure (peptide linker) and IgG antibody. The newly generated band (rectangle at right, lane 13, ~70 kDa) shows that covalent conjugation between the heavy chain (50 kDa) and the peptide linker (18.55 kDa) occurred, while no similar band could be observed in lane 10. Similarly, lanes 12 and 13 show linker (peptide linker) and IgG antibody conjugation gel runs in the absence of reducing agents, with the smear at the top of lane 13 showing the existence of partial conjugation. Lanes 14 and 15 show the conjugation of both the HUH domain and the Fc-block binding motif, joined by the peptide linker. The conjugation between the HUH domain and ssDNA was first performed (refer to protocol), then the resulting mixture was combined with IgG antibody and incubated with UV light. The gel-retarded, boxed band (rectangle, lane 15) shows the conjugation product, with both components (IgG and ssDNA, respectively) conjugated to the 15 122903345.1BN00007.2520 BI-11236 peptide linker, as designed. This assay therefore demonstrated that both domains of the peptide linker were functional.

[0062] FIG. 6 shows the results of further testing of the peptide linker of the disclosure,including a comparison with the commercially available OYO-link product. In lane 5, a conjugate was made using the commercially available OYO-link mouse IgG1 kit, and formation of a conjugate was visualized as the appearance of an observed band shift, yet this commercial kit still exhibited a substantial portion of unconjugated heavy chain antibody. In lanes 6-8, conjugates were made using the peptide linker of the disclosure, at different ratios of IgG to peptide linker. As the binding between the Fc block and protein G achieved an equilibrium, increased amounts of the peptide linker were shown to push this equilibrium towards the conjugated product side, thereby increasing the efficiency of conjugation. Lane 9 shows two bands that were shifted upward from the less distinct, and thus indicative of a small amount, unconjugated unreduced antibody band. This was due to the fact that the peptide linker of the disclosure can bind both heavy chains of an IgG dimer. 100% conjugation would therefore result in observation of a 2:1 ratio between peptide linker and IgG. Less than 100% conjugation but rather a 1:1 conjugation generated the middle band. Lanes 11 and 12 show results for performance of the two-step conjugation between ssDNA, peptide linker and IgG antibody. Further conjugation optimization of the peptide linker of the disclosure showed at least comparable efficiency to OYO-link. Increases in the conjugation ratio significantly increased conjugation efficiency, while partial inactive Fc-block binding domain in product was seen due to purification / DTT, etc. Mostly single or double conjugation products were observed, and the observed overall conjugation ratio was less than 100%.

[0063] FIG. 7 shows results obtained in screening of peptide linker binding of multiple kindsof antibodies from different isotypes and hosts. While it is contemplated that the Fc-block binding motif of the peptide linker(s) of the disclosure might be further optimized to increase the coverage of the peptide linker in conjugating different antibody isotypes and hosts, the current peptide linker was demonstrated to work with multiple IgG isotypes or hosts. It was again observed that equilibriums could be further pushed towards conjugate formation by increasing the ratio of peptide linker to respective immunoglobulins (lanes 2-4). Fig. 7 "Table 1" image is reproduced from Rodrigo et al. Antibodies, 4: 259-277 (2015).

[0064] FIG. 8 shows a version of the peptide linker of the disclosure having an affinity tagmoiety of a Mxe intein and chitin-binding domain (CBD) with three C-terminal Alanine residues, 16 122903345.1BN00007.2520 BI-11236 and associated results showing improved levels of cleavage for this updated format. Two alanine and three alanine forms (the three alanine form having the internal sequence PAAA (SEQ ID NO: 13) as shown) of the PG-Huh-intein-CBD fusion protein showed higher levels of processed PG- HUH protein following cleavage to remove the Mxe intein-CBD domain that was used for peptide linker purification, as compared to the initially tested single alanine form.

[0065] FIG. 9 shows results of further optimization by titration of the peptide linker-antibodyratio, using the updated triple alanine format of the peptide linker. Significantly improved yields of peptide linker-antibody conjugates were observed, as demonstrated by gel-shift assays.

[0066] FIG.10 shows results obtained for attempted purifications of peptide linker-containingconjugates that varied across a range of covalently linked ssDNA and covalently bound Abs, for contemplated multiplexed uses, e.g., CODEX. Lanes 5-10 showed that magnetic bead-based purification of peptide linker-containing conjugates were successful (magnetic beads with Fc blocks could bind and remove excessive linker) and lanes 11-14 show that Amicon 50 KDa ultracentrifugation filters were also effective in removing smaller, non-conjugated contaminants.

[0067] FIG. 11 shows validation of Antibody-peptide linker-ssDNA conjugates, for a firstconjugate that contained rabbit polyclonal anti-Lamin B1 antibody-peptide linker-ssDNA #15, and for a second conjugate that contained mouse monoclonal anti-Vimentin antibody-peptide linker- ssDNA #57. Detection by FISH and by dye markers associated with indicated components of the respective conjugates revealed functional Ab-peptide linker-ssDNA conjugates for both formats tested in HeLa cells.

[0068] FIG. 12 shows results of testing the linker (peptide linker of the disclosure) forconjugation activity in the presence of BSA at a concentration of 0.1 mg / mL. Conjugations under BSA concentrations up to 5 ml / mL were also validated. Testing for conjugation activity was done with anti-FoxP3 monoclonal antibody and anti-CD68 rabbit monoclonal antibody.

[0069] FIG. 13 illustrates a process for creation of a simple linker to easily and covalentlyconjugate proteins to antibodies.

[0070] FIG. 14 shows a gel image of the conjugation of anti-vimentin AB with sfGFP. Thepurified anti-vimentin antibody used herein is described at: https: / / www.biolegend.com / en- gb / products / purified-anti-vimentin-antibody-12022. 17 122903345.1BN00007.2520 BI-11236

[0071] FIGs. 15A-B show IF validation of anti-vimentin sfGFP AB. FIG. 15A shows Ab-SC-ST-sfGFP at ~1:100 final dilution (Contrast - Min: 100, Max: 400). FIG.15B shows standard IF with unconjugated Ab (Primary = 1:200, Secondary = 1:500; Contrast - Min: 100, Max - 20,000)

[0072] FIGs. 16A-C show conjugation and IF with anti-TGN46 AB with ST-GFP. The sameconjugation and staining protocol are used as in FIGs. 15A-B. The TGN46 polyclonal antibody used herein is described at: https: / / www.ptglab.com / products / TGOLN2,TGN46-Antibody-13573- 1-AP.htm. DETAILED DESCRIPTION OF THE DISCLOSURE

[0073] The current disclosure is based, at least in part, on the discovery of compositions andmethods that feature a recombinant protein harboring (i) a domain specifically recognizing ssDNA having a predetermined sequence and (ii) a domain capable of binding and subsequently crosslinking with the Fc domain of antigen-binding polypeptides, such recombinant protein possessing advantageous properties for forming antigen binding polypeptide-peptide linker- ssDNA conjugates capable of resisting common denaturation and reduction reagents, thereby enhancing the stability and utility of such conjugates. Certain conjugates of the disclosure overcome the following limitations of traditional conjugation methods commonly used in the art to join antigen-binding polypeptides (e.g., antibodies) with ssDNAs (e.g., barcode and / or other identifying sequences). Methods in common use tend to require chemically modified oligonucleotides, which are of high cost and low yield, as compared to plain (unmodified) oligos. Additionally, for conjugation purposes, there is a need for antibodies to be pre-purified in commonly used methods, to avoid Bovine serum albumin (BSA) and azide contamination of preparations, which adds additional cost to preparation of ssDNA-conjugated antibodies. Furthermore, some conjugation methods also require antibodies to be chemically modified first, which may be harmful for and antibody's stability and / or affinity, in addition to incurring increased costs and reducing yields. Non-specific conjugation methods are inconsistent, and additionally, such methods are non-specific to the site of conjugation, which introduces additional issues regarding whether a conjugated antibody's affinity might be impacted post-conjugation. For highly multiplexed assays, if multiple antibodies are used together, non-covalent conjugation methods may lead to exchange of components between antibodies, which can negatively impact both data quality and quality control (QC). 18 122903345.1BN00007.2520 BI-11236

[0074] For example, antibody-nucleic acid conjugation methods involving Azide-DBCO orThiol-Maleimide / Amine-NHS require modified oligonucleotides, modified antibodies, and carrier-free antibodies. Further, in these methods, conjugation may alter antibody structure and / or binding affinity. In another example, antibody-nucleic acid conjugation methods involving OYO- link require custom made OYO-oligo conjugates. In another example, methods involving Zenon kits are non-covalent conjugation methods that lead to antibody-nucleic acid products that have non-covalent linkages. In these methods, non-covalent linkages may lead to exchange of components between antibodies and are troublesome for multiplexing. In another example, antibody-nucleic acid conjugation methods involving Glycan based tags, SNAP-tags, or Halo-tags are likely unable to use off-the-shelf antibodies. The preceding list of limitations is provided as exemplary, and is not intended to be interpreted as comprehensive.

[0075] Further, 5’-modified oligos obtained from Integrated DNA Technologies (IDT) cost$300 and yield about 5 nmol of product, as compared to unmodified oligos with only an additional15 bp obtained from IDT costs about $10-20 and yields about 50-80 nmol of product. The oligosinvolved in the methods and compositions disclosed herein are unmodified and only include an additional 15 or more base pairs at the 5’ end. Thus, for each modified oligo that costs easily hundreds of dollars (about $150 for each modification), the equivalent unmodified oligo generally has much higher yield and does not require additional purification (about $70 for each oligo). Further, the conjugation protocol of the instant disclosure is faster than most other chemical conjugation protocols, and highly efficient. The compositions and methods of the disclosure also have significant potential for further multiplexing ability due to the variety of HUH endonucleases available.

[0076] According to the methods and compositions disclosed herein, a recombinant proteinmay function as a universal covalent crosslinker (e.g., peptide linker) between antigen-binding polypeptides and ssDNA. The recombinant protein aims to be a low cost, universal covalent crosslinker for antibody-oligo conjugates, and is highly-efficient, requires minimal purification, requires covalent conjugation, is site-specific, and easy to produce. The recombinant protein may comprise two parts: (i) a histidine-hydrophobic-histidine (HUH) endonuclease domain, which recognizes a specific ssDNA sequence, cleaves the ssDNA sequence and then remains covalently linked to a portion of the cleaved sequence; and (ii) an IgG Fc-block binding motif with a photo- crosslinkable unnatural amino acid, which binds with Fc block of IgG, and then may be photo- 19 122903345.1BN00007.2520 BI-11236 crosslinked to the Fc block to form a covalent linkage. The IgG Fc-block binding motif can specifically bind with the Fc block of the IgG heavy chain, which avoids: (i) the need for antibodies to be pre-purified to remove Bovine serum albumin (BSA) and azide contamination; (ii) the need for antibodies to be chemically modified first; and (iii) the limitations of non-specific conjugation methods, mentioned elsewhere herein. Use of a photo-crosslinkable moiety avoids non-covalent conjugation methods that may otherwise lead to exchange of components between antibodies, which is bad for data quality and QC. Additionally, as described further below, the compositions and methods disclosed herein provide significant benefits over antibody-ssDNA conjugation methods commonly used in the art.

[0077] Various expressly contemplated components of certain compositions and methods ofthe instant disclosure are considered in additional detail below. The peptide linkers of the present disclosure have the ability to covalently crosslink both a ssDNA (via activity of a HUH endonuclease positioned at one end of the peptide linker) and an antigen-binding polypeptide (bound by an Fc domain-binding polypeptide positioned at the opposite end of the peptide linker from the HUH endonuclease domain). In embodiments, light activation of a photo-crosslinkable moiety positioned in the Fc domain-binding polypeptide of a peptide linker of the disclosure results in covalent attachment of the antigen-binding polypeptide (having an Fc domain) with the peptide linker, and therefore with the ssDNA covalently attached to the HUH endonuclease domain at the other end of the peptide linker.

[0078] In certain embodiments, it is contemplated that an ssDNA and an antigen-bindingpolypeptide may be crosslinked via further biorthogonal conjugation methodologies using non- photocrosslinkable motifs which may further involve a second active molecule on the antigen- binding polypeptide for conjugation, and thus a further modification on the antigen-binding polypeptide. Also, to avoid premature conjugation towards the antigen-binding polypeptide, the activation energy of the functional group for conjugation needs to be rather high, which makes photo-crosslinking a more accessible method. Many other enzymatic toolbox approaches for protein-protein conjugation exist, such as split-intein, sortase A, etc.; however, such methods generally also utilize modified antigen-binding polypeptide.

[0079] In addition to their utility as general crosslinking agents for identification and isolationof antigen-binding polypeptide-ssDNA conjugates, the peptide linkers of the disclosure can be used in diagnostic and detection scenarios. These scenarios make use of peptides or compounds 20 122903345.1BN00007.2520 BI-11236 previously modified with the peptide linkers (and therefore having antigen binding polypeptides covalently attached to ssDNAs) as probes that have affinities for defined target molecules. Techniques similar to various immunodetection techniques, including ELISA, Western blotting, immunohistochemistry, and FACS, can be adapted to these novel probes. 1. Cross-Linking Reagents

[0080] A recombinant protein may function as a universal covalent crosslinker (e.g., peptidelinker) between antigen-binding polypeptides and ssDNA as described in the present application. In general, peptide linkers serve to join a ssDNA with an antigen binding polypeptide. 2. Methods of Using Peptide Linkers

[0081] Various uses of peptide linkers are contemplated in the present disclosure. In particularembodiments, the peptide linkers of the disclosure can be used to attach labels to a population of antigen-binding polypeptides (e.g., a library of antibodies) in an efficient, cost-effective manner. Several different types of labels can be used, as detailed herein and as otherwise known in the art. To provide a detecting means, the protein can be designed to have an associated label that allows for detection and / or identification, e.g., of a member of an ssDNA-tagged antibody library. In certain embodiments, this label will be an ssDNA.

[0082] The peptide linker may comprise two parts: (i) a histidine-hydrophobic-histidine(HUH) endonuclease domain, which recognizes a specific ssDNA sequence, cleaves the ssDNA sequence at an internal site but then remains covalently linked to a portion of the cleaved sequence; and (ii) an IgG Fc-block binding polypeptide domain harboring a photo-crosslinkable unnatural amino acid, which binds with the Fc block of IgG, and then may be photo-crosslinked to the Fc block, thereby forming a covalent linkage. HUH Endonucleases and ssDNA Recognition Sequences

[0083] HUH endonucleases contain a characteristic motif comprising a first histidine residue(H), a hydrophobic amino acid residue (U), and a second histidine residue (H). HUH endonucleases are known from archaeans, bacteria, and eukaryotes. Endogenous HUH 21 122903345.1BN00007.2520 BI-11236 endonucleases participate in cellular processes involving a transition from double-stranded to single-stranded DNA, such as rolling-circle replication in viruses and bacterial plasmid conjugation. The HUH endonuclease first nicks single-stranded DNA at a specific sequence at the origin of replication (ori) followed by formation of a covalent phosphotyrosine intermediate, whereby the 5′ end of the DNA strand becomes linked to a specific tyrosine in the HUH-protein. While the phosphotyrosine linkage is an intermediate in vivo, purified HUH-proteins are able to form stable covalent bonds in vitro with synthetic oligonucleotides bearing their ori sequence.

[0084] In an aspect, a HUH endonuclease is selected from the group consisting of a FBNYVHUH endonuclease, a PCV HUH endonuclease, a DCV HUH endonuclease, a RepB, a RepBm, a Tral, a mMobA, and a NES (refer to Lovendahl et al. J. Am. Chem. Soc. 139: 7030-7035 for additional description and exemplary types of HUH endonuclease).

[0085] Sequences of known HUH endonucleases include the following:

[0086] Fava bean necrosis yellow virus (FBNYV): N-MARQVICWCFTLNNPLSPLSLHDSMKYLVYQTEQGEAGNIHFQGYIEMKKRTSLAGMK KLIPGAHFEKRRGTQGEARAYSMKEDTRLEGPWEYGEFVP-C (SEQ ID NO: 3; FBNYV HUH endonuclease – see GenBank accession number ASL05580.1);

[0087] An alternate sequence for fava bean necrosis yellow virus (FBNYV) is: N-ARQVICWCFTLNNPLSPLSLHDSMKYLVYQTEQGEAGNIHFQGYIEMKKRTSLAGMKK LIPGAHFEKRRGTQGEARAYSMKEDTRLEGPWEYGE-C (SEQ ID NO: 14; FBNYV HUH endonuclease – see PDB ID 2HWT_A);

[0088] Porcine circovirus 2 (PCV2): N-PSKKNGRSGPQPHKRWVFTLNNPSEDERKKIRDLPISLFDYFIVGEEGNEEGRTPHLQGF ANFVKKQTFNKVKWYLGARCHIEKAKGTDQQNKEYCSKEGNLLMECGAPRSQGQR-C (SEQ ID NO: 15; PCV2 HUH endonuclease – see PDB ID 2HW0_A);

[0089] Porcine circovirus (PCV): N-SPSKKNGRSGPQPHKRWVFTLNNPSEDERKKIRDLPISLFDYFIVGEEGNEEGRTPHLQG FANFVKKQTFNKVKWYLGARCHIEKAKGTDQQNKEYCSKEGNLLMECGAPRSQGQR- C (SEQ ID NO: 16);

[0090] Duck circovirus (DCV): N-MAKSGNYSYKRWVFTINNPTFEDYVHVLEFCTLDNCKFAIVGEEKGANGTPHLQGFLN LRSNARAAALEESLGGRAWLSRARGSDEDNEEYCAKESTYLRVGEPVSKGRSSDLAEA 22 122903345.1BN00007.2520 BI-11236 TSAVMAGVPLTEVARKFPTTYVIFGRGLERLRHLIVETQRDWKTEVIVLIGPPGTGKSRY AFEFPAENKYYKPRGKWWDGYSGNDVVVMDDFYGWLPYDDLLRITDRYPLRVEFKG GMTQFVAKTLIITSNREPRDWYKSEFDLSALYRRINKYLVYNIDKYEPAQACTLPFPINY- C (SEQ ID NO: 17; DCV HUH endonuclease – see GenBank accession number WRU22358.1);

[0091] An alternate DCV sequence is: N-MAKSGNYSYKRWVFTINNPTFEDYVHVLEFCTLDNCKFAIVGEEKGANGTPHLQGFLN LRSNARAAALEESLGGRAWLSRARGSDEDNEEYCAKESTYLRVGEPVSKGRSSDLAEA TSAV-C (SEQ ID NO: 18);

[0092] Replication protein RepB Streptcoccus agalactiae (RepB): N-MSKDKRSNKWAFLLYQESVPKNYLEVLEELHIPFVLSPWHDKDVNKETGEFKKAHKHG ALFFESLKSYSQVSELLTKHLNTPSHVEVIMSPKGMYDYFIHAENPDKTLYDINDIESGC GFELDKFLTSNNNDKFLSLVIDIIEEQNFTEFNNLVRYARSENPQLLALIMNKTYFFAKYL DSRRYSRRK-C (SEQ ID NO: 19; RepB HUH endonuclease – see GenBank accession number TRM93243.1);

[0093] An alternate sequence for RepB Streptcoccus agalactiae (RepB) is: N-MAKEKARYFTFLLYPESIPSDWELKLETLGVPMAISPLHDKDKSSIKGQKYKKAHYHVL YIAKNPVTADSVRKKIKLLLGEKSLAMVQVVLNVENMYLYLTHESKDAIAKKKHVYDK ADIKLINNFDIDRYVTLDVEEKTELFNVVVSLIRAYTLQNIFDLYDFIDENGETYGLTINL VNEVIAGKTGFMKLLFDGAYQRSKRGTKNEER-C (SEQ ID NO: 20; RepB HUH endonuclease – see PDB ID 3DKY_A);

[0094] An additional alternate sequence for RepB is: N-MAKEKARYFTFLLYPESIPSDWELKLETLGVPMAISPLHDKDKSSIKGQKYKKAHYHVL YIAKNPVTADSVRKKIKLLLGEKSLAMVQVVLNVENMYLYLTHESKDAIAKKKHVYDK ADIKLINNFDIDRY-C (SEQ ID NO: 21);

[0095] Replication protein RepB Fructobacillus tropaeoli (RepBm): N-MTENKNQKGRDWSFILYPESAPKNWREILDETHMRWVESPLHDKDVNPDGEKKKPHW HILLTADGPITYQAVKKIIEPLNAPIPQKVGSARGLVRYFIHMDNPEKYQYSIDEIVGHSG ADVGSYFELTATNRLTVMKDMVQYIYDNDVMNYADFLITCIEKSDDWFSVAINNNTLA INKMIDGVWQLKHKDK-C (SEQ ID NO: 22; RepBm HUH endonuclease – see GenBank accession number GAP05054.1); 23 122903345.1BN00007.2520 BI-11236

[0096] An alternate sequence for RepBm is: N-MSEKKEIVKGRDWTFLVYPESAPENWRTILDETFMRWVESPLHDKDVNADGEIKKPHW HILLSSDGPITQTAVQKIIGPLNAPNAQKVGSAKGLVRYMVHLDNPEKYQYSLDEIVGH NGADVASYFELTA-C (SEQ ID NO: 23);

[0097] Conjugation protein TraI Escherichia coli (TraI): N-MMSIAQVRSAGSAGNYYTDKDNYYVLGSMGERWAGRGAEQLGLQGSVDKDVFTRLL EGRLPDGADLSRMQDGSNRHRPGYDLTFSAPKSVSMMAMLGGDKRLIDAHNQAVDFA VRQVEALASTRVMTDGQSETVLTGNLVMALFNHDTSRDQEPQLHTHAVVANVTQHNG EWKTLSSDKVGKTGFIENVYANQIAFGRLYREKLKEQVEALGYETEVVGKHGMWEMP GVPVEAFSGRSQTIREAVGEDASLKSRDVAALDTRKSKQHVDPEIKMAEWMQTLKETG FDIRAYRDAADQRADLRTLTPGPASQDGPDVQQAVTQAIAGLSER-C (SEQ ID NO: 24; TraI HUH endonuclease – see PDB ID 1P4D_A);

[0098] Mobilization protein A Pseudomonas aeruginosa (mMobA): N-AIYHLTAKTGSRSGGQSARAKADYIQREGKYARDMDEVLHAESGHMPEFVERPADYW DAADLYERANGRLFKEVEFALPVELTLDQQKALASEFAQHLTGAERLPYTLAIHAGGGE NPHCHLMISERINDGIERPAAQWFKRYNGKTPEKGGAQKTEALKPKAWLEQTREAWAD HANRALERAGH-C (SEQ ID NO: 25; mMobA HUH endonuclease – see PDB ID 2NS6_A);

[0099] An alternate sequence for mMobA is: N-MAIYHLTAKTGSRSGGQSARAKADYIQREGKYARDMDEVLHAESGHMPEFVERPADY WDAADLYERANGRLFKEVEFALPVELTLDQQKALASEFAQHLTGAERLPYTLAIHAGG GENPHCHLMISERINDGIERPAAQWFKRYNGKTPEKGGAQKTEALKPKAWLEQTREAW ADHANRALERAGH-C (SEQ ID NO: 26);

[0100] Nicking enzyme Staphylococcus aureus (NES): N-AXYHFQNKFVSKANGQSATAKSAFNSASRIKDFKENEFKDYSNKQCDYSEILLPNNADD KFKDREYLWNKVHDVENRKNSQVAREIIIGLPNEFDPNSNIELAKEFAESLSNEGXIVDL NIHKINEENPHAHLLCTLRGLDKNNEFEPKRKGNDYIRDWNTKEKHNEWRKRWENVQ NKHLEKNGFSVRVSADSYL-C (SEQ ID NO: 27; NES HUH endonuclease – see PDB ID 4HT4_A);

[0101] An alternate NES sequence is: N-MAMYHFQNKFVSKANGQSATAKSAYNSASRIKDFKENEFKDYSNKQCDYSEILLPNNA DDKFKDREYLWNKVHDVENRKNSQVAREIIIGLPNEFDPNSNIELAKEFAESLSNEGMIV 24 122903345.1BN00007.2520 BI-11236 DLNIHKINEENPHAHLLCTLRGLDKNNEFEPKRKGNDYIRDWNTKEKHNEWRKRWENV QNKHLEKNGFSVRVSADSYKNQNIDLEPTKKEGWKARKFEDETG-C (SEQ ID NO: 28);

[0102] Each of the above FBNYV, PCV2 and DCV HUH endonucleases recognizes andcleaves the following ssDNA sequence: 5'-AAGTATT|ACCAGAAA-3' (SEQ ID NO: 8).

[0103] Each of the above RepB and RepBm HUH endonucleases recognizes and cleaves thefollowing ssDNA sequence: 5'-TGCTTCCGTACTACG|ACCCCCCA-3' (SEQ ID NO: 9).

[0104] The above TraI HUH endonuclease recognizes and cleaves the following ssDNAsequence: 5'-TTTGCGTGGGGTGTGGTGCTTT-3' (SEQ ID NO: 10).

[0105] The above mMobA HUH endonucleases each recognizes and cleaves the followingssDNA sequence: 5'-CCAGTTTCTCGAAGAGAAACCGGTAAGTGCA|CCCTCCC-3' (SEQ ID NO: 11).

[0106] The above NES HUH endonucleases each recognize and cleaves the following ssDNAsequence: 5'-ACGCGAACGGAACGTTCGCATAAGTGCG|CCCTTACGGGATTTAAC-3' (SEQ ID NO: 12). HUH-endonuclease domains can be used to covalently link a polypeptide domain capable of binding a target polypeptide and ssDNA. For example, purified HUH proteins form stable covalent bonds in vitro with synthetic oligonucleotides bearing a specific sequence at the origin of replication (ori), and do not require incorporation of modified bases into the DNA to be bound. Accordingly, HUH proteins can be used in a fusion protein with a polypeptide domain capable of binding a target polypeptide without disruption of the polypeptide domain's binding function. By designing ssDNA to contain a HUH-domain target sequence, the ssDNA will bind to the HUH-endonuclease domain of the peptide linker. Domain-Linking Polypeptides of the Peptide Linker (Universal Crosslinking Fusion Protein)

[0107] Certain aspects of the peptide linker of the disclosure feature a domain-linkingpolypeptide sequence that links a HUH endonuclease-containing polypeptide with a domain capable of binding to an antigen-binding polypeptide. Flexible polypeptide sequences are routinely used as such domain-linking polypeptides in fusion proteins known in the art and include, e.g., glycine-rich, serine-rich, and / or proline-rich, etc. sequences. In exemplified embodiments, a peptide linker of the disclosure includes a glycine-serine (GS) repeat sequence that joins the HUH endonuclease domain and the domain capable of binding to an antigen-binding polypeptide of the 25 122903345.1BN00007.2520 BI-11236 peptide linker. Exemplary such sequences include, e.g., N-GSGSGS-C (SEQ ID NO: 1) and N- SGSGSGSGSGS-C (SEQ ID NO: 2). Domains Capable of Binding to an Antigen-Binding Polypeptide

[0108] Exemplified forms of the peptide linkers disclosed herein feature an IgG Fc-blockbinding polypeptide domain as the domain capable of binding to an antigen-binding polypeptide. “An IgG Fc-block binding polypeptide domain” as used herein is meant any part of a protein or polypeptide having a particular function or structure, from any organism that binds to the Fc region of an IgG antibody. Fc ligands include but are not limited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannan binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH), which are a family of Fc receptors that are homologous to the FcγRs (Davis et al., 2002, Immunological Reviews 190: 123-136, entirely incorporated by reference). Fc ligands may include undiscovered molecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gamma receptors. By “Fc ligand” as used herein is meant a molecule, in certain embodiments a polypeptide, from any organism that binds to the Fc region of an antibody.

[0109] In exemplified embodiments, the domain capable of binding to an antigen-bindingpolypeptide is or includes a Protein G B1 domain derived from Streptococcus, e.g., Streptococcus sp. G148. An exemplary Protein G B1 domain derived from Streptococcus has the following N- C amber as : N-26 122903345.1BN00007.2520 BI-11236

[0110] In certain embodiments, the unnatural amino acid (UAA) has a group selected fromamong the following photo-crosslinkable moieties known in the art, e.g., having a phenyl azide, :27 122903345.1BN00007.2520 BI-11236

[0111] In specific, exemplified embodiments, the photo-crosslinkable amino acid is p-benzoyl-L-phenylalanine (pBpa), i.e., having the structure: .

[0112] It is also expressly contemplated that other, variant forms of the Protein G B1 domainsequence can be used in the peptide linkers of the disclosure, and that positioning of the specific site of UAA integration could vary in otherwise functional peptide linkers of the disclosure as contemplated herein.

[0113] For Protein G B1 domain binding and photo-crosslinking to the Fc region of an antigen-binding polypeptide, it is contemplated that when bound to the Fc region of IgG and activated by long wavelength UV light (365nm), a covalent bond is formed between Protein G and IgG. Either one or two Protein Gs can be conjugated onto each Fc region (see Hui et al. Bioconjug Chem. 26(8): 1456–1460). It is further contemplated that a peptide linker of the disclosure may comprise two parts: (i) a histidine-hydrophobic-histidine (HUH) endonuclease domain, which recognizes specific ssDNA sequence, cleaves the ssDNA sequence but then remains covalently linked to a portion of the cleaved sequence; and (ii) a target such as biotin (e.g., streptavidin-HUH fusion), lectins, nanobodies, and antibody subtypes and / or fragments (e.g., Fab, light chain, single domain antibody (Dab), single-chain variable fragment (scFv)).

[0114] The peptide linkers of the disclosure are designed for use with nucleic acids,particularly single-stranded DNAs. Examples of forms of ssDNAs useful for screening methods and other applications include, without limitation, barcode and / or other identifying sequences, as well as functional ssDNAs, such as antisense oligos, as described further below. A. Barcode Sequences

[0115] In certain embodiments, HUH endonuclease-presenting peptide linkers of thedisclosure target and covalently attach to ssDNA sequences that harbor identifying nucleic acid sequences, such as barcode nucleic acids and / or unique molecular identifiers (UMIs). Barcode nucleic acid sequences are short sequences of nucleotides (for example, DNA or RNA) that are 28 122903345.1BN00007.2520 BI-11236 used as an identifier for an associated molecule (e.g., a linked antigen-binding polypeptide), or as an identifier of the source of an associated molecule, such as a cell-of-origin. A barcode may also refer to any unique, non-naturally occurring, nucleic acid sequence that may be used to identify the originating source of a nucleic acid fragment. Barcoding may be performed based on any of the compositions or methods disclosed in patent publication WO 2014047561 A1, Compositions and methods for labeling of agents, incorporated herein in its entirety. In certain embodiments barcoding uses an error correcting scheme (T. K. Moon, Error Correction Coding: Mathematical Methods and Algorithms (Wiley, New York, ed.1, 2005)). Not being bound by a theory, amplified sequences from single cells can be sequenced together and resolved based on the barcode associated with each cell.

[0116] Attaching barcode sequences to nucleic acids is shown in U.S. Patent Application Pub.2008 / 0081330 and International Patent Application Publication WO 2010 / 056728, the content of each of which is incorporated by reference herein in its entirety. Methods for designing sets of barcode sequences and other methods for attaching barcode sequences are shown in U.S. Pat. Nos. 6,138,077; 6,352,828; 5,636,400; 6,172,214; 6235,475; 7,393,665; 7,544,473; 5,846,719; 5,695,934; 5,604,097; 6,150,516; RE39,793; 7,537,897; 6172,218; and 5,863,722, the content of each of which is incorporated by reference herein in its entirety. B. Antisense Oligos

[0117] Antisense methodology takes advantage of the fact that nucleic acids tend to pair with“complementary” sequences. By complementary, it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.

[0118] Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helixformation; targeting RNA will lead to double-helix formation. Antisense polynucleotides, when introduced into a target cell, specifically bind to their target polynucleotide and interfere with 29 122903345.1BN00007.2520 BI-11236 transcription, RNA processing, transport, translation and / or stability. Antisense RNA constructs, or DNA encoding such antisense RNA's, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host-animal, including a human subject.

[0119] Antisense oligos may be designed to bind to the promoter and other control regions,exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense oligos will include regions complementary to intron / exon splice junctions. Thus, it is proposed that certain embodiments include an antisense oligo with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.

[0120] As stated above, “complementary” or “antisense” means polynucleotide sequences thatare substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.

[0121] It may be advantageous to combine portions of genomic DNA with cDNA or syntheticsequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence. 30 122903345.1BN00007.2520 BI-11236

[0122] Particular oncogenes that are targets for antisense constructs are ras, myc, neu, raf, erb,src, fms, jun, trk, ret, hst, gsp, bcl-2 and abl. Also contemplated to be useful will be anti-apoptotic genes and angiogenesis promoters. 3. Purification of Crosslinked Molecules

[0123] The present disclosure also provides techniques for purifying antigen-bindingpolypeptide -ssDNA conjugates following cross-linking with the peptide linker. The term “purified antigen-binding polypeptide-ssDNA conjugates” as used herein, is intended to refer to a composition, isolatable from biologic sources, wherein the crosslinked antigen-binding polypeptide -ssDNA conjugates is purified to any degree relative to its naturally-obtainable state, i.e., relative to its purity within a cellular extract.

[0124] Generally, “purified” will refer to an antigen-binding polypeptide-ssDNA conjugatecomposition that has been subjected to fractionation to remove various non-crosslinked compounds, polypeptides, or peptides. Where the term “substantially purified” is used, this will refer to a composition in which the crosslinked compound, polypeptide, or peptide forms the major component of the composition, such as constituting about 50% of the proteins in the composition or more. In certain embodiments, a substantially purified compound, polypeptide, or peptide will constitute more than 60%, 70%, 80%, 90%, 95%, 99% or even more of the compounds, polypeptides, or peptides in the composition.

[0125] An antibody-ssDNA conjugate that is “purified to homogeneity,” as applied to thepresent disclosure, means that the antibody-ssDNA conjugate has a level of purity where the antibody-ssDNA conjugate is substantially free from other proteins and biological components. For example, a purified antibody-ssDNA conjugate will often be sufficiently free of other components so that it can be identified by various methodologies.

[0126] Various methods for quantifying the degree of purification of crosslinked antibody-ssDNA conjugates will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific amount of a crosslinked protein in a fraction, or assessing the number of polypeptides within a fraction by gel electrophoresis. Assessing the number of polypeptides within a fraction by SDS / PAGE analysis will often be preferred in the 31 122903345.1BN00007.2520 BI-11236 context of the present disclosure as this is straightforward and techniques are known to one of ordinary skill in the art.

[0127] To purify a crosslinked antigen-binding polypeptide-ssDNA conjugate, a natural orrecombinant composition comprising at least some crosslinked proteins, polypeptides, or peptides will be subjected to fractionation to remove various non-crosslinked components from the composition. In addition to those techniques described in detail herein below, various other techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxyapatite, lectin affinity and other affinity chromatography steps; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. Another example is the purification of a crosslinked protein using a specific binding partner. Such purification methods are routine in the art.

[0128] Although preferred for use in certain embodiments, there is no general requirement thatthe crosslinked antigen-binding polypeptide-ssDNA conjugates always be provided in their most purified state. Indeed, it is contemplated that less substantially purified antigen-binding polypeptide-ssDNA conjugates, which are nonetheless enriched in crosslinked antigen-binding polypeptide-ssDNA conjugate compositions, relative to the natural state, will have utility in certain embodiments. These include, for example, antibody generation where subsequent screening assays using purified crosslinked antigen-binding polypeptide-ssDNA conjugates are conducted.

[0129] The disclosed peptide linkers are in certain embodiments purified by reliance on non-covalent interactions for purification. The peptide linker is expressed as a recombinant protein which includes a component that binds to an affinity binding partner (e.g., intein-CBD is included as an affinity tag in certain exemplified embodiments). In some embodiments, the affinity purification system utilizes non-covalent binding. In embodiments, binding of a ligand to its binding partner can occur by intermolecular forces, such as ionic bonds, hydrogen bonds, hydrophobic interactions and Van der Waals forces. Thus, affinity interactions as used herein refers to the combination of non-covalent interactions between a ligand and its binding partner to form a complex. Exemplary affinity tags include the polyhistidine affinity tag, also known as the His-tag or His6, usually consists of six consecutive histidine residues, but can vary in length from two to ten histidine residues; FLAG tags; haemagglutinin (HA); MYC tag; Strep-tag, glutathione 32 122903345.1BN00007.2520 BI-11236 S-transferase (GST); Maltose binding protein (MBP), calmodulin binding peptide (CBP); the intein-chitin binding domain (intein-CBD), the streptavidin tag, etc. Methods of using purification tags to facilitate protein purification are known in the art and include, for example, a chromatography step wherein the tag reversibly binds to a chromatography resin.

[0130] The intein-chitin binding domain (intein-CBD) tag is a combination of a protein self-splicing element (intein) with a chitin-binding domain and allows for the purification of a native recombinant protein without need for a protease. The intein self-cleavage reaction is induced by overnight incubation with 50 mM DTT at 4 °C. 2-mercaptoethanol, cysteine, or hydroxylamine may also be used but are less effective.

[0131] Any of a wide variety of chromatographic procedures may be employed to purifycrosslinked antigen-binding polypeptide-ssDNA conjugates. For example, thin layer chromatography, gas chromatography, high performance liquid chromatography, paper chromatography, affinity chromatography or supercritical flow chromatography may be employed.

[0132] Partition chromatography is based on the theory that if two phases are in contact withone another, and if one or both phases constitute a solute, the solute will distribute itself between the two phases. Usually, partition chromatography employs a column which is filled with a sorbent and a solvent. The solution containing the solute is layered on top of the column. The solvent is then passed through the column, continuously, which permits movement of the solute through the column material. The solute can then be collected based on its movement rate. The two most common types of partition chromatograph are paper chromatograph and thin-layer chromatograph (TLC); together these are called adsorption chromatography. In both cases, the matrix contains a bound liquid. Other examples of partition chromatography are gas-liquid and gel chromatography.

[0133] Paper chromatography is a variant of partition chromatography that is performed oncellulose columns in the form of a paper sheet. Cellulose contains a large amount of bound water even when extensively dried. Partitioning occurs between the bound water and the developing solvent. Frequently, the solvent used is water. Usually, very small volumes of the solution mixture to be separated is placed at top of the paper and allowed to dry. Capillary action draws the solvent through the paper, dissolves the sample, and moves the components in the direction of flow. Paper chromatograms may be developed for either ascending or descending solvent flow. Two dimensional separations are permitted by changing the axis of migration 90° after the first run. 33 122903345.1BN00007.2520 BI-11236

[0134] Thin layer chromatography (TLC) has the advantages of paper chromatography, butallows the use of any substance that can be finely divided and formed into a uniform layer. In TLC, the stationary phase is a layer of sorbent spread uniformly over the surface of a glass or plastic plate. The plates are usually made by forming a slurry of sorbent that is poured onto the surface of the gel after creating a well by placing tape at a selected height along the perimeter of the plate. After the sorbent dries, the tape is removed and the plate is treated just as paper in paper chromatography. The sample is applied and the plate is contacted with a solvent. Once the solvent has almost reached the end of the plate, the plate is removed and dried. Spots can then be identified by fluorescence, immunologic identification, counting of radioactivity, or by spraying varying reagents onto the surface to produce a color change.

[0135] In Gas-Liquid chromatography (GLC), the mobile phase is a gas and the stationaryphase is a liquid adsorbed either to the inner surface of a tube or column or to a solid support. The liquid usually is applied as a solid dissolved in a volatile solvent such as ether. The sample, which may be any sample that can be volatized, is introduced as a liquid with an inert gas, such as helium, argon or nitrogen, and then heated. This gaseous mixture passes through the tubing. The vaporized compounds continually redistribute themselves between the gaseous mobile phase and the liquid stationary phase, according to their partition coefficients.

[0136] The advantage of GLC is in the separation of small molecules. Sensitivity and speedare quite good, with speeds that approach 1000 times that of standard liquid chromatography. By using a non-destructive detector, GLC can be used preparatively to purify grams quantities of material. The principal use of GLC has been in the separation of alcohols, esters, fatty acids and amines.

[0137] Gel chromatography, or molecular sieve chromatography, is a special type of partitionchromatography that is based on molecular size. The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all 34 122903345.1BN00007.2520 BI-11236 other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsorption, less zone spreading and the elution volume is related in a simple matter to molecular weight.

[0138] The gel material for gel chromatography is a three-dimensional network whosestructure is usually random. The gels consist of cross-linked polymers that are generally inert, do not bind or react with the material being analyzed, and are uncharged. The space filled within the gel is filled with liquid and this liquid occupies most of the gel volume. Common gels are dextran, agarose and polyacrylamide; they are used for aqueous solution.

[0139] High Performance Liquid Chromatography (HPLC) is characterized by a very rapidseparation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain and adequate flow rate. Separation can be accomplished in a matter of minutes, or a most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.

[0140] Affinity Chromatography is a chromatographic procedure that relies on the specificaffinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).

[0141] The matrix should be a substance that itself does not adsorb molecules to anysignificant extent and that has a broad range of chemical, physical and thermal stability. The ligand should be coupled in such a way as to not affect its binding properties. The ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand. One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present disclosure are well known to those skilled in the art. 4. Detection Methods 35 122903345.1BN00007.2520 BI-11236

[0142] In still further embodiments, the present disclosure concerns methods for binding,purifying, removing, quantifying or otherwise generally detecting biological components. The antigen-binding polypeptide-ssDNA conjugates prepared in accordance with the present disclosure may be employed to detect target polypeptides or peptides. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Nakamura et al. (1987), incorporated herein by reference.

[0143] It is contemplated that these various immunodetection methods can be modified tomake use of the antigen-binding polypeptide-ssDNA conjugates as a substitute for the primary antibodies that are generally used. Immunoassays based on immunodetection, in their most simple and direct sense, are binding assays. Certain immunoassays that can be modified to use the antigen- binding polypeptide-ssDNA conjugates are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) as well as immunohistochemical detection using tissue sections known in the art. In addition, other detection techniques, such as western blotting, dot blotting, FACS analyses, and the like can also be modified to use peptide- or small molecule- metal ligand conjugates.

[0144] These methods include methods for purifying antigen-binding polypeptide-ssDNAconjugates. In these instances, the antigen-binding polypeptide-ssDNA conjugates removes the target polypeptide or peptide component from a sample. The antigen-binding polypeptide-ssDNA conjugates will in certain embodiments be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the target polypeptide component will be applied to the immobilized antigen-binding polypeptide-ssDNA conjugates. The unwanted components will be washed from the column, leaving the target complexed to the immobilized column, which target is then collected by removal from the column.

[0145] The binding methods also include methods for detecting or quantifying the amount ofa target polypeptide reactive component in a sample, which methods require the detection or quantification of any complexes formed during the binding process. Here, one would obtain a sample suspected of containing a target polypeptide or peptide, and contact the sample with an antigen-binding polypeptide-ssDNA conjugate capable of binding the target, and then detect or quantify the amount of complexes formed under the specific conditions.

[0146] Contacting the chosen biological sample with the antigen-binding polypeptide-ssDNAconjugates under conditions effective and for a period of time sufficient to allow the formation of 36 122903345.1BN00007.2520 BI-11236 complexes is generally a matter of simply adding the antigen-binding polypeptide-ssDNA conjugates to the sample and incubating the mixture for a period of time long enough for the peptide- or small molecule-metal ligand conjugates to form complexes with, i.e., to bind to, any target protein, peptide or polypeptide present. After this time, the sample, such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non-specifically bound species, allowing only those antigen-binding polypeptide-ssDNA conjugates -specifically bound within the complexes to be detected.

[0147] In general, the detection of complex formation is well known in the art and may beachieved through the application of numerous approaches analogous to immunodetection methods. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological or enzymatic tags. U.S. Pat. Nos. concerning the use of such labels include 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated herein by reference. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antigen-binding polypeptide or a biotin / avidin ligand binding arrangement, as is known in the art.

[0148] The antigen-binding polypeptide-ssDNA conjugates employed in the detection mayitself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary complexes in the composition to be determined. Alternatively, the first antibody-ssDNA conjugates that becomes bound within the primary complexes may be detected by means of a second binding ligand that has binding affinity for the antibody-ssDNA conjugates. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand may be an antibody. The primary complexes are contacted with the labeled, secondary binding ligand, or antigen-binding polypeptide, under conditions effective and for a period of time sufficient to allow the formation of secondary complexes. The secondary complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary complexes is then detected.

[0149] Further methods include the detection of primary complexes by a two step approach.A second binding ligand, such as an antigen-binding polypeptide, that has binding affinity for the antigen-binding polypeptide-ssDNA conjugates is used to form secondary complexes, as described above. After washing, the secondary complexes are contacted with a third binding ligand or antigen-binding polypeptide that has binding affinity for the antigen-binding polypeptide, again 37 122903345.1BN00007.2520 BI-11236 under conditions effective and for a period of time sufficient to allow the formation of immune complexes. The third ligand or antigen-binding polypeptide is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired. 5. Detection Kits

[0150] In still further embodiments, the present disclosure concerns detection kits for use withthe detection methods described above. As the antigen-binding polypeptide-ssDNA conjugates are generally used to detect target polypeptides or peptides, the antigen-binding polypeptide-ssDNA conjugates will in certain embodiments be included in the kit. However, kits including both such components may be provided. The detection kits will thus comprise, in suitable container means, a first antigen-binding polypeptide-ssDNA conjugate that binds to a target polypeptide or peptide, and optionally, an immunodetection reagent and further optionally, a target polypeptide or peptide.

[0151] In certain embodiments, the antigen-binding polypeptide-ssDNA conjugate that bindsto the target protein, polypeptide or peptide may be pre-bound to a solid support, such as a column matrix or well of a microtitre plate.

[0152] The detection reagents of the kit may take any one of a variety of forms, including thosedetectable labels that are associated with or linked to the given antigen-binding polypeptide- ssDNA conjugates. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antigen-binding polypeptides that have binding affinity for the first antigen-binding polypeptide-ssDNA conjugates.

[0153] Further suitable detection reagents for use in the present kits include the two-component reagent that comprises a secondary antibody that has binding affinity for the antigen- binding polypeptide-ssDNA conjugates, along with a second antigen-binding polypeptide that has binding affinity for the first antigen-binding polypeptide, the second antigen-binding polypeptide being linked to a detectable label. As noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present disclosure.

[0154] The kits may further comprise a suitably aliquoted composition of the target peptide orpolypeptide, whether labeled or unlabeled, as may be used to prepare a standard curve for a 38 122903345.1BN00007.2520 BI-11236 detection assay. The kits may contain antigen-binding polypeptide-ssDNA conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media or in lyophilized form.

[0155] The container means of the kits will generally include at least one vial, test tube, flask,bottle, syringe or other container means, into which the antigen-binding polypeptide-ssDNA conjugates may be placed, and in certain embodiments, suitably aliquoted. Where target polypeptide or peptide, or a second or third binding ligand or additional component is provided, the kit also will generally contain a second, third or other additional container into which this ligand or component may be placed. The kits of the present disclosure also will typically include a means for containing the antigen-binding polypeptide-ssDNA conjugates, target, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. 6. Construction of Expression Plasmids

[0156] Where the disclosure involves production of expression constructs, exemplaryregulatory regions that can be included include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5′ and 3′ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns. Exemplary promoter sequences that can be used in the expression constructs of the present disclosure include, e.g., promoter sequences capable of driving gene expression in eukaryotic and / or prokaryotic cells. Specifically, constructs can include eukaryotic and prokaryotic promoters. An expression construct can include multiple copies of a single promoter or different promoters, e.g., two or more promoters.

[0157] The practice of the present disclosure employs, unless otherwise indicated,conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory 39 122903345.1BN00007.2520 BI-11236 Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Ausubel et al., 1992), Current Protocols in Molecular Biology (John Wiley & Sons, including periodic updates); Glover, 1985, DNA Cloning (IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds.1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I- IV (D. M. Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ. of Oregon Press, Eugene, 2000).

[0158] Unless otherwise defined, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0159] Reference will now be made in detail to exemplary embodiments of the disclosure.While the disclosure will be described in conjunction with the exemplary embodiments, it will be understood that it is not intended to limit the disclosure to those embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the 40 122903345.1BN00007.2520 BI-11236 spirit and scope of the disclosure as defined by the appended claims. Standard techniques well known in the art or the techniques specifically described below were utilized. EXAMPLES Example 1: Antibody-Oligonucleotide Conjugates

[0160] Given the combined features of a vast variety of nucleotide sequences being readily(and cheaply) available as compared to other types of tagging molecules and the still increasing capacity of nucleic acid sequencing, antibody-oligonucleotide ("antibody-oligo") conjugates are one of the most useful tools for performing highly multiplexed assays. Antibody-oligo conjugates are fundamental and necessary components for multiple biotechniques, including but not limited to Olink, Duolink, CODEX, and CITE-seq. The quality of the conjugates directly determines the outcome of those techniques, while hundreds of or even up to tens of thousands of antibodies are generated for those assays, which means that a vast and ever-increasing demand for antibody-oligo conjugates exists. However, in considering how best to acquire such conjugates, conventional antibody conjugation methods (such as those shown in FIG. 1) have the following drawbacks. They require chemically modified oligonucleotides (e.g., Thiol-Maleimide / Amine-NHS / Azide- DBCO), which are of high cost and low yield, as compared with plain oligos. For conjugation purposes, antibodies need to be pre-purified to avoid inclusion of Bovine serum albumin (BSA) and azide in antibody preparations, which adds additional cost to obtaining usable antibodies. Some conjugation methods need antibodies to be chemically modified first, which may negatively impact an antibody's stability and / or affinity. Non-specific conjugation methods currently available in the art have less consistency, and additionally, such methods are not specific to the site of conjugation, which introduces an additional issue that could potentially impact an antibody's affinity post-conjugation. For highly multiplexed assays, if multiple antibodies are used together, non-covalent conjugation methods may lead to exchange of components between antibodies, which is bad for data quality and quality control (QC). Example 2: Design of a Universal Covalent Crosslinker (Peptide Linker) for Antibody-Oligo Conjugation

[0161] A low-cost, universal covalent crosslinker (peptide linker) for high-throughput, costeffective production of antibody-oligo conjugates was designed, that also requires minimal purification, performs covalent conjugation, is site-specific, and is cheap and easy to make. Certain 41 122903345.1BN00007.2520 BI-11236 peptide linkers described herein consist of two parts: (i) a HUH endonuclease domain, which recognizes a specific ssDNA sequence, cleaves the ss DNA sequence at a specific recognition sequence but then remains covalently linked to the remaining sequence; and (ii) an IgG Fc-block binding motif harboring a photo-crosslinkable unnatural amino acid, which binds the Fc block (Fc domain) of IgG, then can be photo-crosslinked to the Fc domain to form a covalent linkage. The IgG Fc-block binding motif can specifically bind with the Fc block of the IgG heavy chain, which avoids: (i) the need for antibodies to be pre-purified to eliminate Bovine serum albumin (BSA) and azide contamination; (ii) the need for antibodies to be chemically modified first; and (iii) the other negative qualities of currently available non-specific conjugation methods, mentioned above. The photo crosslinkable property provides advantage over non-covalent conjugation methods that may lead to exchange of components between antibodies, which is bad for data quality and QC.

[0162] A specifically exemplified precursor form of a peptide linker of the disclosure has thestructure N-Met-Protein G B1 domain-GS polypeptide linker-HUH endonuclease-proline (or other cleavable spacer sequence)-Mxe intein-chitin-binding domain (CBD)-C (FIG. 2), with Mxe intein-CBD sequences used for an initial purification of expressed precursor forms of the peptide linker, and such Mxe-intein-CBD sequences are subsequently cleaved, to liberate a fully active peptide linker of the disclosure having a first domain capable of binding the Fc domain of an antigen-binding moiety (e.g., an IgG antibody) harboring a photo-crosslinkable unnatural amino acid, a polypeptide joining sequence (e.g., a GS-rich joining sequence) and a second domain harboring a HUH endonuclease capable of cleaving and covalently attaching a ssDNA in a sequence-specific manner.

[0163] Polypeptides harboring a HUH endonuclease domain can be employed to createpolypeptide-ssDNA fusions, as disclosed herein and as previously described in the art, which avoids reliance upon chemically modified oligos for linking ssDNAs to polypeptides. HUH endonucleases have been previously described to break and covalently link to specific ssDNA sequences, with high efficiency (15 minutes, 37 degrees °C) (FIGs.3A-3C).

[0164] Exemplary 5’-modified oligos obtained from Integrated DNA Technologies (IDT) cost$300 and yielded about 5 nmol of product, as compared to plain (unmodified) oligos having anadditional 15 bp of sequence, which were obtained from IDT at a cost of $10-20 per oligo and atyields of 50-80 nmol of product. 42 122903345.1BN00007.2520 BI-11236

[0165] Plain (unmodified) oligos could be used for conjugation with the universal crosslinkerof the disclosure (peptide linker) and, as noted above, harbor an additional 15 or more base pairs at the 5’ end of the prospective oligos. For each modified oligo that costs easily hundreds of dollars (about $150 for each modification), the equivalent plain oligo generally has much higher yield and does not require additional purification (about $70 for each oligo). The conjugation protocol employed for the peptide linkers of the disclosure is also faster than most oligonucleotide chemical conjugation protocols commonly used in the art and is highly efficient. The instant method also offers the potential for further multiplexing, given the available variation of known HUH endonucleases (see, e.g., Lovendahl, K. N., et al. JACS, 2017). Example 3: Expression and Purification of the Universal Crosslinker (Peptide Linker)

[0166] The universal crosslinker (peptide linker) of the above example was expressed andpurified using an NEBTMIMPACT kit (FIG.4). Fusion protein construction employed the pTXB1 system, it was demonstrated that the fusion protein could be separated from cell lysates and then cleaved from the Mxe intein-CBD component, thereby eluting a Protein G B1 domain-G5 linker- HUH-containing peptide linker of 18.55 kDa. This initial construct after on-column cleavage still possessed a large portion of uncleaved precursor bound to the post-DTT cleavage column resin (FIG. 4, lane 14), which prompted subsequent attempts to further optimize constructs for better cleavage and higher yield. Example 4: Universal Crosslinker (Peptide Linker) Function

[0167] Peptide linkers of the disclosure were next tested for conjugation activity. Gel shiftassays were performed upon various combinations of peptide linker, and results revealed that directed covalent binding events between peptide linker and target ssDNA, as well as between peptide linker and antibody, and between all three of target ssDNA, peptide linker, and Fc- containing antibody occurred (FIG. 5). The peptide linker of the disclosure was therefore identified as functional, and the reduction in and even absence of the original 18.55 kDa form in certain lanes exposed to targeted ssDNAs demonstrated the successful and highly efficient conjugation between target ssDNA oligos and the universal crosslinker (peptide linker). Covalent conjugation of the peptide linker to Abs was observed to be successful and only happened when UV light was used for crosslinking. 43 122903345.1BN00007.2520 BI-11236

[0168] The conjugation between the HUH domain and ssDNA was first performed (refer toprotocol), then the resulting mixture was combined with IgG antibody and incubated with UV light. Conjugation products were obtained, with both components (IgG and ssDNA, respectively) conjugated to the peptide linker, as designed. Such assays therefore demonstrated that both domains of the peptide linker were functional for promoting covalent binding to target (ssDNA or Fc region).

[0169] Further testing of the peptide linker, including a comparison with the commerciallyavailable OYO-link product, was performed (FIG. 6). A conjugate was made using the commercially available OYO-link mouse IgG1 kit, and formation of a conjugate was visualized as the appearance of an observed band shift, yet this commercial kit still exhibited a substantial portion of unconjugated heavy chain antibody. In comparison, conjugates were made using the peptide linker of the disclosure, at different ratios of IgG to peptide linker. As the binding between the Fc block and protein G achieved an equilibrium, increased amounts of the peptide linker were shown to push this equilibrium towards the conjugated product side, thereby increasing the efficiency of conjugation (FIG.6).

[0170] Screening of peptide linker binding of multiple kinds of antibodies from differentisotypes and hosts was next attempted. While the Fc-block binding motif of the peptide linker(s) disclosed herein should be able to be further optimized to increase the coverage of the peptide linker in conjugating different antibody isotypes and hosts, the current peptide linker was demonstrated to work with multiple IgG isotypes or hosts (FIG.7). A modified Fc-block binding motif may be of interest for better isotype coverage. It was again observed that equilibriums could be further pushed towards conjugate formation by increasing the ratio of peptide linker to respective immunoglobulins.

[0171] An updated version of the peptide linker of the disclosure having an affinity tag moietyof a Mxe intein and chitin-binding domain (CBD) with three C-terminal alanine residues was next assessed, and results showed improved levels of cleavage for this updated format (FIG.8). Two alanine and three alanine formats of the PG-Huh-intein-CBD fusion protein showed higher levels of processed PG-HUH protein following cleavage to remove the Mxe intein-CBD domain than was used for peptide linker purification, as compared to the initially tested single alanine form (FIG.8; 50-60 mg of product was obtained from 1 liter, for a 50x yield improvement). 44 122903345.1BN00007.2520 BI-11236

[0172] Further optimization was performed by titration of the peptide linker-antibody ratio,using the updated triple alanine format of the peptide linker (FIG. 9). Significantly improved yields of peptide linker-antibody conjugates were observed, as demonstrated by gel-shift assays.

[0173] Attempted purifications of peptide linker-containing conjugates that varied across arange of covalently linked ssDNA and covalently bound Abs, for the goal of multiplexed uses, e.g., CODEX, were performed and results were assessed. Both magnetic bead-based purification of peptide linker-containing conjugates and Amicon 50 KDa ultracentrifugation filters were successful (magnetic beads with Fc blocks could bind and remove excessive linker, while the Amicon filters were effective in removing smaller, non-conjugated contaminants) (FIG.10).

[0174] Antibody-peptide linker-ssDNA conjugates were also validated, for a first conjugatethat contained rabbit polyclonal anti-Lamin B1 antibody-peptide linker-ssDNA #15, and for a second conjugate that contained mouse monoclonal anti-Vimentin antibody-peptide linker-ssDNA #57 (FIG. 11). Detection by FISH and by imaging of dye markers associated with indicated components of the respective conjugates revealed functional Ab-peptide linker-ssDNA conjugates for both formats tested in HeLa cells (FIG.11).

[0175] The peptide linker of the disclosure was also confirmed to work in the presence of BSAconcentrations up to 5 mg / mL. Exemplary assessments of peptide linker-antibody conjugations (for anti-FoxP3 and anti-CD68 antibodies) were unaffected by the presence of BSA (FIG.12). Example 5: Universal Crosslinker Protocol

[0176] Reagents:5 M Sodium chloride solution (Sigma, cat. S5150) 1 M Tris·HCl buffer (pH = 7.5; Invitrogen, cat.15567-027) 1 M MgCl2solution (Invitrogen, cat. AM9530G) 1 M M Cl l ti Th t 63150AD45 122903345.1BN00007.2520 BI-11236 Water bath or dry incubator

[0178] Buffer setup:2x reaction buffer: 100 mM Tris·HCl (pH = 7.5), 300 mM NaCl, 2 mM MgCl2, 2 mM MnCl2

[0179] Procedure:Conjugation between universal crosslinker and ssDNA oligo, Timing 45 min

[0180] The ssDNA oligo was diluted with a sequence that can be recognized by HUHendonuclease to 100 μM in NF water (for example, for HUH endonucleases PCV2, DCV, and at its 5mLComponent Stock conc. Final conc. VolumeUniversal crosslinker 40 μM 10.0 μM 5.0 μL ssDNA 100 μM 25.0 μM (2.5x) 5.0 μL 2x reaction buffer 2x 1x 10.0 μL NF water Total 20.0 μL 1. The components were mixed and incubated at 37 ℃ for 30 min.2. The product was rested on ice for the following conjugation steps, and some of the productwas saved for later SDS-PAGE gel validation.

[0182] Conjugation between ssDNA-universal crosslinker and antibody, Timing 2.5hours 1. The reaction was assembled according to the following with antibody and product fromthe last step: 46 122903345.1BN00007.2520 BI-11236 Component Stock Final conc. Volume (8x)Product from last step 10.0 μM (protein) 6.4 μM 12.8 μL Antibody 0.5 mg / mL (~3.3 μM) 0.8 μM 4.8 μL (2.4 μg) Total 20.0 μL 2. The mixture was immediately placed on an ice bath and irradiated for 2 hours with 365 nmUV light using an OYO-link UV crosslinker. 3. Some of the product was saved for reduced SDS-PAGE gel validation.4. Optionally, purification was done to remove excessive ssDNA-universal crosslinker:a. Magnetic beads with IgG Fc domain were used to bind and remove the excessiveunconjugated ssDNA-universal crosslinker. b. Amicon ultra filter of 50 kDa Molecular Weight Cut-off (MWCO) was used toremove the excessive unconjugated ssDNA-universal crosslinker. c. For batch applications, products like 96-well MWCO filter plates were tested forconjugation then purification in batch. REFERENCES

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[0241] Szarowski, D.H., Andersen, M.D., Retterer, S., Spence, A.J., Isaacson, M., Craighead,H.G., Turner, J.N., and Shain, W. (2003). Brain responses to micro-machined silicon devices. Brain Res 983, 23-35.

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[0251] Zhao, Y.J., Yu, T.T., Zhang, C., Li, Z., Luo, Q.M., Xu, T.H., and Zhu, D. (2018). Skulloptical clearing window for in vivo imaging of the mouse cortex at synaptic resolution. Light-Sci Appl 7. 53 122903345.1BN00007.2520 BI-11236

[0252] All patents and publications mentioned in the specification are indicative of the levelsof skill of those skilled in the art to which the disclosure pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

[0253] One skilled in the art would readily appreciate that the present disclosure is welladapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the disclosure. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the disclosure, are defined by the scope of the claims.

[0254] In addition, where features or aspects of the disclosure are described in terms ofMarkush groups or other grouping of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

[0255] All methods described herein can be performed in any suitable order unless otherwiseindicated 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 disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0256] Embodiments of this disclosure are described herein, including the best mode knownto the inventors for carrying out the embodiments presented in the disclosure. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description.

[0257] The disclosure illustratively described herein suitably can be practiced in the absenceof any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of", and "consisting of" may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications 54 122903345.1BN00007.2520 BI-11236 are possible within the scope of the embodiments presented in the disclosure. Thus, it should be understood that although the present disclosure provides preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the description and the appended claims.

[0258] It will be readily apparent to one skilled in the art that varying substitutions andmodifications can be made to the embodiments presented in the disclosure herein without departing from the scope and spirit of the presented embodiments in the disclosure. Thus, such additional embodiments are within the scope of the present disclosure and the following claims. The present disclosure teaches one skilled in the art to test various combinations and / or substitutions of chemical modifications described herein toward generating conjugates possessing improved contrast, diagnostic and / or imaging activity. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying conjugates possessing improved contrast, diagnostic and / or imaging activity.

[0259] The inventors expect skilled artisans to employ such variations as appropriate, and theinventors intend for the disclosure to be practiced otherwise than as specifically described herein. Accordingly, this disclosure 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 disclosure unless otherwise indicated herein or otherwise clearly contradicted by context. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.

[0260] Features described above as well as those claimed below may be combined in variousways without departing from the scope thereof. The following examples illustrate some possible, non-limiting combinations:

[0261] (A1) A peptide linker for joining a target polypeptide and a target oligonucleotide, thepeptide linker including: a first polypeptide domain capable of binding the target polypeptide; a second polypeptide domain including a linking polypeptide sequence; and a third polypeptide 55 122903345.1BN00007.2520 BI-11236 domain including a histidine-hydrophobic-histidine (HUH) endonuclease, wherein the third polypeptide domain is capable of binding the target oligonucleotide.

[0262] (A2) For the peptide linker denoted as (A1), the target polypeptide is an antigen-binding polypeptide.

[0263] (A3) For the peptide linker denoted as any one of (A1) through (A2), the target antigen-binding polypeptide is an antibody having a fragment crystallizable region (Fc region).

[0264] (A4) For the peptide linker denoted as (A3), the antibody having the Fc region is anIgG isotype antibody.

[0265] (A5) For the peptide linker denoted as any one of (A1) through (A4), the firstpolypeptide domain is capable of binding the Fc region of the antibody.

[0266] (A6) For the peptide linker denoted as any one of (A1) through (A5), the firstpolypeptide domain includes a crosslinkable moiety.

[0267] (A7) For the peptide linker denoted as (A6), the crosslinkable moiety is photo-crosslinkable.

[0268] (A8) For the peptide linker denoted as (A6), the crosslinkable moiety includes a photo-crosslinking unnatural amino acid (UAA).

[0269] (A9) For the peptide linker denoted as (A8), the photo-crosslinking UAA is p-benzoyl-L-phenylalanine (pBpa).

[0270] (A10) For the peptide linker denoted as any one of (A1) through (A9), the crosslinkablemoiety covalently binds the first polypeptide domain and the target polypeptide.

[0271] (A11) For the peptide linker denoted as any one of (A1) through (A10), the firstpolypeptide domain includes an immunoglobulin-binding domain of streptococcal protein G. Optionally, the first polypeptide domain includes a B1 domain of streptococcal protein G.

[0272] (A12) For the peptide linker denoted as any one of (A1) through (A11), the linkingpolypeptide sequence of the second polypeptide domain includes a glycine-serine (GS) repeat sequence. Optionally, the linking polypeptide sequence of the second polypeptide domain is N- GSGSGS-C (SEQ ID NO: 1). Optionally, the linking polypeptide sequence of the second polypeptide domain is N-SGSGSGSGSGS-C (SEQ ID NO: 2).

[0273] (A13) For the peptide linker denoted as any one of (A1) through (A12), the thirdpolypeptide domain includes a histidine-hydrophobic-histidine (HUH) endonuclease having a sequence at least 90% identical to the sequence N- 56 122903345.1BN00007.2520 BI-11236 MARQVICWCFTLNNPLSPLSLHDSMKYLVYQTEQGEAGNIHFQGYIEMKKRTSLAGMK KLIPGAHFEKRRGTQGEARAYSMKEDTRLEGPWEYGEFVP-C (SEQ ID NO: 3).

[0274] (A14) For the peptide linker denoted as any one of (A1) through (A13), the peptidelinker further includes an affinity tag moiety.

[0275] (A15) For the peptide linker denoted as any one of (A1) through (A14), the affinity tagmoiety is a Mxe intein and a chitin-binding domain (CBD).

[0276] (A16) For the peptide linker denoted as any one of (A1) through (A15), the peptidelinker having the Mxe intein and the chitin-binding domain (CBD) further includes an alanine residue at the C-terminus of the HUH endonuclease domain, which can act to promote cleavage and removal of the Mxe intein and the chitin-binding domain (CBD) once affinity purification has been performed.

[0277] (A17) For the peptide linker denoted as any one of (A1) through (A16), one or morealanine residues are located at the C-terminus of the HUH endonuclease domain of the peptide linker.

[0278] (A18) For the peptide linker denoted as any one of (A1) through (A17), two or morealanine residues are located at the C-terminus of the HUH endonuclease domain of the peptide linker.

[0279] (A19) For the peptide linker denoted as any one of (A1) through (A18), three or morealanine residues are located at the C-terminus of the HUH endonuclease domain of the peptide linker.

[0280] (A20) For the peptide linker denoted as any one of (A1) through (A19), three alanineresidues are located at the C-terminus of the HUH endonuclease domain of the peptide linker, and promote cleavage and removal of the affinity tag domain (in exemplified embodiments, the Mxe intein and chitin-binding domain (CBD)).

[0281] (A21) For the peptide linker denoted as any one of (A1) through (A20), the targetoligonucleotide is a single-stranded DNA (ssDNA).

[0282] (A22) For the peptide linker denoted as any one of (A1) through (A21), the peptidelinker has a sequence at least 95% identical to N- MTFKLIINGKTLKGEITIEAVDA[UAA]EAEKIFKQYANDYGIDGEWTYDDATKTFTVTES GSGSGSGSGSMARQVICWCFTLNNPLSPLSLHDSMKYLVYQTEQGEAGNIHFQGYIEMK 57 122903345.1BN00007.2520 BI-11236 KRTSLAGMKKLIPGAHFEKRRGTQGEARAYSMKEDTRLEGPWEYGEFVP-C (SEQ ID NO: 4), wherein [UAA] is an unnatural amino acid,

[0283] (A23) For the peptide linker denoted as any one of (A1) or (A22), the [UAA] is pBpa.

[0284] (A24) For the peptide linker denoted as (A15) or (A17), the peptide linker having anaffinity tag moiety of a Mxe intein and chitin-binding domain (CBD) with one C-terminal Alanine residue has a sequence at least 95% identical to: MTFKLIINGKTLKGEITIEAVDA[*pBPA]EAEKIFKQYANDYGIDGEWTYDDATKTFTVT ESGSGSGSGSGSMARQVICWCFTLNNPLSPLSLHDSMKYLVYQTEQGEAGNIHFQGYIE MKKRTSLAGMKKLIPGAHFEKRRGTQGEARAYSMKEDTRLEGPWEYGEFVPAACITGD ALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTV EGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFARGKPE FAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLR VDTADHAFITNGFVSHATGLTGLNSGLTTNPGVSAWQVNTAYTAGQLVTYNGKTYKCL QPHTSLAGWEPSNVPALWQLQ* (SEQ ID NO: 31).

[0285] (A25) For the peptide linker denoted as any one of (A15) through (A17), the peptidelinker having an affinity tag moiety of a Mxe intein and chitin-binding domain (CBD) with two C-terminal Alanine residues has a sequence at least 95% identical to: MTFKLIINGKTLKGEITIEAVDA[*pBPA]EAEKIFKQYANDYGIDGEWTYDDATKTFTVT ESGSGSGSGSGSMARQVICWCFTLNNPLSPLSLHDSMKYLVYQTEQGEAGNIHFQGYIE MKKRTSLAGMKKLIPGAHFEKRRGTQGEARAYSMKEDTRLEGPWEYGEFVPAACITGD ALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTV EGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFARGKPE FAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLR VDTADHAFITNGFVSHATGLTGLNSGLTTNPGVSAWQVNTAYTAGQLVTYNGKTYKCL QPHTSLAGWEPSNVPALWQLQ* (SEQ ID NO: 33).

[0286] (A26) For the peptide linker denoted as any one of (A15) through (A20), the peptidelinker having an affinity tag moiety of a Mxe intein and chitin-binding domain (CBD) with three C-terminal Alanine residues has a sequence at least 95% identical to: MTFKLIINGKTLKGEITIEAVDA[*pBPA]EAEKIFKQYANDYGIDGEWTYDDATKTFTVT ESGSGSGSGSGSMARQVICWCFTLNNPLSPLSLHDSMKYLVYQTEQGEAGNIHFQGYIE MKKRTSLAGMKKLIPGAHFEKRRGTQGEARAYSMKEDTRLEGPWEYGEFVPAAACITG 58 122903345.1BN00007.2520 BI-11236 DALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRT VEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFARGKP EFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSL RVDTADHAFITNGFVSHATGLTGLNSGLTTNPGVSAWQVNTAYTAGQLVTYNGKTYK CLQPHTSLAGWEPSNVPALWQLQ* (SEQ ID NO: 35).

[0287] (B1) An isolated nucleic acid that encodes the peptide linker.

[0288] (B2) For the isolated nucleic acid denoted as (B1), the isolated nucleic acid is 5'-ATGACCTTTAAACTGATTATTAACGGCAAAACCCTGAAAGGCGAAATTACCATTGAA GCGGTGGATGCGTAGGAAGCGGAAAAAATTTTTAAACAGTATGCGAACGATTATGG CATTGATGGCGAATGGACCTATGATGATGCGACCAAAACCTTTACCGTGACCGAATC TGGCAGCGGTAGTGGGTCGGGATCAGGCTCCATGGCGCGCCAGGTGATTTGCTGGT GCTTTACCCTGAACAACCCGCTGAGCCCGCTGAGCCTGCATGATAGCATGAAATATC TGGTGTATCAGACCGAACAGGGCGAAGCGGGCAACATTCATTTTCAGGGCTATATTG AAATGAAAAAACGCACCAGCCTGGCGGGCATGAAAAAACTGATTCCGGGCGCGCAT TTTGAAAAACGCCGCGGCACCCAGGGCGAAGCGCGCGCGTATAGCATGAAAGAAG ATACCCGCCTGGAAGGCCCGTGGGAATATGGCGAATTTGTGCCGTGA-3' (SEQ ID NO: 5).

[0289] (B3) For the isolated nucleic acid denoted as (B1), the TAG stop codon at residues 70-72 of SEQ ID NO: 5 is capable of introducing an unnatural amino acid during translation of SEQ ID NO: 5.

[0290] (B4) For the isolated nucleic acid denoted as (B1), the isolated nucleic acid encodingthe peptide linker having an affinity tag moiety of a Mxe intein and chitin-binding domain (CBD) with one C-terminal Alanine residue is 5'- ATGACCTTTAAACTGATTATTAACGGCAAAACCCTGAAAGGCGAAATTACCATTGAA GCGGTGGATGCGTAGGAAGCGGAAAAAATTTTTAAACAGTATGCGAACGATTATGG CATTGATGGCGAATGGACCTATGATGATGCGACCAAAACCTTTACCGTGACCGAATC TGGCAGCGGTAGTGGGTCGGGATCAGGCTCCATGGCGCGCCAGGTGATTTGCTGGT GCTTTACCCTGAACAACCCGCTGAGCCCGCTGAGCCTGCATGATAGCATGAAATATC TGGTGTATCAGACCGAACAGGGCGAAGCGGGCAACATTCATTTTCAGGGCTATATTG AAATGAAAAAACGCACCAGCCTGGCGGGCATGAAAAAACTGATTCCGGGCGCGCAT TTTGAAAAACGCCGCGGCACCCAGGGCGAAGCGCGCGCGTATAGCATGAAAGAAG 59 122903345.1BN00007.2520 BI-11236 ATACCCGCCTGGAAGGCCCGTGGGAATATGGCGAATTTGTGCCGGCATGCATCACG GGAGATGCACTAGTTGCCCTACCCGAGGGCGAGTCGGTACGCATCGCCGACATCGT GCCGGGTGCGCGGCCCAACAGTGACAACGCCATCGACCTGAAAGTCCTTGACCGGC ATGGCAATCCCGTGCTCGCCGACCGGCTGTTCCACTCCGGCGAGCATCCGGTGTACA CGGTGCGTACGGTCGAAGGTCTGCGTGTGACGGGCACCGCGAACCACCCGTTGTTGT GTTTGGTCGACGTCGCCGGGGTGCCGACCCTGCTGTGGAAGCTGATCGACGAAATCA AGCCGGGCGATTACGCGGTGATTCAACGCAGCGCATTCAGCGTCGACTGTGCAGGTT TTGCCCGCGGGAAACCCGAATTTGCGCCCACAACCTACACAGTCGGCGTCCCTGGAC TGGTGCGTTTCTTGGAAGCACACCACCGAGACCCGGACGCCCAAGCTATCGCCGAC GAGCTGACCGACGGGCGGTTCTACTACGCGAAAGTCGCCAGTGTCACCGACGCCGG CGTGCAGCCGGTGTATAGCCTTCGTGTCGACACGGCAGACCACGCGTTTATCACGAA CGGGTTCGTCAGCCACGCTACTGGCCTCACCGGTCTGAACTCAGGCCTCACGACAAA TCCTGGTGTATCCGCTTGGCAGGTCAACACAGCTTATACTGCGGGACAATTGGTCAC ATATAACGGCAAGACGTATAAATGTTTGCAGCCCCACACCTCCTTGGCAGGATGGG AACCATCCAACGTTCCTGCCTTGTGGCAGCTTCAATGA-3' (SEQ ID NO: 32).

[0291] (B5) For the isolated nucleic acid denoted as (B1), the isolated nucleic acid encodingthe peptide linker having an affinity tag moiety of a Mxe intein and chitin-binding domain (CBD) with two C-terminal Alanine residues is 5'- ATGACCTTTAAACTGATTATTAACGGCAAAACCCTGAAAGGCGAAATTACCATTGAA GCGGTGGATGCGTAGGAAGCGGAAAAAATTTTTAAACAGTATGCGAACGATTATGG CATTGATGGCGAATGGACCTATGATGATGCGACCAAAACCTTTACCGTGACCGAATC TGGCAGCGGTAGTGGGTCGGGATCAGGCTCCATGGCGCGCCAGGTGATTTGCTGGT GCTTTACCCTGAACAACCCGCTGAGCCCGCTGAGCCTGCATGATAGCATGAAATATC TGGTGTATCAGACCGAACAGGGCGAAGCGGGCAACATTCATTTTCAGGGCTATATTG AAATGAAAAAACGCACCAGCCTGGCGGGCATGAAAAAACTGATTCCGGGCGCGCAT TTTGAAAAACGCCGCGGCACCCAGGGCGAAGCGCGCGCGTATAGCATGAAAGAAG ATACCCGCCTGGAAGGCCCGTGGGAATATGGCGAATTTGTGCCGGCAGCTTGCATCA CGGGAGATGCACTAGTTGCCCTACCCGAGGGCGAGTCGGTACGCATCGCCGACATC GTGCCGGGTGCGCGGCCCAACAGTGACAACGCCATCGACCTGAAAGTCCTTGACCG GCATGGCAATCCCGTGCTCGCCGACCGGCTGTTCCACTCCGGCGAGCATCCGGTGTA CACGGTGCGTACGGTCGAAGGTCTGCGTGTGACGGGCACCGCGAACCACCCGTTGT 60 122903345.1BN00007.2520 BI-11236 TGTGTTTGGTCGACGTCGCCGGGGTGCCGACCCTGCTGTGGAAGCTGATCGACGAAA TCAAGCCGGGCGATTACGCGGTGATTCAACGCAGCGCATTCAGCGTCGACTGTGCA GGTTTTGCCCGCGGGAAACCCGAATTTGCGCCCACAACCTACACAGTCGGCGTCCCT GGACTGGTGCGTTTCTTGGAAGCACACCACCGAGACCCGGACGCCCAAGCTATCGC CGACGAGCTGACCGACGGGCGGTTCTACTACGCGAAAGTCGCCAGTGTCACCGACG CCGGCGTGCAGCCGGTGTATAGCCTTCGTGTCGACACGGCAGACCACGCGTTTATCA CGAACGGGTTCGTCAGCCACGCTACTGGCCTCACCGGTCTGAACTCAGGCCTCACGA CAAATCCTGGTGTATCCGCTTGGCAGGTCAACACAGCTTATACTGCGGGACAATTGG TCACATATAACGGCAAGACGTATAAATGTTTGCAGCCCCACACCTCCTTGGCAGGAT GGGAACCATCCAACGTTCCTGCCTTGTGGCAGCTTCAATGA-3' (SEQ ID NO: 34).

[0292] (B6) For the isolated nucleic acid denoted as (B1), the isolated nucleic acid encodingthe peptide linker having an affinity tag moiety of a Mxe intein and chitin-binding domain (CBD) with three C-terminal Alanine residues is 5'- ATGACCTTTAAACTGATTATTAACGGCAAAACCCTGAAAGGCGAAATTACCATTGAA GCGGTGGATGCGTAGGAAGCGGAAAAAATTTTTAAACAGTATGCGAACGATTATGG CATTGATGGCGAATGGACCTATGATGATGCGACCAAAACCTTTACCGTGACCGAATC TGGCAGCGGTAGTGGGTCGGGATCAGGCTCCATGGCGCGCCAGGTGATTTGCTGGT GCTTTACCCTGAACAACCCGCTGAGCCCGCTGAGCCTGCATGATAGCATGAAATATC TGGTGTATCAGACCGAACAGGGCGAAGCGGGCAACATTCATTTTCAGGGCTATATTG AAATGAAAAAACGCACCAGCCTGGCGGGCATGAAAAAACTGATTCCGGGCGCGCAT TTTGAAAAACGCCGCGGCACCCAGGGCGAAGCGCGCGCGTATAGCATGAAAGAAG ATACCCGCCTGGAAGGCCCGTGGGAATATGGCGAATTTGTGCCGGCAGCTGCCTGC ATCACGGGAGATGCACTAGTTGCCCTACCCGAGGGCGAGTCGGTACGCATCGCCGA CATCGTGCCGGGTGCGCGGCCCAACAGTGACAACGCCATCGACCTGAAAGTCCTTG ACCGGCATGGCAATCCCGTGCTCGCCGACCGGCTGTTCCACTCCGGCGAGCATCCGG TGTACACGGTGCGTACGGTCGAAGGTCTGCGTGTGACGGGCACCGCGAACCACCCG TTGTTGTGTTTGGTCGACGTCGCCGGGGTGCCGACCCTGCTGTGGAAGCTGATCGAC GAAATCAAGCCGGGCGATTACGCGGTGATTCAACGCAGCGCATTCAGCGTCGACTG TGCAGGTTTTGCCCGCGGGAAACCCGAATTTGCGCCCACAACCTACACAGTCGGCGT CCCTGGACTGGTGCGTTTCTTGGAAGCACACCACCGAGACCCGGACGCCCAAGCTAT CGCCGACGAGCTGACCGACGGGCGGTTCTACTACGCGAAAGTCGCCAGTGTCACCG 61 122903345.1BN00007.2520 BI-11236 ACGCCGGCGTGCAGCCGGTGTATAGCCTTCGTGTCGACACGGCAGACCACGCGTTTA TCACGAACGGGTTCGTCAGCCACGCTACTGGCCTCACCGGTCTGAACTCAGGCCTCA CGACAAATCCTGGTGTATCCGCTTGGCAGGTCAACACAGCTTATACTGCGGGACAAT TGGTCACATATAACGGCAAGACGTATAAATGTTTGCAGCCCCACACCTCCTTGGCAG GATGGGAACCATCCAACGTTCCTGCCTTGTGGCAGCTTCAATGA-3' (SEQ ID NO: 36).

[0293] (C1) An expression vector having the isolated nucleic acid that encodes the peptidelinker.

[0294] (C2) For the expression vector denoted as (C1), the expression vector is pTXB1.

[0295] (D1) A method of conjugating a target polypeptide and a target oligonucleotideincluding a histidine-hydrophobic-histidine (HUH) endonuclease recognition sequence, the method involving: (a) contacting the target oligonucleotide having the HUH endonuclease recognition sequence with a peptide linker, said peptide linker having: a first polypeptide domain capable of binding the target polypeptide and having a crosslinkable moiety; a second polypeptide domain having a linking polypeptide sequence; and a third polypeptide domain having a HUH endonuclease, thereby forming a first complex comprising the target oligonucleotide and the peptide linker; and (b) contacting the target polypeptide with the first complex under conditions suitable for crosslinking of the target polypeptide and the first complex, thereby conjugating the target polypeptide and the target oligonucleotide.

[0296] (E1) A method of conjugating a target polypeptide and a target oligonucleotide havinga histidine-hydrophobic-histidine (HUH) endonuclease recognition sequence, the method involving: (a) contacting the target polypeptide with a peptide linker, said peptide linker comprising: a first polypeptide domain capable of binding the target polypeptide and having a crosslinkable moiety; a second polypeptide domain having a linking polypeptide sequence; and a third polypeptide domain having a HUH endonuclease, under conditions suitable for crosslinking of the target polypeptide and the peptide linker, thereby forming a first complex having the target polypeptide and the peptide linker; and (b) contacting the target oligonucleotide having the HUH endonuclease recognition sequence with the first complex; thereby conjugating the target polypeptide and the target oligonucleotide.

[0297] (E2) For the method denoted as (E1), the crosslinkable moiety is photo-crosslinkable.

[0298] (E3) For the method denoted as (E2), the crosslinkable moiety is a photo-crosslinkingunnatural amino acid (UAA). 62 122903345.1BN00007.2520 BI-11236

[0299] (E4) For the method denoted as (E3), the photo-crosslinking UAA is p-benzoyl-L-phenylalanine (pBpa).

[0300] (E5) For the method denoted as any one of (E1) through (E4), the target polypeptide isan antigen-binding polypeptide.

[0301] (E6) For the method denoted as any one of (E1) through (E5), the target antigen-bindingpolypeptide is an antibody having a fragment crystallizable region (Fc region).

[0302] (E7) For the method denoted as any one of (E1) through (E6), the antibody having theFc region is an IgG isotype antibody.

[0303] (E8) For the method denoted as any one of (E1) through (E7), the target oligonucleotideis a single-stranded DNA (ssDNA).

[0304] (E9) For the method denoted as any one of (E1) through (E8), the peptide linkercomprises a sequence at least 95% identical to N- MTFKLIINGKTLKGEITIEAVDA[UAA]EAEKIFKQYANDYGIDGEWTYDDATKTFTVTES GSGSGSGSGSMARQVICWCFTLNNPLSPLSLHDSMKYLVYQTEQGEAGNIHFQGYIEMK KRTSLAGMKKLIPGAHFEKRRGTQGEARAYSMKEDTRLEGPWEYGEFVP-C (SEQ ID NO: 4), wherein [UAA] is an unnatural amino acid.

[0305] (E10) For the method denoted as any one of (E1) through (E9), the [UAA] is pBpa.

[0306] (F1) A composition including: (i) an antigen-binding polypeptide; (ii) a peptide linkerhaving: a first polypeptide domain covalently attached to the antigen-binding polypeptide, a second polypeptide domain having a linking polypeptide sequence, and a third polypeptide domain having a histidine-hydrophobic-histidine (HUH) endonuclease; and (iii) a target oligonucleotide, wherein the third polypeptide domain of the peptide linker is covalently attached to the target oligonucleotide.

[0307] Although the invention has been described with reference to the embodimentsillustrated in the attached drawing figures, it is noted that equivalents may be employed and substitutions made herein without departing from the scope of the invention as recited in the claims. 63 122903345.1

Claims

BN00007.2520 BI-11236 We Claim:

1. A peptide linker for joining a target polypeptide and a target oligonucleotide, the peptide linker comprising: a first polypeptide domain capable of binding the target polypeptide; a second polypeptide domain comprising a linking polypeptide sequence; and a third polypeptide domain comprising a histidine-hydrophobic-histidine (HUH) endonuclease, wherein the third polypeptide domain is capable of binding the target oligonucleotide.

2. The peptide linker of claim 1, wherein the target polypeptide is an antigen-binding polypeptide.

3. The peptide linker of claim 2, wherein the target antigen-binding polypeptide is an antibody having a fragment crystallizable region (Fc region), optionally wherein the antibody having the Fc region is an IgG isotype antibody.

4. The peptide linker of claim 3, wherein the first polypeptide domain is capable of binding the Fc region of the antibody.

5. The peptide linker of claim 1, wherein the first polypeptide domain comprises a crosslinkable moiety.

6. The peptide linker of claim 5, wherein the crosslinkable moiety is photo-crosslinkable, optionally wherein the crosslinkable moiety comprises a photo-crosslinking unnatural amino acid (UAA), optionally wherein the photo-crosslinking UAA is p-benzoyl-L-phenylalanine (pBpa).

7. The peptide linker of claim 5 or claim 6, wherein the crosslinkable moiety covalently binds the first polypeptide domain and the target polypeptide.

8. The peptide linker of claim 1, wherein the first polypeptide domain comprises an immunoglobulin-binding domain of streptococcal protein G, optionally wherein the first polypeptide domain comprises a B1 domain of streptococcal protein G. 64 122903345.1BN00007.2520 BI-11236 9. The peptide linker of claim 1, wherein the linking polypeptide sequence of the second polypeptide domain comprises a glycine-serine (GS) repeat sequence, optionally wherein the linking polypeptide sequence of the second polypeptide domain comprises N-GSGSGS-C (SEQ ID NO: 1), optionally wherein the linking polypeptide sequence of the second polypeptide domain comprises N-SGSGSGSGSGS-C (SEQ ID NO: 2).

10. The peptide linker of claim 1, wherein the third polypeptide domain comprising a histidine- hydrophobic-histidine (HUH) endonuclease comprises a sequence at least 90% identical to the sequence N- MARQVICWCFTLNNPLSPLSLHDSMKYLVYQTEQGEAGNIHFQGYIEMKKRTSLAGMK KLIPGAHFEKRRGTQGEARAYSMKEDTRLEGPWEYGEFVP-C (SEQ ID NO: 3).

11. The peptide linker of claim 1, further comprising an affinity tag moiety.

12. The peptide linker of claim 11, wherein the affinity tag moiety comprises a Mxe intein and a chitin-binding domain (CBD), optionally wherein the peptide linker comprising the affinity tag moiety comprises one or more alanine residue(s) positioned at the C-terminus of the HUH endonuclease domain and upstream of the affinity tag moiety comprising the Mxe intein and the chitin-binding domain (CBD), optionally wherein the peptide linker comprising the affinity tag moiety comprises two or more alanine residue(s) positioned at the C-terminus of the HUH endonuclease domain and upstream of the affinity tag moiety comprising the Mxe intein and the chitin-binding domain (CBD), optionally wherein the peptide linker comprising the affinity tag moiety comprises three alanine residue(s) positioned at the C-terminus of the HUH endonuclease domain and upstream of the affinity tag moiety comprising the Mxe intein and the chitin-binding domain (CBD).

13. The peptide linker of claim 1, wherein the target oligonucleotide is a single-stranded DNA (ssDNA). 65 122903345.1BN00007.2520 BI-11236 14. The peptide linker of claim 1, comprising a sequence at least 95% identical to N- MTFKLIINGKTLKGEITIEAVDA[UAA]EAEKIFKQYANDYGIDGEWTYDDATKTFTVTES GSGSGSGSGSMARQVICWCFTLNNPLSPLSLHDSMKYLVYQTEQGEAGNIHFQGYIEMK KRTSLAGMKKLIPGAHFEKRRGTQGEARAYSMKEDTRLEGPWEYGEFVP-C (SEQ ID NO: 4), wherein [UAA] is an unnatural amino acid, optionally wherein [UAA] is pBpa.

15. An isolated nucleic acid that encodes the peptide linker of claim 1.

16. The isolated nucleic acid of claim 14, wherein the isolated nucleic acid comprises 5'- ATGACCTTTAAACTGATTATTAACGGCAAAACCCTGAAAGGCGAAATTACCATTGAA GCGGTGGATGCGTAGGAAGCGGAAAAAATTTTTAAACAGTATGCGAACGATTATGG CATTGATGGCGAATGGACCTATGATGATGCGACCAAAACCTTTACCGTGACCGAATC TGGCAGCGGTAGTGGGTCGGGATCAGGCTCCATGGCGCGCCAGGTGATTTGCTGGT GCTTTACCCTGAACAACCCGCTGAGCCCGCTGAGCCTGCATGATAGCATGAAATATC TGGTGTATCAGACCGAACAGGGCGAAGCGGGCAACATTCATTTTCAGGGCTATATTG AAATGAAAAAACGCACCAGCCTGGCGGGCATGAAAAAACTGATTCCGGGCGCGCAT TTTGAAAAACGCCGCGGCACCCAGGGCGAAGCGCGCGCGTATAGCATGAAAGAAG ATACCCGCCTGGAAGGCCCGTGGGAATATGGCGAATTTGTGCCGTGA-3' (SEQ ID NO: 5), optionally wherein the TAG stop codon at residues 70-72 of SEQ ID NO: 5 is capable of introducing an unnatural amino acid during translation of SEQ ID NO:

5.

17. An expression vector comprising the isolated nucleic acid of claim 15 or 16.

18. The expression vector of claim 17, wherein the expression vector is pTXB1.

19. A method for conjugating a target polypeptide and a target oligonucleotide comprising a histidine-hydrophobic-histidine (HUH) endonuclease recognition sequence, the method comprising: (a) contacting the target oligonucleotide comprising the HUH endonuclease recognition sequence with a peptide linker comprising a first polypeptide domain capable of binding the target polypeptide and comprising a crosslinkable moiety; a second polypeptide domain comprising a linking polypeptide sequence; and a third polypeptide domain comprising a HUH endonuclease, thereby forming a first complex comprising the target oligonucleotide and the peptide linker; and 66 122903345.1BN00007.2520 BI-11236 (b) contacting the target polypeptide with the first complex under conditions suitable for crosslinking of the target polypeptide and the first complex, thereby conjugating the target polypeptide and the target oligonucleotide.

20. A method of conjugating a target polypeptide and a target oligonucleotide comprising a histidine-hydrophobic-histidine (HUH) endonuclease recognition sequence, the method comprising: (a) contacting the target polypeptide with a peptide linker comprising a first polypeptide domain capable of binding the target polypeptide and comprising a crosslinkable moiety; a second polypeptide domain comprising a linking polypeptide sequence; and a third polypeptide domain comprising a HUH endonuclease, under conditions suitable for crosslinking of the target polypeptide and the peptide linker, thereby forming a first complex comprising the target polypeptide and the peptide linker; and (b) contacting the target oligonucleotide comprising the HUH endonuclease recognition sequence with the first complex; thereby conjugating the target polypeptide and the target oligonucleotide.

21. The method of claim 19 or 20, wherein the crosslinkable moiety is photo-crosslinkable, optionally wherein the crosslinkable moiety comprises a photo-crosslinking unnatural amino acid (UAA), optionally wherein the photo-crosslinking UAA is p-benzoyl-L-phenylalanine (pBpa).

22. The method of claim 19 or 20, wherein the target polypeptide is an antigen-binding polypeptide.

23. The method of claim 22, wherein the target antigen-binding polypeptide is an antibody having a fragment crystallizable region (Fc region), optionally wherein the antibody having the Fc region is an IgG isotype antibody.

24. The method of claim 19 or 20, wherein the target oligonucleotide is a single-stranded DNA (ssDNA).

25. The method of claim 19 or 20, wherein the peptide linker comprises a sequence at least 95% identical to N- 67 122903345.1BN00007.2520 BI-11236 MTFKLIINGKTLKGEITIEAVDA[UAA]EAEKIFKQYANDYGIDGEWTYDDATKTFTVTES GSGSGSGSGSMARQVICWCFTLNNPLSPLSLHDSMKYLVYQTEQGEAGNIHFQGYIEMK KRTSLAGMKKLIPGAHFEKRRGTQGEARAYSMKEDTRLEGPWEYGEFVP-C (SEQ ID NO: 4), wherein [UAA] is an unnatural amino acid, optionally wherein [UAA] is pBpa.

26. A composition comprising: (i) an antigen-binding polypeptide; (ii) a peptide linker comprising: a first polypeptide domain covalently attached to the antigen-binding polypeptide; a second polypeptide domain comprising a linking polypeptide sequence; and a third polypeptide domain comprising a histidine-hydrophobic-histidine (HUH) endonuclease; and (iii) a target oligonucleotide, wherein the third polypeptide domain of the peptide linker is covalently attached to the target oligonucleotide.

27. A peptide linker comprising a sequence at least 95% identical to SEQ ID NO: 31, 33, or 35.

28. An isolated nucleic acid comprising a sequence at least 95% identical to SEQ ID NO: 32, 34, or 36. 68 122903345.1

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