Antibody-polynucleotide conjugates via inverse electron demand diels-alder reactions

Abstract

Provided herein are polynucleotide linker-payloads, and conjugates thereof, and pharmaceutical compositions thereof, and of their use for therapy. Also provided herein are methods of making the polynucleotide linker payloads and conjugates thereof.

Description

250298.000925 11911WO01ANTIBODY-POLYNUCLEOTIDE CONJUGATES VIA INVERSE ELECTRON DEMAND DIELS-ALDER REACTIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Application No.63 / 683,065, filed on August 14, 2024, the disclosure of which is incorporated by reference herein in its entirety.FIELD

[0002] Provided herein are polynucleotide linker-payloads, and conjugates thereof, and pharmaceutical compositions thereof, and of their use for therapy.BACKGROUND

[0003] Antibody-drug conjugates (ADCs) are antibodies that are covalently linked to biologically active small molecule drugs, often referred to as payloads, thus combining the targeting specificity of antibodies with the mode-of-action and potency of small molecule drugs. The therapeutic utility of ADC(s) has been validated in cancer treatment and is a major ongoing focus of study. ADCETRIS® (bentruximab vedotin) and KADCYLA® (ado-trastuzumab emtansine) are ADCs approved for the treatment of certain cancer types, and several other ADCs are currently in clinical development.

[0004] Chemical site-selective protein modification has become increasingly popular for antibody-based bio-conjugates. Amongst all biorthogonal reactions developed to date, the [4+2] cycloaddition of 1,2,4,5-tetrazines (tetrazines, Tz) with various dienophiles, referred as inverse electron demand Diels-Alder (IEDDA) reactions, is one that satisfies most of the biorthogonal criteria (e.g., fast, selective, biocompatible, and catalyst-free) necessary for the conjugations (Deb at al., “Mechanisms and Substituent Effects of Metal-Free Bioorthogonal Reactions,” Chem. Rev. 121(12): 6850-6914 (2021)).

[0005] Polynucleotides have been developed with therapeutic potential. For instance, small interfering RNAs (siRNAs) are capable of interfering with the expression of specific genes in vivo. Approved siRNA therapeutics include patisiran, givosiran, lumasiran, and inclisiran. Antisense oligonucleotides (ASOs) target and inhibit specific messenger RNAs in vivo. Approved ASO therapeutics include fomivirsen, mipomersen, eteplirsen, nusinersen, inotersen, volanesorsen, i313810218250298.00092511911WO01 golodirsen, viltolarsen, and casimersen. Therapeutic siRNAs and antisense oligonucleotides often require complex formulation and administration strategies for stability and delivery. Further constructs are needed for improved delivery and targeting of polynucleotide therapeutics such as siRNAs and ASOs.

[0006] The foregoing discussion is presented solely to provide a better understanding of the nature of the problems confronting the art and should not be construed in any way as an admission as to prior art nor should the citation of any reference herein be construed as an admission that such reference constitutes “prior art” to the instant application.SUMMARY

[0007] Various non-limiting aspects and embodiments of the disclosure are described below.

[0008] Provided herein are polynucleotide linker payloads and conjugates thereof, and pharmaceutical compositions thereof, and methods of their use for treating a variety of diseases, disorders, and conditions. Also provided herein are methods of making the polynucleotide linker payloads and conjugates thereof.

[0009] In one aspect, provided herein is a compound of the following formula:or a pharmaceutically acceptable salt thereof, where BA is an antigen-binding protein; RG' comprises a reactive group residue following reaction of a first reactive group RG1 with second reactive group RG2; RG1 comprises a reactive group chosen fromRG2 comprises a reactive group chosen from313810218250298.00092511911WO01 where each R1is independently hydrogen or Ci-Cio alkyl; T is a polynucleotide; and k is 1 or 2.

[0010] In some embodiments, each RG' comprises

[0011] In some embodiments, each RG' is chosen from

[0012] In some embodiments, each RG' comprises

[0013] In some embodiments, each RG' is chosen from

[0014] In another aspect, provided herein is a compound according to Formula (I):pharmaceutically acceptable salt thereof, or regioisomer thereof, where each L is independently a linker.313810218250298.00092511911WO01

[0015] In some embodiments, provided herein is a compound according to Formula (la) orthereof, or regioisomer thereof, wherein each L is independently a linker.

[0016] In another aspect, provided herein is a compound according to Formula (II):thereof, or regioisomer thereof, wherein L is a linker.

[0017] In some embodiments, provided herein is a compound according to Formula (Ila) or(Hb):313810218250298.00092511911WO01(lib), or a pharmaceutically acceptable salt thereof, or regioisomer thereof, wherein each L is independently a linker.

[0018] In another aspect, provided herein is a compound according to Formula (III):thereof, or regioisomer thereof, wherein each L is independently a linker.

[0019] In some embodiments, provided herein is a compound according to Formula (Illa) orthereof, or regioisomer thereof, wherein each L is independently a linker.313810218250298.00092511911WO01

[0020] In some embodiments, provided herein is a compound according to Formula (IV):pharmaceutically acceptable salt thereof, or regioisomer thereof, wherein L is a linker.

[0021] In some embodiments, provided herein is a compound according to Formula (IVa) or(IVb):pharmaceutically acceptable salt thereof, or a regioisomer thereof, wherein L is a linker.

[0022] In some embodiments, L is -N(R2)-Ci-Cioalkylene or -N(R2)-Ci-Cioalkylene-P03S-; and R2is hydrogen or Ci-Cio alkyl.

[0023] In some embodiments, R1is methyl and R2is hydrogen.

[0024] In some embodiments, the reaction of RG1 and RG2 comprises an inverse electron demand Diels-Alder reaction.

[0025] In some embodiments, RG' comprises a Diels-Alder adduct.

[0026] In some embodiments, provided herein is a compound selected from the group consisting of313810218250298.00092511911WO01or pharmaceutically acceptable salt and / or regioisomer thereof.

[0027] In some embodiments, BA comprises an antibody or an antigen binding fragment thereof.

[0028] In some embodiments, BA comprises an antigen binding fragment chosen from Fab fragments; F(ab')2 fragments; Fd fragments; Fv fragments; single-chain Fv (scFv) molecules; and dAb fragments.

[0029] In some embodiments, BA an antigen-binding protein or a fragment thereof.

[0030] In some embodiments, BA binds specifically to human transferrin receptor or a variant or an antigenic fragment thereof.

[0031] In some embodiments, BA comprises one or two RG2 groups.313810218250298.00092511911WO01

[0032] In some embodiments, BA comprises one or two residues according to the formula

[0034] In some embodiments, one of the residues is at position 295 or 297 of BA, numbered according to the EU index of Kabat.

[0035] In some embodiments, one of the residues is at the C-terminus of BA.

[0036] In some embodiments, one of the modified glutamine residues is represented by Q* in the sequence LLQ*GA at the C-terminus of BA.

[0037] In another aspect, provided herein is a linker-payload compound of the following formula:RG1 — L— T or a pharmaceutically acceptable salt thereof, where L is a linker covalently bound to RG1 and to T ; RG1 comprises a reactive group chosenCio alkyl; and T is a polynucleotide.313810218250298.00092511911WO01

[0038] In some embodiments, RG1 comprises

[0039] In some embodiments, RG1 comprises

[0040] In some embodiments, RG1 comprises

[0041] In some embodiments, the linker-payload compound has the following formula:

[0043] In some embodiments, the linker-payload compound has the following formula:

[0044] In some embodiments, L is -N(R2)-CI-CIO alkylene or -N(R2)-CI-CIO alkylene-PChS-; and R2is hydrogen or Ci-Cio alkyl.

[0045] In some embodiments, R2is hydrogen.

[0046] In some embodiments, the linker-payload compound is selected from313810218250298.00092511911WO01or a pharmaceutically acceptable salt thereof.

[0047] In some embodiments, the polynucleotide is an interfering nucleic acid molecule, a guide RNA, a ribozyme, an aptamer, a gapmer, a mixmer, a multimer, or an mRNA.

[0048] In some embodiments, the polynucleotide is an siRNA, an shRNA, a miRNA, or an antisense oligonucleotide.

[0049] In some embodiments, the polynucleotide is an siRNA. In other embodiments, the polynucleotide is an antisense oligonucleotide.

[0050] In some embodiments, the polynucleotide comprises one or more modified nucleotides.

[0051] In another aspect, provided herein is a pharmaceutical composition comprising the compound as described herein and a pharmaceutically acceptable carrier.

[0052] In another aspect, provided herein is a composition or kit comprising the compound or pharmaceutical composition as described herein in association with a further therapeutic agent.

[0053] In another aspect, provided herein is a method of making a conjugate. This method comprises (a) treating an antigen-bidning protein313810218250298.00092511911WO01 transglutaminase-modified or sortase-modified binding agent. This method further comprises (b) treating the transglutaminase-modified or sortase-modified binding agent with the linker payload of any of the previous claims. This method further comprises (c) purifying the conjugate.

[0054] In further embodiments, provided herein are methods of using the compounds in therapy. In additional embodiments, provided herein are the compounds for use in therapy. In additional embodiments, provided herein are the compounds for use the manufacture of medicaments in therapy.BRIEF DESCRIPTION OF THE FIGURES

[0055] FIG. 1 shows the preparation of an exemplary bivalent mAb conjugate.

[0056] FIGS. 2A-2B show the preparation of an exemplary single-arm Ab conjugate.

[0057] FIGS. 3A-3B show the preparation of an exemplary Fab conjugate.

[0058] FIG. 4 shows the preparation of an exemplary bivalent mAb conjugate.

[0059] FIG. 5 shows the preparation of an exemplary single-arm Ab conjugate.

[0060] FIG. 6 shows the preparation of an exemplary Fab conjugate.

[0061] FIG. 7 shows a purification trace for an exemplary Antigen-Binding Protein-Linker.

[0062] FIG. 8 shows that antibody-siRNA conjugates exhibited dose dependent KD in 3T3- hTfRs by lipofection. Potent KD was not observed at lOnM.

[0063] FIG. 9 shows that antibody-siRNA conjugates showed dose dependent KD in 3T3- hTfRs by self delivery.

[0064] FIG. 10 shows that one-arm ab - mSODl siRNA conjugates showed dose-dependent knockdown of mSODl mRNA by lipofection in N2A cells.

[0065] FIG. 11 shows that hTfR Ab - mSODl siRNA conjugate showed dose-dependent knockdown of mSODl mRNA by self-delivery, indicating receptor-mediated uptake.

[0066] FIG. 12 shows stability data for siRNA conjugates.

[0067] FIG. 13 shows the conjugation of the linker to the antigen-binding protein.

[0068] FIG. 14 shows the conjugation of the antibody with tetrazine linker to siRNA withALO handle. n313810218250298.000925 11911WO01DETAILED DESCRIPTIONI. Overview

[0069] Provided herein are polynucleotide linker payloads and conjugates thereof. Such compounds are useful, for example, for delivery of the polynucleotide to various tissues in the body.II. Definitions

[0070] When referring to the compounds provided herein, the following terms have the following meanings unless indicated otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. In the event that there is a plurality of definitions for a term provided herein, these Definitions prevail unless stated otherwise.

[0071] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure.

[0072] The phrase “pharmaceutically acceptable salt”, as used in connection with compositions of the disclosure, refers to any salt suitable for administration to a patient. Suitable salts include, but are not limited to, those disclosed in. Berge et al., "Pharmaceutical Salts", J. Pharm. Sci., 1977, 66:1, incorporated herein by reference. Examples of salts include, but are not limited to, acid derived, base derived, organic, inorganic, amine, and alkali or alkaline earth metal salts, including but not limited to calcium salts, magnesium salts, potassium salts, sodium salts, salts of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methane sulfonic acid, ethane sulfonic acid, para-toluene sulfonic acid, salicylic acid, and the like.

[0073] As used herein, the term “about,” when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.). Ranges can be expressed herein as from “about” or “approximately” one particular value and / or to “about” or “approximately” another particular12313810218250298.00092511911WO01 value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value.

[0074] By “comprising” or “containing” or “including” is meant that at least the named compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, or method steps, even if the other such compounds, material, particles, or method steps have the same function as what is named.

[0075] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the disclosure.

[0076] Unless otherwise stated, cyclic adducts, e.g., products of a cycloaddition reaction, e.g., an azide-acetylene cycloaddition reaction, also referred to as Click reaction, depicted herein include all regioisomers, i.e., structural isomers that differ only in the position of a functional group or a substituent. By way of an example, the following structures represent triazole regioisomers, which differ only in the position of the substituent on the triazole ring:Triazole regioisomers may also be represented by the following structure:

[0077] Unless otherwise stated, all tautomeric forms of the compounds of the disclosure are within the scope of the disclosure.

[0078] Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by313810218250298.00092511911WO01 deuterium or tritium, or the replacement of a carbon by anC- or13C- or14C -enriched carbon are within the scope of this disclosure.

[0079] It is also to be understood that the mention of one or more method steps does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified. Similarly, it is also to be understood that the mention of one or more components in a device or system does not preclude the presence of additional components or intervening components between those components expressly identified.

[0080] Certain groups, moieties, substituents, and atoms are depicted with a wavy line. The wavy line can intersect or cap a bond or bonds. The wavy line indicates the atom through which the groups, moieties, substituents, or atoms are bonded. For example, a phenyl group that is-KCCHHSHCCHH3substituted with a propyl group depicted as:3 3has the following structure:

[0081] All amino acid abbreviations used in this disclosure are those accepted by the United States Patent and Trademark Office as set forth in 37 C.F.R. § 1 .822 (B)(J).

[0082] The amino acid sequence of an antibody can be numbered using any known numbering schemes, including those described by Kabat et al., (“Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001 , 309:657-70 (“AHo” numbering scheme). Unless otherwise specified, the numbering scheme used herein is the Kabat numbering scheme. However, selection of a numbering scheme is not intended to imply differences in sequences where they do not exist, and one of skill in the art can readily confirm a sequence position by examining the amino acid sequence of one or more antibodies. Unless stated otherwise, the “EU numbering scheme” is generally used when referring to a residue in an antibody heavy chain constant region (e.g., as reported in Kabat et al., supra).

[0083] The term “glutaminyl-modified antibody” refers to an antibody with at least one covalent linkage from a glutamine side chain to a primary amine compound of the present disclosure. In particular embodiments, the primary amine compound is linked through an amide linkage on the glutamine side chain. In certain embodiments, the glutamine is an endogenous313810218250298.000925 11911WO01 glutamine. In other embodiments, the glutamine is an endogenous glutamine made reactive by polypeptide engineering (e.g., via amino acid deletion, insertion, substitution, or mutation on the polypeptide). In additional embodiments, the glutamine is polypeptide engineered with an acyl donor glutamine-containing tag (e.g., glutamine-containing peptide tags, Q-tags, or TGase recognition tag).

[0084] As used herein, “alkyl” refers to a monovalent and saturated hydrocarbon radical moiety. Alkyl is optionally substituted and can be linear, branched, or cyclic, (i.e., cycloalkyl). Alkyl includes, but is not limited to, those radicals having one to twenty carbon atoms, for example, Ci-20 alkyl; one to twelve carbon atoms, for example, Ci-12 alkyl; one to eight carbon atoms, for example, Ci-8 alkyl; one to six carbon atoms, for example, Ci-6 alkyl; and one to three carbon atoms for example, C1-3 alkyl. Examples of alkyl moieties include, but are not limited to, methyl, ethyl, n-propyl, z-propyl, n-butyl, s-butyl, / -butyl, z’-butyl, a pentyl moiety, a hexyl moiety, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. A pentyl moiety includes, but is not limited to, n-pentyl and z’-pentyl. A hexyl moiety includes, but is not limited to, n-hexyl.

[0085] As used herein, “alkylene” refers to a divalent alkyl group. Unless specified otherwise, alkylene includes, but is not limited to, one to twenty carbon atoms. The alkylene group is optionally substituted as described herein for alkyl. In some embodiments, alkylene is unsubstituted. Examples of alkylene moieties include -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and the like.

[0086] As used herein, “heteroalkyl” refers to an alkyl in which one or more carbon atoms are replaced by heteroatoms. As used herein, “heteroalkenyl” refers to an alkenyl in which one or more carbon atoms are replaced by heteroatoms. As used herein, “heteroalkynyl” refers to an alkynyl in which one or more carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen, oxygen, and sulfur atoms. Heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted. Examples of heteroalkyl moieties include, but are not limited to, aminoalkyl, sulfonylalkyl, and sulfinylalkyl. Examples of heteroalkyl moieties also include, but are not limited to, methylamino, methylsulfonyl, and methylsulfinyl.

[0087] As used herein, “heteroalkylene” refers to a divalent alkyl group wherein one or more carbon atoms is replaced with a heteroatom. Unless specified otherwise, heteroalkylene includes, but is not limited to, one to twenty total atoms (i.e., carbons and heteroatoms). The heteroalkylene group is optionally substituted as described herein for alkyl. In some embodiments, heteroalkylene15313810218250298.00092511911WO01 is unsubstituted. In some embodiments, heteroatoms contemplated within heteroalkylene moieties include oxygen, nitrogen, sulfur (i.e., including sulfoxide, sulphite, sulfate, and sulfone), silicon, and phosphorous (i.e., including phosphite and phosphate), and / or combinations thereof. Nonlimiting exemplary embodiments of heteroalkylene moieties include -CH2O-, -CH2OCH2-, - CH2OCH2CH2-, -CH2CH2CH2OCH2-, and the like; -CH2NR-, -CH2NRCH2-, -CH2NRCH2CH2-, -CH2CH2CH2NRCH2-, and the like; and -CH2S-, -CH2SCH2-, -CH2SCH2CH2-, -CH2CH2CH2SCH2-, and the like wherein R includes, but is not limited to, hydrogen or alkyl.

[0088] As used herein, “binding agent” refers to any molecule, for example, protein, antibody, or antigen binding fragment thereof, capable of binding with specificity to a given binding partner, for example, an antigen.

[0089] As used herein, the term “residue” refers to the chemical moiety within a compound that remains after a chemical reaction. For example, the term “amino acid residue,” “peptide residue,” “0-amino acid residue,” “HO-amino acid residue,” or ‘W-alkyl amino acid residue” refers to the product of an amide coupling or peptide coupling of an amino acid, 0-amino acid, OH-amino acid, or a A-alkyl amino acid to a suitable coupling partner; wherein, for example, a water molecule is expelled after the amide or peptide coupling of the amino acid, peptide, 0-amino acid, OH-amino acid, or the A-alkylamino acid, resulting in the product having the amino acid residue, 0-amino acid residue, OH-amino acid residue, or A-alkyl amino acid residue incorporated therein.

[0090] As used herein, the phrase “reactive linker,” or the abbreviation “RL” refers to a monovalent group that includes a reactive group (“RG”) and spacer group (“SP”), depicted for example as, wherein RG is the reactive group and SP is the spacer group. The spacer group is any divalent moiety that bridges the reactive group to another group, such as a payload (e.g., a polynucleotide). The reactive linkers (RLs), together with the payloads to which they are bonded, provide intermediates (“linker-payloads” or LPs) useful as synthetic precursors for the preparation of the conjugates described herein. The reactive linker includes a reactive group, which is a functional group or moiety that is capable of reacting with a reactive portion of another group, for instance, a binding agent, an antibody, modified antibody, or antigen binding fragment thereof, or an enhancement group. The moiety resulting from the reaction of the reactive group with the binding agent, antibody, modified antibody, or antigen binding fragment thereof, together with the16313810218250298.00092511911WO01 linking group, include the “binding agent linker” (“BL”) portion of the conjugate, described herein. In certain embodiments, the “reactive group” is a functional group or moiety (e.g., maleimide or A-hydroxysuccinimide (NHS) ester) that reacts with a cysteine or lysine residue of an antibody or antigen-binding fragment thereof. In certain embodiments, the “reactive group” is a functional group or moiety that is capable of undergoing a click chemistry reaction (see, e.g., click chemistry, Huisgen “1,3-Dipolar Cycloaddition,” Proc. Chem. Soc. 357-396 (1961); Wang et al., “Bioconjugation by Copper(I)-Catalyzed Azide- Alkyne [3 + 2] Cycloaddition” J. Am. Chem. Soc. 125(11 ):3192-3193 (2003); and Agard et al., “A Strain-Promoted [3 + 2] Azide-Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems,” J. Am. Chem. Soc. 126(46): 15046-15047 (2004)). In certain embodiments, the “reactive group” is a functional group or moiety that is capable of undergoing an inverse electron demand Diels-Alder reaction (see, e.g., Deb at al., “Mechanisms and Substituent Effects of Metal-Free Bioorthogonal Reactions, ” Chem. Rev. 121(12):6850— 6914 (2021)). In some embodiments of said inverse electron demand Diels- Alder reaction, the reactive group is a dienophile (e.g., alkene or alkyne) that is capable of undergoing an inverse electron demand Diels-Alder reaction with a diene (e.g., a tetrazine). Such suitable reactive groups include, but are not limited to, dienophiles or strained dienophiles suitable for the inverse electron demand Diels-Alder reaction, for example,. Linker-payloads including such reactive groups are useful for conjugating antibodies that have been functionalized with diene groups. Such functionalized antibodies include antibodies functionalized with compounds described herein. In certain embodiments, such a functionalized antibody is derived by treating an antibody having at least one glutamine residue, for example, heavy chain Gln295, with a compound bearing an amino group and a diene group (e.g., a tetrazine), in the presence of the enzyme transglutaminase. In certain embodiments, the “reactive group” is a functional group or moiety that is capable of undergoing an inverse electron demand Diels-Alder reaction (see, e.g., Deb at al., “Mechanisms and Substituent Effects of Metal-Free Bioorthogonal Reactions, ” Chem. Rev. 121(12):6850— 6914 (2021)). In some embodiments of said inverse electron demand Diels- Alder reaction, the reactive group is a diene (e.g., a tetrazine) that is capable of undergoing an inverse electron demand Diels- Alder reaction with a dieneophile (e.g., alkene or alkyne). Such suitable reactive groups include, but are not limited to, dienes suitable for the inverse313810218250298.00092511911WO01 electron demand Diels-Alder reaction, for example,Linker-payloads including such reactive groups are useful for conjugating antibodies that have been functionalized with dieneophile groups. Such functionalized antibodies include antibodies functionalized with compounds described herein. In certain embodiments, such a functionalized antibody is derived by treating an antibody having at least one glutamine residue, for example, heavy chain Gln295, with a compound bearing an amino group and a dienophile group, in the presence of the enzyme transglutaminase.

[0091] In some examples, the reactive group is an alkyne, for example,, which can react via click chemistry with an azide, for example,forma click chemistry product, for example,some examples, the group reacts with an azide on a modified antibody or antigen binding fragment thereof. In some examples, the reactive group is an alkyne, for example,, which can react via click chemistry with an azide, for example,form aclick chemistry product, for example,313810218250298.00092511911WO01. In some examples, the reactive group is a dienophile, for example,o, which can react via an inverse electron demand Diels-Adler reaction with a. p , g p (e.g., a tetrazine) on a modified antibody or antigen binding fragment thereof. In some examples, the reactive group is a diene, for example,, which can react via an inverse electron demand Diels-Alder reaction with a dienophile, for example,to form a Diels-Alder product, for example,

[0092] As used herein, the term “regioisomer,” “regioisomers,” or “mixture of regioisomers” refers to the product(s) of 1,3 -cycloadditions or strain-promoted alkyne-azide cycloadditions (SPAACs) — otherwise known as click reactions — that derive from suitable azides (e.g., -N3, or - PEG-N3 derivatized antibodies) treated with suitable alkynes; or refers to the product(s) of inverse electron demand Diels-Alder reactions that derive from suitable dienophiles (e.g., alkenes or alkynes) treated with suitable dienes (e.g., tetrazines), or suitable dienes (e.g., tetrazines) treated313810218250298.00092511911WO01 with suitable dienophiles. In certain embodiments, for example, regioisomers and mixtures of regioisomers are characterized by the click reaction products shown below:A wherein represents attachment to a binding agent as described elsewhere herein. In certain embodiments, more than one suitable azide and more than one suitable alkyne can be utilized within a synthetic scheme en route to a product, where each pair of azide-alkyne can participate in one or more independent click reactions to generate a mixture of regioisomeric click reaction products. For example, a person of skill will recognize that a first suitable azide may independently react with a first suitable alkyne, and a second suitable azide may independently react with a second suitable alkyne, en route to a product, resulting in the generation of four possible click reaction regioisomers or a mixture of the four possible click reaction regioisomers. In certain embodiments, for example, regioisomers and mixtures of regioisomers are characterized by the inverse electron demand Diels-Alder reaction products below:A wherein represents attachment to a binding agent as described elsewhere herein. In certain embodiments, more than one suitable dienophile and more than one suitable diene can be utilized within a synthetic scheme en route to a product, where each pair of dienophile-diene can participate in one or more independent inverse electron demand Diels-Alder reactions to generate a mixture20313810218250298.00092511911WO01 of regioisomeric Diels-Alder reaction products. For example, a person of skill will recognize that a first suitable dienophile may independently react with a first suitable diene, and a second suitable dienophile may independently react with a second suitable diene, en route to a product, resulting in the generation of four possible Diels-Alder reaction regioisomers or a mixture of the four possible Diels-Alder reaction regioisomers.

[0093] As used herein, “amino acid side chain” refers to the additional chemical moiety on the same carbon that bears a primary or secondary amine and a carboxylic acid of an amino acid. As would be appreciated by a person of skill in the art, there are twenty-one “standard” amino acids. Exemplary “standard” amino acids include, without limitation, alanine, serine, proline, arginine, and aspartic acid. Other amino acids include, cysteine, selenocysteine, and glycine (e.g., wherein the additional chemical moiety on the same carbon that bears the primary amine and carboxylic acid of glycine is hydrogen). Exemplary amino acid side chains include, without limitation, methyl (i.e., alanine), sec-buytl (i.e., isoleucine), zso-butyl (i.e., leucine),-CH2CH2SCH3 (i.e., methionine), -CFEPh (i.e., phenylalanine),tryptophan),(i.e., tyrosine), zso-propyl (i.e., valine), hydroxymethyl (i.e., serine), -CH(OH)CH3 (i.e., threonine), -CH2C(O)NH2 (i.e., asparagine), -CH2CH2C(O)NH2 (i.e., glutamine), -CH2SH (i.e., cysteine), -CFESeH (i.e., selenocysteine), -CH2NH2 (i.e., glycine), propylene or -CH2CH2CH2- (i.e., proline), -CH2CH2CH2NHC(=NH)NH2 (i.e., arginine),(i.e., histidine), -CH2CH2CH2CH2NH2 (i.e., lysine), -CH2COOH (i.e., aspartic acid), and -CH2CH2COOH (i.e., glutamic acid).

[0094] As used herein, “biologically active compound” refers to a compound, prodrug, or payload that elicits a biological response when administered to a biological entity. Exemplary biological responses include, without limitation, increase or decrease in DNA or protein synthesis, up-regulation or down-regulation of signaling pathways, and increase or decrease in cell proliferation, and the like.

[0095] As used herein, the term “0-amino acid” or “HO-amino acid” designates an amino acid wherein the native amino group at the N-terminus of an amino acid or an amino acid sequence has21313810218250298.00092511911WO01 been replaced with an oxygen or hydroxyl group, respectively. For example, “O-AAAA” or “HO- AAAA” is intended to designate an amino acid sequence (AAAA) wherein the native amino group at the N-terminus has been replaced with an oxygen or hydroxyl group, respectively (e.g.,, where each R is an amino acid side chain). Similarly, the terms “0-amino acid residue” or “HO-amino acid residue” refers to the chemical moiety within a compound that remains after a chemical reaction. For example, “0-amino acid residue” or “HO- amino acid residue” refers to the product of an amide coupling or peptide coupling of an 0-amino acid or a HO-amino acid to a suitable coupling partner; wherein, for example, a water molecule is expelled after the amide or peptide coupling of the 0-amino acid or a HO-amino acid, resulting in the product having the 0-amino acid residue or a HO-amino acid residue incorporated therein.

[0096] There may be employed herein conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein "Sambrook, et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985)); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel, et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0097] A polynucleotide includes DNA and RNA. Provided herein is any polynucleotide disclosed herein which is operably linked to a promoter or other expression control sequence.

[0098] An oligonucleotide is a polynucleotide of up to about 30 nucleotides in length, e.g., about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

[0099] A symptom is a manifestation of disease apparent to the patient himself, while a sign is a manifestation of disease that the physician perceives. Reduction, fully or in part, of a sign or symptom may be referred to as alleviation of the sign or symptom.III. Conjugate Compounds

[0100] In one aspect, provided herein is a compound of the following formula:22313810218250298.00092511911WO01or a pharmaceutically acceptable salt thereof, where BA is an antigen-binding protein; RG' comprises a reactive group residue following reaction of a first reactive group RG1 with a second reactive group RG2; RG1 comprises a reactive group chosen fromRG2 comprises a reactive group chosen fromeach R1is independently hydrogen or Ci-Cio alkyl; T is a polynucleotide; and k is 1 or 2.

[0101] According to the present disclosure, k represents an average number of units of RG'- T conjugated to the antigen-binding protein (BA), k is also known as Drug Antibody Ratio (DAR). In certain embodiments, k is from about 1 to about 2.

[0102] In some embodiments, BA an antigen-binding protein or a fragment thereof.

[0103] In some embodiments, BA is an antibody, or an antigen binding fragment thereof. In some embodiments, BA is an IgG, e.g. IgGl, IgG2, IgG3 or IgG4.

[0104] In some embodiments, BA is an antigen binding fragment of an antibody. In certain embodiments, BA is a single chain fragment variable (scFv). In some embodiments, BA is a Fab. In some embodiments, BA comprises an antigen binding fragment chosen from Fab fragments; F(ab')2 fragments; Fd fragments; Fv fragments; single-chain Fv (scFv) molecules; and dAb fragments.

[0105] In some embodiments, BA binds specifically to human transferrin receptor or a variant or an antigenic fragment thereof.

[0106] In another aspect, provided herein is a compound of the following formula:23313810218250298.00092511911WO01or a pharmaceutically acceptable salt thereof, where BA is an antigen-binding protein, or a fragment thereof; RG' comprises a reactive group residue following reaction of a first reactive group RG1 with a second reactive group RG2; RG1 comprises a reactive group chosen fromRG2 comprises a reactive group chosen fromwherein each R1is independently hydrogen or Ci-Cio alkyl; T is a polynucleotide; and k ranges from about one to about two, representing an average number of units of RG'-T conjugated to the antibody.

[0107] In some embodiments, BA and RG' are linked directly, or via a linker, as described herein.

[0108] In some embodiments, RG' and T are linked directly, or via a linker, as described herein.

[0109] In some embodiments, k is 1. In certain embodiments, k is 2.

[0110] In some embodiments, BA comprises a residue of RG1, and T comprises a residue of RG2. In some embodiments, BA comprises a residue of RG2, and T comprises a residue of RG2. In some embodiments, BA is bonded to a residue of RG1, and T is bonded to a residue of RG2. In some embodiments, BA is bonded to a residue of RG2, and T is bonded to a residue of RG2. The bonds can be direct, or via a linker, as described herein.

[0111] In some embodiments, RG1 comprises or is chosen from:24313810218

[0112] In some embodiments, RG2 comprises or is chosen from:

[0113] In some embodiments, RG1 comprises or is chosen from:25313810218250298.00092511911WO01

[0114] In some embodiments, RG2 comprises or is chosen from:

[0115] In some embodiments, RG1 is a reactive group described in WO 2024 / 168199 to Han et al., which is hereby incorporated by reference in its entirety. In some embodiments, RG2 is a reactive group described in WO 2024 / 168199 to Han et al.

[0116] In some embodiments, each RG' is comprises26313810218250298.00092511911WO01

[0117] In some embodiments, each RG' is chosen from

[0118] In some embodiments, each RG' is comprises

[0119] In some embodiments, each RG' is chosen from

[0120] In another aspect, provided herein is a compound according to Formula (I):27313810218250298.00092511911WO01or a pharmaceutically acceptable salt thereof, or regioisomer thereof, where each L is independently a linker.

[0121] In some embodiments, provided herein is a compound according to Formula (la) or (lb):or a pharmaceutically acceptable salt thereof, or regioisomer thereof, where each L is independently a linker.

[0122] In some embodiments, provided herein is a compound according to Formula (II):or a pharmaceutically acceptable salt thereof, or regioisomer thereof, where L is a linker.313810218250298.00092511911WO01

[0123] In some embodiments, provided herein is a compound according to Formula (Ila) or(lib):or a pharmaceutically acceptable salt thereof, or regioisomer thereof, where each L is independently a linker.

[0124] In another aspect, provided herein is a compound according to Formula (III):or a pharmaceutically acceptable salt thereof, or regioisomer thereof, where each L is independently a linker.

[0125] In some embodiments, provided herein is a compound according to Formula (Illa) or (Illb):29313810218250298.00092511911WO01or a pharmaceutically acceptable salt thereof, or regioisomer thereof, wherein each L is independently a linker.

[0126] In another aspect, provided herein is a compound according to Formula (IV):or a pharmaceutically acceptable salt thereof, or regioisomer thereof, where L is a linker.

[0127] In some embodiments, provided herein is a compound according to Formula (IVa) or(IVb):313810218250298.00092511911WO01or a pharmaceutically acceptable salt thereof, or regioisomer thereof, where L is a linker.

[0128] In some embodiments, L is -N(R2)-Ci-Cioalkylene or -N(R2)-Ci-Cioalkylene-P03S-; and R2is hydrogen or C1-C10 alkyl.

[0129] In some embodiments, R1is methyl and R2is hydrogen.

[0130] In some embodiments, the reaction of RG1 and RG2 comprises an inverse electron demand Diels-Alder reaction.

[0131] In some embodiments, RG' comprises a Diels-Alder adduct.

[0132] In some embodiments, the compound is selected from the group consisting of313810218250298.00092511911WO01or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0133] In some embodiments, BA comprises one or two RG2 groups. In some embodiments, BA comprises one RG2 group. In other embodiments, BA comprises two RG2 groups.

[0134] In some embodiments, BA comprises one or two residues according to the formulaIn some embodiments, BA comprises one residue accordingcomprises two residues according to the formula313810218250298.00092511911WO01

[0135] In some embodiments, BA comprises one or two residues according to the formula. In some embodiments, BA comprises one residue according to the formulacomprises two residues according to the formula

[0136] In some embodiments, one of the residues is at position 295 or 297 of BA, numbered according to the EU index of Kabat.

[0137] In some embodiments, one of the residues is at the C-terminus of BA.

[0138] In some embodiments, one of the modified glutamine residues is represented by Q* in the sequence LLQ*GA at the C-terminus of BA.IV. Linker Pay load Compounds

[0139] In another aspect, provided herein is a compound of the following formula:RG1 — L— T or a pharmaceutically acceptable salt thereof, where L is a linker covalently bound to RG1 and toT ; RG1 comprises a reactive group chosen from313810218250298.00092511911WO01where each R1is independently hydrogen or C1-C10 alkyl; and T is a polynucleotide.

[0142]

[0143] In some embodiments, the linker payload compound has a structure of formula313810218250298.00092511911WO01

[0145] In some embodiments, the linker payload compound has a structure of formula, or a pharmaceutically acceptable salt thereof.

[0146] In some embodiments, L is -N(R2)-CI-CIO alkylene or -N(R2)-CI-CIO alkylene-PChS-; and R2is hydrogen or C1-C10 alkyl.

[0147] In some embodiments, R2is hydrogen.

[0148] In some embodiments, the linker payload compound is selected from the group consisting ofor a pharmaceutically acceptable salt thereof.

[0149] In some embodiments, the polynucleotide is an interfering nucleic acid molecule, a guide RNA, a ribozyme, an aptamer, a gapmer, a mixmer, a multimer, or an mRNA.

[0150] In some embodiments, the polynucleotide is an siRNA, an shRNA, a miRNA, or an antisense oligonucleotide.

[0151] In some embodiments, the polynucleotide is an siRNA.

[0152] In some embodiments, the polynucleotide is an antisense oligonucleotide.

[0153] In some embodiments, the polynucleotide comprises one or more modified nucleotides.35313810218250298.00092511911WO01V. Linkers

[0154] In certain embodiments, the linker L portion of the compounds described herein is a moiety, for instance a divalent moiety, that covalently links i) an antigen-binding protein (BA) to a RG1reactive group; ii) an antigen-binding protein (BA) to a RG2reactive group; iii) a polynucleotide (T) to a RG1reactive group; or iv) a polynucleotide (T) to a RG2reactive group. Suitable linkers may be found, for example, in Antibody-Drug Conjugates and Immunotoxins', Phillips, G. L., Ed.; Springer Verlag: New York, 2013; Antibody-Drug Conjugates; Ducry, L., Ed.; Humana Press, 2013; Antibody-Drug Conjugates; Wang, J., Shen, W.-C., and Zaro, J. L., Eds.; Springer International Publishing, 2015, the contents of each incorporated herein in their entirety by reference. In certain embodiments, the linker L portion of the linker-payloads described herein is a moiety capable of divalently and covalently linking a RG1reactive group to a polynucleotide (T) described herein. The linker- polynucleotides can be further bonded to an antigen-binding protein (BA) such as antibodies or antigen binding fragments thereof to form conjugates.

[0155] In certain embodiments, the linkers are stable in physiological conditions. In certain embodiments, the linkers are cleavable, for instance, able to release a polynucleotide (T) in the presence of an enzyme or at a particular pH range or value. In some embodiments, a linker comprises an enzyme-cleavable moiety. Illustrative enzyme-cleavable moieties include, but are not limited to, peptide bonds, ester linkages, hydrazones, and disulfide linkages. In some embodiments, the linker comprises a cathepsin-cleavable linker. In some embodiments, the linker comprises a moiety that is stable at certain pHs and cleavable to release the polynucleotide (T) at other pHs. For instance, in certain embodiments, the linker is stable at physiological pH and capable of releasing the polynucleotide (T) at a local pH in the vicinity of a target.

[0156] In certain embodiments, the linker is an alkylene or heteroalkylene. In certain embodiments, the linker is Ci-20 alkylene. In certain embodiments, the linker is Ci-12 alkylene. In certain embodiments, the linker is Ci-10 alkylene. In certain embodiments, the linker is Ci-8 alkylene. In certain embodiments, the linker is Ci-20 heteroalkylene. In certain embodiments, the linker is Ci-12 heteroalkylene. In certain embodiments, the linker is Ci-10 heteroalkylene. In certain embodiments, the linker is Ci-8 heteroalkylene. Additional suitable linkers include, but are not limited to, acid-labile linkers, hydrolytically-labile linkers, enzymatically cleavable linkers, reduction-labile linkers, self-immolative linkers, and non-cleavable linkers. Suitable linkers also include, but are not limited to, those that are or comprise peptides, glucuronides, succinimide-36313810218250298.00092511911WO01 thioethers, polyethylene glycol (PEG) units (e.g., PEGn, (CH2CH2O)n, and (CH2OCH2)n, and used interchangeably herein), hydrazones, mal-caproyl units, dipeptide units, valine-citruline units, para-aminobenzyloxycarbonyl (PABC), and para-aminobenzyl (PAB) units.

[0157] In some embodiments, the linker iswhereinSP1is a spacer;SP2is a spacer;-f-< is one or more bonds to the RG1 reactive group; is one or more bonds to the polynucleotide (T); each AA is an amino acid residue; and n is an integer from zero to ten.

[0158] In some embodiments, the SP1spacer comprises an alkylene. In some embodiments, the SP1spacer comprises a C5-7 alkylene. In some embodiments, the SP1spacer comprises a polyether. In some embodiments, the SP1spacer comprises a polymer of ethylene oxide such as polyethylene glycol.

[0159] In some embodiments, the SP1spacer iswhereRG' is a reactive group residue following reaction of a reactive group RG with a binding agent;-f «- is a bond to the binding agent; is a bond to (AA)nwherein n is an integer from zero to ten; and b is an integer from two to eight.313810218250298.000925 11911WO01

[0160] In certain embodiments, linker L is a linker described in WO 2024 / 168199 to Han et al., which is hereby incorporated by reference in its entirety.VI. Polynucleotide Pay loads

[0161] The polynucleotide payload can be any polynucleotide deemed suitable by the person of skill in the art. In some embodiments, the polynucleotide molecule is an interfering nucleic acid molecule, a guide RNA, a ribozyme, an aptamer, a gapmer, a mixmer, a multimer, or an mRNA.

[0162] In some embodiments, the interfering nucleic acid is an siRNA, an shRNA, a miRNA, or an antisense oligonucleotide. In some embodiments, the interfering nucleic acid is an siRNA. In some embodiments, the interfering nucleic acid is an antisense oligonucleotide. In some embodiments, the polynucleotide molecule is a guide RNA. In various embodiments, the polynucleotide molecule comprises one or more modified nucleotides.

[0163] In some embodiments, the molecular cargo of the protein-drug conjugate is an interfering RNA (e.g., siRNA) selected from the group consisting of an interfering RNA (e.g., siRNA) that inhibits the DMPK, CNBP, Dystrophin, DUX4, MTM1, LAMA2 or FKRP gene or a mutant thereof.

[0164] Non-limiting examples of polynucleotide molecules that are useful as molecular cargoes in the protein-drug conjugates of the present invention include, but are not limited to, interfering nucleic acids (e.g., shRNAs, siRNAs, microRNAs, antisense oligonucleotides), gapmers, mixmers, ribozymes, phosphorodiamidite morpholinos, peptide nucleic acids, aptamers, and guide nucleic acids (e.g., Cas9 guide RNAs), mRNAs, etc. In various embodiments, a polynucleotide may comprise one or more modified nucleotides. In various embodiments, a polynucleotide may comprise one or more modified inter-nucleotide linkage. Polynucleotides may be single-stranded or double-stranded.

[0165] In some embodiments, the polynucleotide molecule is DNA. In some embodiments, the polynucleotide molecule is RNA.

[0166] In various embodiments, a polynucleotide described herein (e.g., interfering nucleic acid or guide RNA) may comprise a region of complementarity to a target nucleic acid which can be in the range of 8 to 15, 8 to 30, 8 to 40, 10 to 50, 5 to 50, or 5 to 40 nucleotides in length. In certain embodiments, a region of complementarity of a polynucleotide to a target nucleic acid may be 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 nucleotides in length. In38313810218250298.00092511911WO01 some embodiments, the region of complementarity may be complementary with at least 10 consecutive nucleotides of a target nucleic acid. In some embodiments, a polynucleotide may contain 1, 2, 3, 4, or 5 base mismatches compared to the portion of the consecutive nucleotides of target nucleic acid. In some embodiments the polynucleotide may have up to 3 mismatches over 15 bases, or up to 4 mismatches over 10 bases. In some embodiments, the polynucleotide is complementary (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or 100%) to a target sequence of any one of the polynucleotides of the present disclosure. In various embodiments, such target sequence may be 100% complementary to the polynucleotide described herein. In some embodiments, any one or more of the thymine bases (T's) in any one of the polynucleotides described herein may be uracil bases (U's), and / or any one or more of the U's may be T's. A target sequence described herein may comprise a sequence of nucleic acid in a target gene that has complementarity to the guide sequence of the gRNA. The interaction of the target sequence and the guide sequence directs an RNA-guided DNA-binding agent (e.g., Cas protein) to bind, and potentially nick or cleave (depending on the activity of the agent), within the target sequence.

[0167] The polynucleotides described herein may be modified, e.g., comprise a modified nucleotide, a modified internucleoside linkage, and / or a modified sugar moiety, or combinations thereof. In addition, polynucleotides can possess one or more of the following properties: have improved cell uptake compared to unmodified polynucleotides; are not toxic to cells or mammals; are not immune stimulatory; avoid pattern recognition receptors; do not mediate alternative splicing; are nuclease resistant; have improved endosomal exit internally in a cell; or minimize TLR stimulation. Any of the various modified chemistries or formats of polynucleotides disclosed herein may be combined with each other. As a non-limiting example, one, two, three, four, five, six, seven, eight or more different types of modifications may be included within the same polynucleotide.

[0168] In various embodiments, particular nucleotide modification(s) may be used that render a polynucleotide into which the modification(s) are incorporated more resistant to nuclease digestion than the native oligoribonucleotide or oligodeoxy nucleotide molecules; such modified polynucleotides stay intacted for a longer time than unmodified polynucleotides. Exemplary modified polynucleotides include those comprising modified backbones, for example, modified internucleoside linkages such as, methyl phosphonates, phospho triesters, phosphorothioates short chain alkyl or cycloalkyl intersugar linkages, heterocyclic intersugar linkages, or short chain39313810218250298.000925 11911WO01 heteroatomic linkages. As such, polynucleotides described herein may be stabilized against nucleolytic degradation, e.g., via incorporation of a modification such as a nucleotide modification.

[0169] In various embodiments, a polynucleotide may be of up to 50 nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, or 2 to 45 nucleotides of the polynucleotide may be modified nucleotides. The polynucleotide may be of 8 to 30 nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, or 2 to 30 nucleotides of the polynucleotide can be modified nucleotides. In some embodiments, the polynucleotide may be of 8 to 15 nucleotides in length in which 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, or 2 to 14 nucleotides of the polynucleotide are modified nucleotides. In some embodiments, the polynucleotides can have every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides are modified.

[0170] In various embodiments, the polynucleotide disclosed herein may comprise at least one nucleoside, e.g., modified at the 2' position of the sugar. In some embodiments, all of the nucleosides in the polynucleotide are 2’-modified nucleosides. In some embodiments, a polynucleotide comprises at least one 2'-modified nucleoside.

[0171] In various embodiments, the polynucleotide disclosed herein may one or more non- bicyclic 2’-modified nucleosides, e.g., 2’-O- dimethylaminoethyloxyethyl (2’-0-DMAE0E), 2’- O-methyl (2’- O-Me), 2’-O- dimethylaminoethyl (2’-0-DMA0E), 2’-O-methoxyethyl (2’-M0E), 2’-deoxy, 2’-O-N-methylacetamido (2’-0-NMA) modified nucleoside, 2’-fluoro (2’-F), 2’-O- aminopropyl (2’-O-AP), or 2’-O-dimethylaminopropyl (2’-0-DMAP).

[0172] In some embodiments, the polynucleotide of the present disclosure may comprise one or more 2’ -4’ bicyclic nucleosides in which the ribose ring may comprise a bridge moiety, e.g., connecting two atoms in the ring (e.g., connecting the 2’-0 atom to the 4’-C atom via an ethylene (ENA) bridge, a methylene (LNA) bridge, or a (S)-constrained ethyl (cEt) bridge). Non-limiting examples of ENAs are disclosed in WO 2005 / 042777; Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Koizumi, Curr. Opin. Mol. Ther., 8: 144-149, 2006, Surono et al., Hum. Gene Ther., 15:749-757, 2004; and Horie et al., Nucleic Acids Symp. Ser (Oxf), 49: 171-172, 2005; the disclosures of which are incorporated herein by reference in their entireties. Non-limiting examples of LNAs are disclosed in W02008 / 043753, the contents of which is incorporated herein by reference in its entirety. Non-limiting examples of cEt are disclosed in in U.S. Patent Nos40313810218250298.00092511911WO017,569,686, 7,101,993, and 7,399,845 each of which is herein incorporated by reference in its entirety.

[0173] In various embodiments, the polynucleotide described herein may comprise a modified nucleoside disclosed in, for example, US Patent Nos. 8,022,193; 7,569,686; 7,399,845; 7,741,457; 7,335,765; 7,816,333; 8,957,201; 7,314,923, the entire contents of each of which are incorporated herein by reference for all purposes.

[0174] In various embodiments, the polynucleotide comprises at least one modified nucleoside that results in an increase in Tm of the polynucleotide in a range of 1°C to 10°C compared with a polynucleotide that does not have the at least one modified nucleoside. The polynucleotide may have a plurality of modified nucleosides that result in a total increase in Tm of the polynucleotide in a range of 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C or more as compared to a polynucleotide which does not have the modified nucleoside.

[0175] In some embodiments, the polynucleotide may comprise a mix of nucleosides of different kinds. A polynucleotide may comprise a mix of deoxyribonucleosides or ribonucleosides and 2’-0-Me modified nucleosides. A polynucleotide may comprise a mix of 2’-4’ bicyclic nucleosides and 2’- MOE, 2’-fluoro, or 2’-0-Me modified nucleosides. A polynucleotide may comprise a mix of non-bicyclic 2’-modified nucleosides (e.g., 2’-M0E, 2’-fluoro, or 2’-0-Me) and 2’-4’ bicyclic nucleosides (e.g., LNA, ENA, cEt). A polynucleotide may comprise a mix of 2’ -deoxyribonucleosides or ribonucleosides and 2’-fluoro modified nucleosides. A polynucleotide may comprise a mix of 2’-fluoro modified nucleosides and 2’-0-Me modified nucleosides.

[0176] In various embodiments, the oligonucleotide may comprise alternating nucleosides of different types. In certain embodiments, the oligonucleotide may comprise alternating deoxyribonucleosides or ribonucleosides and 2’-0-Me modified nucleosides. In certain embodiments, a polynucleotide may comprise alternating 2’-deoxyribonucleosides or ribonucleosides and 2’ -fluoro modified nucleosides. In certain embodiments, the oligonucleotide may comprise alternating 2’-fluoro modified nucleosides and 2’-0-Me modified nucleosides. In certain embodiments, the oligonucleotide may comprise alternating 2’-4’ bicyclic nucleosides and 2’ -MOE, 2’ -fluoro, or 2’-0-Me modified nucleosides. In certain embodiments, the oligonucleotide may comprise alternating non-bicyclic 2’-modified nucleosides (e.g., 2’-M0E, 2’-fluoro, or 2’-O- Me) and 2’- 4’ bicyclic nucleosides (e.g., LNA, ENA, cEt).41313810218250298.00092511911WO01

[0177] In various embodiments, a polynucleotide of the present disclosure may comprise one or more abasic residues, a 5 - vinylphosphonate modification, and / or one or more inverted abasic residues.

[0178] In various embodiments, the oligonucleotide may comprise a phosphorothioate or other modified internucleoside linkage. In various embodiments, the oligonucleotide may comprise phosphorothioate internucleoside linkages. In various embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages between at least two nucleotides. In various embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages between all nucleotides. By way of a non-limiting example, in certain embodiments, oligonucleotides comprise modified internucleoside linkages at the first, second, and / or (e.g., and) third internucleoside linkage at the 5' or 3' end of the nucleotide sequence.

[0179] Non-limiting examples of phosphorus-containing linkages include aminoalkylphosphotriesters, phosphorothioates, chiral phosphorothioates, phosphotriesters, phosphorodithioates, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3'-amino phosphoramidate and aminoalky Iphosphoramidates, thionoalkylphosphonates, thionophosphoramidates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see U.S. Pat. Nos. 5,625,050; 4,469,863; 4,476,301; 5,023,243; 5,550,111; 5,177,196; 5,587,361; 5,188,897; 5,264,423; 5,276,019; 5,519,126; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925; 5,536,821; 5,541,306; 5,563, 253; 5,571,799; and 3,687,808.

[0180] In various embodiments, a polynucleotide of the present disclosure may have heteroatom backbones, e.g., or peptide nucleic acid (PNA) backbones (wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497), morpholino backbones (see Summerton and Weller, U.S. Patent No. 5,034,506); amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); or MMI or methylene(methylimino) backbones.

[0181] Nitrogenous bases can be conventional bases (A, G, C, T, U), analogs thereof (e.g., modified uridines such as 5-methoxyuridine, pseudouridine, or N1 -methylpseudouridine, or42313810218250298.00092511911WO01 others); inosine; derivatives of purines or pyrimidines (e.g., N4-methyl deoxy guanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position (e.g., 5 -methylcytosine), purine bases with a substituent at the 2, 6, or 8 positions, 2- amino-6-methylaminopurine, 6-0 -methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4- dimethylhydrazine-pyrimidines, and 4-O-alkyl-pyrimidines; U.S. Patent No. 5,378,825 and WO 93 / 13121). For general discussion see Adams et al, The Biochemistry of the Nucleic Acids 5-36, 11th ed., 1992. Nucleic acids can include one or more “abasic” residues where the backbone includes no nitrogenous base for position(s) of the polymer (U.S. Patent No. 5,585,481). A nucleic acid can comprise only conventional RNA or DNA sugars, bases and linkages, or can include both conventional components and substitutions (e.g., conventional nucleosides with 2’ methoxy substituents, or polymers containing both conventional nucleotides and one or more nucleotide analogs). Nucleic acid includes “locked nucleic acid” (LNA), an analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhance hybridization affinity toward complementary RNA and DNA sequences (Vester and Wengel, “LNA (Locked Nucleic Acid): High-Affinity Targeting of Complementary RNA and DNA,” Biochemistry 43(42): 13233-13241 (2004)). RNA and DNA have different sugar moieties and can differ by the presence of uracil or analogs thereof in RNA and thymine or analogs thereof in DNA.Interfering Nucleic Acids

[0182] In some embodiments, a conjugated molecular cargo may comprise a polynucleotide molecule(s) which is capable of modifying expression of one more gene in a target cell. In some embodiments, the polynucleotide molecule may be an interfering nucleic acid molecule, e.g., an siRNA, an shRNA, a miRNA, or an antisense oligonucleotide, that targets, e.g., an RNA (e.g., an mRNA).

[0183] In certain embodiments, interfering nucleic acid molecules that selectively target and inhibit the activity or expression of a product (e.g., an mRNA product) of a targeted gene are used in compositions and methods described herein. An interfering nucleic acid molecule may inhibit the expression or activity of a product (e.g., an mRNA product) of at least one targeted gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. An agent disclosed herein may43313810218250298.000925 11911WO01 comprise at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementarity to a product (e.g., an mRNA product) of at least targeted gene. Without wishing to be bound by theory, “complementarity ” of nucleic acids can mean that a nucleotide sequence in one strand of nucleic acid, due to orientation of its nucleobase groups, forms hydrogen bonds with another sequence on an opposing nucleic acid strand. The complementary bases in DNA are typically A with T and C with G. In RNA, they are typically C with G and U with A. Complementarity can be perfect or substantial / sufficient. Perfect complementarity between two nucleic acids means that the two nucleic acids can form a duplex in which every base in the duplex is bonded to a complementary base by Watson-Crick pairing. “Substantial” or “sufficient” complementary means that a sequence in one strand is not completely and / or perfectly complementary to a sequence in an opposing strand, but that sufficient bonding occurs between bases on the two strands to form a stable hybrid complex in set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by using the sequences and standard mathematical calculations to predict the Tm (melting temperature) of hybridized strands, or by empirical determination of Tm by using routine methods. Tm includes the temperature at which a population of hybridization complexes formed between two nucleic acid strands are 50% denatured (i.e., a population of double-stranded nucleic acid molecules becomes half dissociated into single strands). At a temperature below the Tm, formation of a hybridization complex is favored, whereas at a temperature above the Tm, melting or separation of the strands in the hybridization complex is favored. Tm may be estimated for a nucleic acid having a known G+C content in an aqueous 1 M NaCl solution by using, e.g., Tm=81.5+0.41(% G+C), although other known Tm computations take into account nucleic acid structural characteristics.

[0184] Interfering nucleic acids can include a sequence of cyclic subunits, each bearing a basepairing moiety, linked by intersubunit linkages that allow the base-pairing moieties to hybridize to a target sequence in a nucleic acid (typically an RNA) by Watson-Crick base pairing, to form a nucleic acid: oligomer heteroduplex within the target sequence.

[0185] Typically, at least 17, 18, 19, 20, 21, 22, or 23 nucleotides of the complement of the target mRNA sequence are sufficient to mediate inhibition of a target transcript. Perfect complementarity is not necessary. In some embodiments, the interfering nucleic acid molecule is44313810218250298.000925 11911WO01 single-stranded RNA. In some embodiments, the interfering nucleic acid molecule is doublestranded RNA. The double-stranded RNA molecule may have a 2 nucleotide 3 ’ overhang. In some embodiments, the two RNA strands are connected via a hairpin structure, forming a shRNA molecule. shRNA molecules can contain hairpins derived from microRNA molecules.

[0186] Interfering nucleic acid molecules described herein can contain RNA bases, non-RNA bases or a mixture of RNA bases and non-RNA bases. For example, interfering nucleic acid molecules described herein can be primarily composed of RNA bases but also contain DNA bases or non-naturally occurring nucleotides. The term “ribonucleotide” or “nucleotide” can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions.

[0187] In some embodiments, the interfering nucleic acid molecule is a small interfering RNAs (siRNA), also known as short interfering RNA or silencing RNA. siRNA, is a class of doublestranded RNA molecules, typically about 20-25 base pairs in length that target nucleic acids (e.g., mRNAs) for degradation via the RNA interference (RNAi) pathway in cells. Such siRNA molecules typically include a region of sufficient homology to the target region, and be of sufficient length in terms of nucleotides, such that the siRNA molecule down-regulate target nucleic acid. It is not necessary that there be perfect complementarity between the siRNA molecule and the target, but the correspondence must be sufficient to enable the siRNA molecule to direct sequence-specific silencing, such as by RNAi cleavage of the target RNA. In some embodiments, the sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double-strand character of the molecule.

[0188] Specificity of siRNA molecules may be measured via the binding of the antisense strand of the molecule to its target RNA. Effective siRNA molecules are often fewer than 30 to 35 base pairs in length, e.g., to prevent stimulation of non-specific RNA interference pathways in the cell by way of the interferon response, however longer siRNA may also be effective. In various embodiments, the siRNA molecules are 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, 35, 40, 45, 50, 55, 60, 65, 70, or more base pairs in length. In some embodiments, the siRNA molecules are 8 to 40 base pairs in length, 10 to 20 base pairs in length, 10 to 30 base pairs in length, 15 to 20 base pairs in length, 19 to 23 base pairs in length, or 21 to 24 base pairs in length.45313810218250298.00092511911WO01

[0189] After selection of a suitable target RNA sequence, siRNA molecules that comprise a nucleotide sequence complementary to all or a portion of the target sequence, i.e., an antisense sequence, may be designed and prepared using suitable methods (see, e.g., U.S. Patent Publication Nos. 2004 / 0077574 and 2008 / 0081791 and WO 2004 / 016735). In some embodiments, the siRNA molecule may be single-stranded (i.e. a ssRNA molecule comprising just an antisense strand) or double stranded (i.e. a dsRNA molecule comprising an antisense strand and a complementary sense strand that hybridizes to form the dsRNA). In various embodiments, the siRNA molecules may comprise a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, comprising self-complementary sense and / or antisense strands.

[0190] In various embodiments, the antisense strand of the siRNA molecule is 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, 35, 40, 45, 50, 55, 60, 65, 70, or more nucleotides in length. In some embodiments, the antisense strand is 8 to 40 nucleotides in length, 10 to 20 nucleotides in length, 10 to 30 nucleotides in length, 15 to 20 nucleotides in length, 19 to 23 nucleotides in length, 21 to 24 nucleotides in length.

[0191] In some embodiments, the sense strand of the siRNA molecule is 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, 35, 40, 45, 50, 55, 60, 65, 70, or more nucleotides in length. In some embodiments, the sense strand is 8 to 40 nucleotides in length, 10 to 20 nucleotides in length, 10 to 30 nucleotides in length, 15 to 20 nucleotides in length, 19 to 23 nucleotides in length, or 21 to 24 nucleotides in length.

[0192] In various embodiments, siRNA molecules can comprise an antisense strand comprising a region of complementarity to a target region in a target mRNA. In some embodiments, the region of complementarity is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to a target region in a target mRNA. In some embodiments, the target region may comprise a region of consecutive nucleotides in the target mRNA. In some embodiments, it may not be requisite for a complementary nucleotide sequence to be 100% complementary to that of its target to be specifically hybridizable or specific for a target RNA sequence.

[0193] In some embodiments, siRNA molecules disclosed herein may comprise an antisense strand that comprises a region of complementarity to a target RNA sequence and the region of complementarity is in the range of 8 to 20, 8 to 35, 8 to 45, 10 to 50, 5 to 55, or 5 to 40 nucleotides46313810218250298.00092511911WO01 in length. In some embodiments, a region of complementarity is 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 nucleotides in length. In some embodiments, the region of complementarity is complementary with at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, or more consecutive nucleotides of a target RNA sequence. In some embodiments, siRNA molecules comprise a nucleotide sequence that contains no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base mismatches compared to the portion of the consecutive nucleotides of target RNA sequence. In some embodiments, siRNA molecules comprise a nucleotide sequence that has up to 3 mismatches over 15 bases, or up to 4 mismatches over 10 bases.

[0194] In various embodiments, siRNA molecules may comprise an antisense strand comprising a nucleotide sequence that is complementary (e.g., at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, or 100%) to the target RNA sequence of the oligonucleotides disclosed herein. In some embodiments, siRNA molecules comprise an antisense strand comprising a nucleotide sequence that is at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, or 100% identical to the oligonucleotides provided herein. In some embodiments, siRNA molecules comprise an antisense strand comprising at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, or more consecutive nucleotides of the oligonucleotides provided herein.

[0195] In some embodiments, double-stranded siRNA can comprise sense and anti-sense RNA strands that are different lengths or the same length. In some embodiments, double-stranded siRNA molecules may also be generated from a single oligonucleotide in a stem-loop structure and the self-complementary sense and antisense regions of the siRNA molecule may be linked by means of a nucleic acid based or non-nucleic acid-based linker(s), and / or by a circular single-stranded RNA having two or more loop structures and a stem comprising self-complementary sense and antisense strands. In some embodiments, the circular RNA may be processed in vivo or in vitro to produce an active siRNA molecule which may be capable of mediating RNAi. Small hairpin RNA (shRNA) molecules are therefore also contemplated in the present disclosure. Such molecules may comprise a specific antisense sequence together with the reverse complement (sense) sequence,47313810218250298.00092511911WO01 which may be separated by a spacer or loop sequence in some instances. A reverse complement described herein may comprise a sequence that is a complement sequence of a reference sequence, wherein the complement sequence is written in the reverse orientation. Due to codon usage redundancy, a reverse complement can diverge from a reference sequence that encodes the same polypeptide. As used herein, “reverse complement” also includes sequences that are, e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the reverse complement sequence of a reference sequence. Cleavage of the spacer or loop can provide a single- stranded RNA molecule and its reverse complement, such that they may anneal to form a dsRNA molecule. In various embodiments, additional optional processing steps may result in removal or addition of 1, 2, 3, 4, 5 or more nucleotides from the 3' end and / or the 5' end of one or both strands. A spacer may be of a suitable length to allow the antisense and sense sequences to anneal and form a double- stranded structure or stem prior to cleavage of the spacer. In certain embodiments subsequent optional processing steps may result in removal or addition of 1, 2, 3, 4, 5 or more nucleotides from the 3' end and / or the 5' end of one or both strands. In some embodiments, a spacer sequence can be an unrelated nucleotide sequence that may be, e.g., situated between two complementary nucleotide sequence regions that, when annealed into a double-stranded nucleic acid, can comprise a shRNA.

[0196] The length of the siRNA molecules can vary from about 10 to about 120 nucleotides depending on the type of siRNA molecule being designed. Generally, between about 10 and about 55 of these nucleotides may be complementary to the RNA target sequence. For instance, when the siRNA is a double-stranded siRNA or single-stranded siRNA, the length can vary from about 10 to about 55 nucleotides, whereas when the siRNA is a shRNA or circular molecule, the length can vary from about 30 nucleotides to about 100 nucleotides.

[0197] In various embodiments, an siRNA molecule can comprise a 3' overhang at one end of the molecule. In some embodiments, the other end can be blunt-ended or may also comprise an overhang (e.g., 5' and / or 3'). When the siRNA molecule comprises an overhang at both ends of the molecule, the length of the overhangs may be different or the same. In some embodiments, an siRNA molecule described herein may comprises 3' overhangs of about 1 to about 3 nucleotides on both ends of the molecule. In some embodiments, the siRNA molecule comprises 3’ overhangs of about 1 to about 3 nucleotides on both the sense strand and the antisense strand. In some embodiments, the siRNA molecule comprises 3’ overhangs of about 1 to about 3 nucleotides on48313810218250298.00092511911WO01 the antisense strand. In some embodiments, the siRNA molecule may comprise 3’ overhangs of about 1 to about 3 nucleotides on the sense strand.

[0198] In various embodiments, the siRNA molecule comprises one or more modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more). In certain embodiments, the siRNA molecule can comprise one or more modified nucleotides and / or one or more modified internucleotide linkages. In some embodiments, the modified nucleotide may comprise a modified sugar moiety (e.g., a 2' modified nucleotide). In some embodiments, the siRNA molecule can comprise one or more 2’ modified nucleotides, e.g., a 2'-deoxy, 2'-fluoro (2’-F), 2'-O-methyl (2’- O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'- O- DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O- DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA). In various embodiments, each nucleotide of the siRNA molecule can a modified nucleotide (e.g., a 2'-modified nucleotide). In some embodiments, the siRNA molecule may comprise one or more phosphorodiamidate morpholinos. In some embodiments, each nucleotide of the siRNA molecule consists of a phosphorodiamidate morpholino.

[0199] In various embodiments, the siRNA molecule may comprise a phosphorothioate or other modified internucleotide linkage. In various embodiments, the siRNA molecule may comprise, e.g., a phosphorothioate internucleoside linkage(s). In some embodiments, the siRNA molecule may comprise a phosphorothioate internucleoside linkage(s) between two or more nucleotides. In some embodiments, the siRNA molecule may comprise a phosphorothioate internucleoside linkage(s) between all nucleotides. In some embodiments, the siRNA molecule may comprise modified intemucleotide linkages at the first, second, and / or third internucleoside linkage at the 5' or 3' end of the siRNA molecule.

[0200] In various embodiments, the modified internucleotide linkages may comprise phosphorus-containing linkages. In some embodiments, phosphorus-containing linkages which may be used in the practice of the present disclosure include, without limitation, chiral phosphorothioates, phosphorothioates, phosphorodithioates, aminoalkylphosphotriesters, phosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphoramidates comprising 3 '-amino phosphoramidate and aminoalky Iphosphoramidates, phosphinates, thionoalkylphosphonates, thionophosphoramidates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked49313810218250298.00092511911WO01 analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see US Patent Nos. 5,625,050; 3,687,808; 4,469,863; 4,476,301; 5,177,196; 5,455, 233; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,519,126; 5,453,496; 5,466,677; 5,476,925; 5,536,821; 5,023,243; 5,541,306; 5,550,111; 5,563, 253; 5,571,799; 5,587,361; and 5,188,897.

[0201] Any of the various modified formats or chemistries of siRNA molecules disclosed herein may be combined together. For example, without limitation, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different types of modifications may be included within the same siRNA molecule.

[0202] In various embodiments, the antisense strand may comprise one or more modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more). In some embodiments, the antisense strand may comprise one or more modified nucleotides and / or one or more modified internucleotide linkage(s). In some embodiments, the modified nucleotide may comprise a modified sugar moiety (e.g., a 2' modified nucleotide). In some embodiments, the antisense strand comprises one or more 2' modified nucleotides, e.g., a 2'-deoxy, 2'-fluoro (2’-F), 2'-O-methyl (2’- O-Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-aminopropyl (2'-O-AP), 2'-O- dimethylaminoethyl (2'- O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O- dimethylaminoethyloxyethyl (2'-O- DMAEOE), or 2'-O-N-methylacetamido (2'-0-NMA). In various embodiments, each nucleotide of the antisense strand can be a modified nucleotide (e.g., a 2'-modified nucleotide). In some embodiments, the antisense strand may comprise one or more phosphorodiamidate morpholinos. In some embodiments, the antisense strand consists of a phosphorodiamidate morpholino oligomer (PMO).

[0203] In some embodiments, antisense strand contains a phosphorothioate or other modified internucleotide linkage. In some embodiments, the antisense strand may comprise phosphorothioate internucleoside linkage(s). In some embodiments, the antisense strand may comprise phosphorothioate internucleoside linkage(s) between two or more nucleotides. In some embodiments, the antisense strand may comprise phosphorothioate internucleoside linkage(s) between all nucleotides. In some embodiments, the antisense strand may comprise modified intemucleotide linkages at the first, second, and / or third nucleotide at the 5' or 3' end of the siRNA molecule.

[0204] In various embodiments, the modified internucleotide linkages may comprise phosphorus-containing linkages. In some embodiments, phosphorus-containing linkages which50313810218250298.00092511911WO01 may be used in the practice of the present disclosure include, without limitation, chiral phosphorothioates, phosphorothioates, phosphorodithioates, aminoalkylphosphotriesters, phosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphoramidates comprising 3 '-amino phosphoramidate and aminoalky Iphosphoramidates, phosphinates, thionoalkylphosphonates, thionophosphoramidates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see US Patent Nos. 5,625,050; 3,687,808; 4,469,863; 4,476,301; 5,177,196; 5,455, 233; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,519,126; 5,453,496; 5,466,677; 5,476,925; 5,536,821; 5,023,243; 5,541,306; 5,550,111; 5,563, 253; 5,571,799; 5,587,361; and 5,188,897.

[0205] Any of the modified formats or chemistries of the antisense strand disclosed herein may be combined together. For example, without limitation, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different types of modifications may be included within the same antisense strand.

[0206] In some embodiments, the sense strand comprises one or more modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15 or more). In some embodiments, the antisense strand may comprise one or more modified nucleotides and / or one or more modified internucleotide linkage(s). In some embodiments, the modified nucleotide may comprise a modified sugar moiety (e.g., a 2' modified nucleotide). In some embodiments, the antisense strand comprises one or more 2' modified nucleotides, e.g., a 2'-deoxy, 2'-fluoro (2’-F), 2'-O-methyl (2’-0-Me), 2'-O- methoxyethyl (2'-M0E), 2'-O-aminopropyl (2'-O-AP), 2'-O- dimethylaminoethyl (2'-0-DMA0E), 2'-O-dimethylaminopropyl (2'-0-DMAP), 2'-O- dimethylaminoethyloxyethyl (2'-0-DMAE0E), or 2'-O-N-methylacetamido (2'-0-NMA). In various embodiments, each nucleotide of the antisense strand can be a modified nucleotide (e.g., a 2'-modified nucleotide). In some embodiments, the antisense strand may comprise one or more phosphorodiamidate morpholinos. In some embodiments, the antisense strand consists of a phosphorodiamidate morpholino oligomer (PMO).

[0207] In some embodiments, the sense strand contains a phosphorothioate or other modified internucleotide linkage. In some embodiments, the sense strand may comprise phosphorothioate internucleoside linkage(s). In some embodiments, the sense strand may comprise phosphorothioate internucleoside linkage(s) between two or more nucleotides. In some embodiments, the sense51313810218250298.00092511911WO01 strand may comprise phosphorothioate internucleoside linkages between all nucleotides. For example, in some embodiments, the sense strand comprises modified internucleotide linkages at the first, second, and / or third nucleotide at the 5' or 3' end of the sense strand.

[0208] In various embodiments, the modified internucleotide linkages may comprise phosphorus-containing linkages. In some embodiments, phosphorus-containing linkages which may be used in the practice of the present disclosure include, without limitation, chiral phosphorothioates, phosphorothioates, phosphorodithioates, aminoalkylphosphotriesters, phosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphoramidates comprising 3'-amino phosphoramidate and aminoalky Iphosphoramidates, phosphinates, thionoalkylphosphonates, thionophosphoramidates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see U.S. Pat. Nos. 5,625,050; 3,687,808; 4,469,863; 4,476,301; 5,177,196; 5,455, 233; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,519,126; 5,453,496; 5,466,677; 5,476,925; 5,536,821; 5,023,243; 5,541,306; 5,550,111; 5,563, 253; 5,571,799; 5,587,361; and 5,188,897. In particular embodiments, a phophorothioate group at one terminus of the polynucleotide T bonds to L.

[0209] Any of the modified chemistries or formats of the sense strand described herein can be combined together. For example, without limitation, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different types of modifications may be included within the same sense strand.

[0210] In various embodiments, the antisense or sense strand of the siRNA molecule may comprise a modification(s) capable of enhancing or reducing, e.g., RNA-induced silencing complex (RISC) loading. In some embodiments, the antisense strand of the siRNA molecule may comprise a modification(s) capable of enhancing RISC loading. In various embodiments, the sense strand of the siRNA molecule may comprise(s) a modification(s) capable of reducing RISC loading and / or reducing off-target effects. In various embodiments, the antisense strand of the siRNA molecule may comprise a 2'-O- methoxy ethyl (2’ -MOE) modification. In some embodiments, the addition of the 2'-O-methoxyethyl (2’ -MOE) group, e.g., at the cleavage site may improve the silencing activity and / or specificity of siRNAs, e.g., by facilitating the oriented RNA-induced silencing complex (RISC) loading of the modified strand, e.g., as disclosed in Song et al., (2017) Mol Ther Nucleic Acids 9:242-250, incorporated herein by reference in its entirety.52313810218250298.00092511911WO01In various embodiments, the antisense strand of the siRNA molecule may comprise a 2'-0-Me- phosphorodithioate modification. In some embodiment, the 2'-O-Me-phosphorodithioate modification may increase RISC loading, e.g., as disclosed in Wu et al., (2014) Nat Commun 5:3459, incorporated herein by reference in its entirety.

[0211] In various embodiments, the sense strand of the siRNA molecule may comprise a 5'- nitroindole modification. In some embodiments, the 5 '-nitroindole modification may decrease the RNAi potency of the sense strand and / or reduces off-target effects, e.g., as disclosed in Zhang et al., (2012) Chembiochem 13(13): 1940-1945, incorporated herein by reference in its entirety. In various embodiments, the sense strand may comprise a 2’-O-methyl (2'-0-Me) modification. In some embodiments, the 2'- O-Me modification may reduce RISC loading and / or the off-target effects of the sense strand, e.g., as disclosed in Zheng et al., FASEB (2013) 27(10): 4017-4026, incorporated herein by reference in its entirety. In various embodiments, the sense strand of the siRNA molecule may be fully substituted with morpholino, 2'-M0E and / or 2'-0-Me residues, and may not be recognized by RISC, e.g., as disclosed in Kole et al., (2012) Nature reviews. Drug Discovery 11(2): 125- 140, incorporated herein by reference in its entirety.

[0212] In various embodiments, the sense strand of the siRNA molecule may comprise a 5'- morpholino modification. In various embodiments, the 5'-morpholino modification may reduce RISC loading of the sense strand and / or improves RNAi activity and / or antisense strand selection, e.g., as disclosed in Kumar et al., (2019) Chem Commun (Camb) 55(35):5139-5142, incorporated herein by reference in its entirety. In various embodiments, the sense strand of the siRNA molecule may be modified, for example, with a synthetic RNA-like high affinity nucleotide analogue called Locked Nucleic Acid (LNA) that may reduce RISC loading of the sense strand and promote antisense strand incorporation into RISC, e.g., as disclosed in Elman et al., (2005) Nucleic Acids Res. 33(1): 439-447, incorporated herein by reference in its entirety. In various embodiments, the sense strand of the siRNA molecule may comprise a 5' unlocked nucleic acid (UNA) modification. In various embodiments, the 5' unlocked nucleic acid (UNA) modification may reduce RISC loading of the sense strand and / or improve silencing capability of the antisense strand, e.g., as disclosed in Snead et al., (2013) Mol Ther Nucleic Acids 2(7):el03, incorporated herein by reference in its entirety.

[0213] In some embodiments, the antisense strand of the siRNA molecule may comprise a 2’- MOE modification and / or the sense strand may comprise a 2’-0-Me modification (see e.g., Song53313810218250298.00092511911WO01 et al., (2017) Mol Ther Nucleic Acids 9:242-250). In some embodiments at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 5, at least 8, at least 9, at least 10 or more) siRNA molecule may be conjugated, for example, covalently to an anti-TfR antigen-binding protein described herein. In some embodiments, the anti-TfR antigen-binding protein may be conjugated to the 5’ end of the sense strand of the siRNA molecule. In some embodiments, the anti-TfR antigen-binding protein may be conjugated to the 3’ end of the sense strand of the siRNA molecule. In some embodiments, the anti-TfR antigen-binding protein may be conjugated internally to the sense strand of the siRNA molecule. In some embodiments, the anti-TfR antigenbinding protein may be conjugated to the 5’ end of the antisense strand of the siRNA molecule. In some embodiments, the anti-TfR antigen-binding protein may be conjugated to the 3’ end of the antisense strand of the siRNA molecule. In some embodiments, the anti-TfR antigen-binding protein be conjugated internally to the antisense strand of the siRNA molecule.

[0214] In addition, an siRNA molecule may be modified or include nucleoside surrogates. Single stranded regions of an siRNA molecule may be modified or include nucleoside surrogates, e.g., the unpaired region or regions of a hairpin structure, e.g., a region which links two complementary regions, can have modifications or nucleoside surrogates. Modification to stabilize one or more 3'- or 5 '-terminus of an siRNA molecule, e.g., against exonucleases, or to favor the antisense siRNA agent to enter into RISC are also useful. Modifications can include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic, tri ethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that come as phosphoramidites and that have another DMT-protected hydroxyl group, allowing multiple couplings during RNA synthesis.

[0215] In some embodiments, the interfering nucleic acid molecule is a short hairpin RNA (shRNA). A “ small hairpin RNA ” or “short hairpin RNA” or “shRNA” described herein may include a short RNA sequence that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. The shRNAs provided herein may be chemically synthesized or transcribed from a transcriptional cassette in a DNA plasmid. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC).

[0216] Non-limiting examples of shRNAs include a double-stranded polynucleotide molecule assembled from a single- stranded molecule, where the sense and antisense regions are linked by a54313810218250298.000925 11911WO01 nucleic acid-based or non-nucleic acid-based linker; and a double-stranded polynucleotide molecule with a hairpin secondary structure having self-complementary sense and antisense regions. In some embodiments, the sense and antisense strands of the shRNA are linked by a loop structure comprising from about 1 to about 25 nucleotides, from about 2 to about 20 nucleotides, from about 4 to about 15 nucleotides, from about 5 to about 12 nucleotides, or 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, or more nucleotides.

[0217] Additional embodiments related to the shRNAs, as well as methods of designing and synthesizing such shRNAs, are described in U.S. Patent Publication No. 2011 / 0071208, the disclosure of which is herein incorporated by reference in its entirety for all purposes.

[0218] In some embodiments, the interfering nucleic acid molecule is a micro RNA (miRNA). miRNAs represent a large group of small RNAs produced naturally in organisms, some of which regulate the expression of target genes. Typically, miRNAs are generated from large RNA precursors (termed pri-miRNAs) that are processed in the nucleus into approximately 70 nucleotide pre-miRNAs, which fold into imperfect stem-loop structures. These pre-miRNAs typically undergo an additional processing step within the cytoplasm where mature miRNAs of 18-25 nucleotides in length are excised from one side of the pre-miRNA hairpin by an RNase III enzyme, Dicer. miRNAs are not translated into proteins, but instead bind to specific messenger RNAs, thereby blocking translation. In some embodiments, miRNAs base-pair imprecisely with their targets to inhibit translation.

[0219] miRNAs as described herein can include pri-miRNA, pre-miRNA, mature miRNA or fragments of variants thereof that retain the biological activity of mature miRNA. In some embodiments, the size range of the miRNA can be from 21 nucleotides to 170 nucleotides. In one embodiment, the size range of the miRNA is from 70 to 170 nucleotides in length. In another embodiment, mature miRNAs of from 21 to 25 nucleotides in length can be used.

[0220] In certain embodiments, the interfering nucleic acid molecule is an antisense oligonucleotide. An antisense oligonucleotide typically comprises a short nucleotide sequence which is substantially complementary to a target nucleotide sequence in a pre-mRNA molecule, heterogeneous nuclear RNA (hnRNA) or mRNA molecule. The degree of complementarity (or substantial complementarity) of the antisense sequence is preferably such that a molecule comprising the antisense sequence can form a stable double stranded hybrid with the target55313810218250298.00092511911WO01 nucleotide sequence in the RNA molecule under physiological conditions. Antisense oligonucleotides are often synthetic and chemically modified.

[0221] Antisense oligonucleotides may be 100% complementary to the target sequence, or may include mismatches, e.g., to improve selective targeting of allele containing the disease- associated mutation, as long as a heteroduplex formed between the oligonucleotide and target sequence is sufficiently stable to withstand the action of cellular nucleases and other modes of degradation which may occur in vivo. Hence, certain oligonucleotides may have about or at least about 70% sequence complementarity, e.g, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity, between the oligonucleotide and the target sequence. Oligonucleotide backbones that are less susceptible to cleavage by nucleases are discussed herein. Mismatches, if present, are typically less destabilizing toward the end regions of the hybrid duplex than in the middle. The number of mismatches allowed will depend on the length of the oligonucleotide, the percentage of G:C base pairs in the duplex, and the position of the mismatch(es) in the duplex, according to well understood principles of duplex stability.

[0222] The interfering nucleic acids can employ a variety of oligonucleotide chemistries. Examples of oligonucleotide chemistries include, without limitation, peptide nucleic acid (PNA), linked nucleic acid (LNA), phosphorothioate, 2’-O-Me-modified oligonucleotides, and morpholino chemistries, including combinations of any of the foregoing. In general, PNA and LNA chemistries can utilize shorter targeting sequences because of their relatively high target binding strength relative to 2’-0-Me oligonucleotides. Phosphorothioate and 2’-O-Me-modified chemistries are often combined to generate 2’-O-Me-modified oligonucleotides having a phosphorothioate backbone. See, e.g., WO / 2013 / 112053 and WO / 2009 / 008725, incorporated by reference in their entireties.

[0223] Peptide nucleic acids (PNAs) are analogs of DNA in which the backbone is structurally homomorphous with a deoxyribose backbone, consisting of N-(2-aminoethyl) glycine units to which pyrimidine or purine bases are attached. PNAs containing natural pyrimidine and purine bases hybridize to complementary oligonucleotides obeying Watson-Crick base-pairing rules, and mimic DNA in terms of base pair recognition (Egholm, Buchardt et al. 1993). The backbone of PNAs is formed by peptide bonds rather than phosphodiester bonds, making them well-suited for antisense applications. The backbone is uncharged, resulting in PNA / DNA or PNA / RNA duplexes56313810218250298.00092511911WO01 that exhibit greater than normal thermal stability. PNAs are not recognized by nucleases or proteases.

[0224] Despite a radical structural change to the natural structure, PNAs are capable of sequence-specific binding in a helix form to DNA or RNA. Characteristics of PNAs include a high binding affinity to complementary DNA or RNA, a destabilizing effect caused by single-base mismatch, resistance to nucleases and proteases, hybridization with DNA or RNA independent of salt concentration and triplex formation with homopurine DNA. PANAGENE™ has developed its proprietary Bts PNA monomers (Bts; benzothiazole-2-sulfonyl group) and proprietary oligomerization process. The PNA oligomerization using Bts PNA monomers is composed of repetitive cycles of deprotection, coupling and capping. PNAs can be produced synthetically using any technique known in the art. See, e.g., U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, 6,969,766, 7,211,668, 7,022,851, 7,125,994, 7,145,006 and 7,179,896. See also U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262 for the preparation of PNAs. Further teaching of PNA compounds can be found in Nielsen et al., Science, 254: 1497-1500, 1991. Each of the foregoing is incorporated by reference in its entirety.

[0225] Interfering nucleic acids described herein may also contain “locked nucleic acid” subunits (LNAs). “LNAs” are a member of a class of modifications called bridged nucleic acid (BNA). BNA is characterized by a covalent linkage that locks the conformation of the ribose ring in a C30-endo (northern) sugar pucker. For LNA, the bridge is composed of a methylene between the 2’-0 and the 4’-C positions. LNA enhances backbone preorganization and base stacking to increase hybridization and thermal stability.

[0226] The structures of LNAs can be found, for example, in Wengel, et al., Chemical Communications (1998) 455; Tetrahedron (1998) 54:3607, and Accounts of Chem. Research (1999) 32:301); Obika, et al., Tetrahedron Letters (1997) 38:8735; (1998) 39:5401, and Bioorganic Medicinal Chemistry (2008) 16:9230. Compounds provided herein may incorporate one or more LNAs; in some cases, the compounds may be entirely composed of LNAs. Methods for the synthesis of individual LNA nucleoside subunits and their incorporation into oligonucleotides are described, for example, in U.S. Pat. Nos. 7,572,582, 7,569,575, 7,084,125, 7,060,809, 7,053,207, 7,034,133, 6,794,499, and 6,670,461, each of which is incorporated by reference in its entirety. Typical intersubunit linkers include phosphodiester and phosphorothioate moieties; alternatively, non-phosphorous containing linkers may be employed. One embodiment is an LNA containing57313810218250298.000925 11911WO01 compound where each LNA subunit is separated by a DNA subunit. Certain compounds are composed of alternating LNA and DNA subunits where the intersubunit linker is phosphorothioate.

[0227] “Phosphorothioates” (or S-oligos) are a variant of normal DNA in which one of the nonbridging oxygens is replaced by a sulfur. The sulfurization of the internucleotide bond reduces the action of endo-and exonucleases including 5’ to 3’ and 3’ to 5’ DNA POL 1 exonuclease, nucleases SI and PI, RNases, serum nucleases and snake venom phosphodiesterase. Phosphorothioates are made by two principal routes: by the action of a solution of elemental sulfur in carbon disulfide on a hydrogen phosphonate, or by the method of sulfurizing phosphite triesters with either tetraethylthiuram disulfide (TETD) or 3H-1, 2-bensodithiol-3-one 1, 1 -dioxide (BDTD) (see, e.g., Iyer et al., J. Org. Chem. 55, 4693-4699, 1990). The latter methods avoid the problem of elemental sulfur’s insolubility in most organic solvents and the toxicity of carbon disulfide. The TETD and BDTD methods also yield higher purity phosphorothioates.

[0228] “2’ O-Me oligonucleotides” molecules carry a methyl group at the 2’ -OH residue of the ribose molecule. 2’-O-Me-RNAs show the same (or similar) behavior as DNA, but are protected against nuclease degradation. 2’-O-Me-RNAs can also be combined with phosphothioate oligonucleotides (PTOs) for further stabilization. 2’ -O-Me oligonucleotides (phosphodiester or phosphothioate) can be synthesized according to routine techniques in the art (see, e.g., Yoo et al., Nucleic Acids Res. 32:2008-16, 2004).

[0229] Interfering nucleic acid molecules can be prepared, for example, by chemical synthesis, in vitro transcription, or digestion of long dsRNA by RNase III or Dicer. These can be introduced into cells by transfection, electroporation, or other methods known in the art. See Hannon, GJ, 2002, Nature 418: 244- 251; Bernstein E et al., 2002, RNA 7: 1509-1521; Hutvagner G et al., Curr. Opin. Genetics & Development 12: 225-232; Brummelkamp, 2002, Science 296: 550-553; Lee NS, et al. 2002. Nature Biotechnol. 20:500-505; Miyagishi M, and Taira K. 2002. Nature Biotechnol. 20:497-500; Paddison PJ, et al., 2002. Genes & Dev. 16:948-958; Paul CP, et al., 2002. Nature Biotechnol. 20:505-508; Sui G et al., 2002. Proc. Natl. Acad. Sci. USA 99(6):5515-5520; Yu J-Y et al., 2002. Proc. Natl. Acad. Sci. USA 99(9):6047-6052. Each of the foregoing is incorporated by reference in its entirety.58313810218250298.00092511911WO01VII. Binding Agents

[0230] In some embodiments, the binding agent is an antibody or an antigen-binding fragment thereof. The antibody can be in any form known to those of skill in the art. The term “antibody,” as used herein, refers to any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen. The term “antibody” includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CLI). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments disclosed herein, the FRs of the antibodies (or antigen-binding portion thereof) suitable for the compounds herein may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. The term “antibody,” as used herein, also includes antigen-binding fragments of full antibody molecules. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, for example, from full antibody molecules using any suitable, standard technique(s) such as proteolytic digestion or recombinant genetic engineering technique(s) involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, for example, commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a59313810218250298.00092511911WO01 suitable configuration, or to introduce codons, create cysteine residues, modify, add, or delete amino acids, etc. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab’)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated CDR such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR- grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein. An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL, or VL- VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain. In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Nonlimiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of this disclosure include: (i) VH-CHI ; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (V) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CHI ; (ix) VL-CH2; (X) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL- In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least two (e.g., five, ten, fifteen, twenty, forty, sixty, or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. As with full antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable60313810218250298.00092511911WO01 domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art. In certain embodiments described herein, antibodies described herein are human antibodies. The term “human antibody,” as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of this disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDRs and in particular CDR3. However, the term “human antibody,” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The term “human antibody” does not include naturally occurring molecules that normally exist without modification or human intervention / manipulation, in a naturally occurring, unmodified living organism. The antibodies of this disclosure may, in some embodiments, be recombinant human antibodies. The term “recombinant human antibody,” as used herein, is intended to include all human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created, or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. Human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable61313810218250298.00092511911WO01 four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked via inter-chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody). These forms have been extremely difficult to separate, even after affinity purification. The frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30: 105) to levels typically observed using a human IgGl hinge. The instant disclosure encompasses antibodies having one or more mutations in the hinge, CH2, or CH3 region which may be desirable, for example, in production, to improve the yield of the desired antibody form. The antibodies described herein may be isolated antibodies. An “isolated antibody,” as used herein, refers to an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an “isolated antibody” for purposes of the instant disclosure. An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals. The antibodies used herein can comprise one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. This disclosure includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”). A person of ordinary skill in the art, starting with the heavy62313810218250298.00092511911WO01 and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues within the VH and / or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only the mutated residues found within the first eight amino acids of FR1 or within the last eight amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and / or CDR regions, for example, wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigenbinding fragments obtained in this general manner are encompassed within the present disclosure. Antibodies useful for the compounds herein also include antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. The term “epitope” refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.63313810218250298.00092511911WO01

[0231] In certain embodiments, the antibody comprises a light chain. In certain embodiments, the light chain is a kappa light chain. In certain embodiments, the light chain is a lambda light chain. In certain embodiments, the antibody comprises a heavy chain. In some embodiments, the heavy chain is an IgA. In some embodiments, the heavy chain is an IgD. In some embodiments, the heavy chain is an IgE. In some embodiments, the heavy chain is an IgG. In some embodiments, the heavy chain is an IgM. In some embodiments, the heavy chain is an IgGl. In some embodiments, the heavy chain is an IgG2. In some embodiments, the heavy chain is an IgG3. In some embodiments, the heavy chain is an IgG4. In some embodiments, the heavy chain is an IgAl. In some embodiments, the heavy chain is an IgA2.

[0232] In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment is an Fv fragment. In some embodiments, the antibody fragment is a Fab fragment. In some embodiments, the antibody fragment is a F(ab')2 fragment. In some embodiments, the antibody fragment is a Fab' fragment. In some embodiments, the antibody fragment is an scFv (sFv) fragment. In some embodiments, the antibody fragment is an scFv-Fc fragment.

[0233] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a bispecific antibody including a first antigen-binding domain (also referred to herein as “DI”), and a second antigenbinding domain (also referred to herein as “D2”).

[0234] As used herein, the expression “antigen-binding domain” means any peptide, polypeptide, nucleic acid molecule, scaffold-type molecule, peptide display molecule, or polypeptide-containing construct that is capable of specifically binding a particular antigen of interest (e.g., PRER, STEAP2, HER2, FelDl, and / or FGFR2). The term “specifically binds” or the like, as used herein, means that the antigen-binding domain forms a complex with a particular antigen characterized by a dissociation constant (KD) of 1 pM or less, and does not bind other unrelated antigens under ordinary test conditions. “Unrelated antigens” are proteins, peptides, or polypeptides that have less than 95% amino acid identity to one another.

[0235] Exemplary categories of antigen-binding domains that can be used in the context of the present disclosure include antibodies, antigen-binding portions of antibodies, peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, proteins comprising a ligand-binding portion of a64313810218250298.00092511911WO01 receptor that specifically binds a particular antigen, antigen-binding scaffolds (e.g., DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, and other scaffolds based on naturally occurring repeat proteins, etc., [see, e.g., Boersma and Pluckthun, 2011, Curr. Opin. Biotechnol. 22:849-857, and references cited therein]), and aptamers or portions thereof.

[0236] Methods for determining whether two molecules specifically bind one another are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antigen-binding domain, as used in the context of the present disclosure, includes polypeptides that bind a particular antigen (e.g., a target molecule [T] or an internalizing effector protein [E]) or a portion thereof with a KD of less than about 1 pM, less than about 500 nM, less than about 250 nM, less than about 125 nM, less than about 60 nM, less than about 30 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 4 pM, less than about 2 pM, less than about 1 pM, less than about 0.5 pM, less than about 0.2 pM, less than about 0.1 pM, or less than about 0.05 pM, as measured in a surface plasmon resonance assay.

[0237] In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody.

[0238] In certain embodiments, the antibody comprises a glutamine residue at one or more heavy chain positions numbered 295 in the EU numbering system. In the present disclosure, this position is referred to as glutamine 295, or as Gln295, or as Q295. Those of skill will recognize that this is a conserved glutamine residue in the wild type sequence of many antibodies. In other useful embodiments, the antibody can be engineered to comprise a glutamine residue. In certain embodiments, the antibody is glycosylated, for instance at N297. In certain embodiments, the antibody is deglycosylated. In certain embodiments, the antibody is aglycosylated. In certain embodiments, the antibody comprises one or more N297 mutations. In certain embodiments, the antibody comprises one or more N297Q mutations. Techniques for modifying an antibody sequence to include a glutamine residue are within the skill of those in the art (see, e.g., Ausubel et al. Current Protoc. Mol. Biol. .65313810218250298.00092511911WO01

[0239] In certain embodiments, the antibody comprises one or more QTAGs. In certain embodiments, the QTAG is at the C-terminus of one or more antibody polypeptide chains. In certain embodiments, the QTAG has the sequence LLQGA.

[0240] In an embodiment, the assignment of amino acids to each framework or CDR domain in an immunoglobulin is in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat et al., National Institutes of Health, Bethesda, Md.; 5thed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia et al., (1987) J Mol. Biol. 196:901-917 or Chothia et al., (1989) Nature 342: 878-883.

[0241] The BA provided herein may be an antigen-binding fragment of an antibody. The terms "antigen-binding portion" or "antigen-binding fragment" of an antibody, as used herein, refers to an immunoglobulin molecule that binds antigen but that does not include all of the sequences of a full antibody (preferably, the full antibody is an IgG). Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; and (vi) dAb fragments; consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3- CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies and small modular immunopharmaceuticals (SMIPs), are also encompassed within the expression "antigen-binding fragment," as used herein.

[0242] As mentioned, BA provided herein may be an scFv. An scFv (single chain fragment variable) has variable regions of heavy (VH) and light (VL) domains (in either order), which, preferably, are joined together by a flexible linker (e.g., peptide linker). The length of the flexible linker used to link both of the V regions may be important for yielding the correct folding of the polypeptide chain. Previously, it has been estimated that the peptide linker must span 3.5 nm (35 A) between the carboxy terminus of the variable domain and the amino terminus of the other domain without affecting the ability of the domains to fold and form an intact antigen-binding site (Huston et al., Protein engineering of single-chain Fv analogs and fusion proteins. Methods313810218250298.00092511911WO01 in Enzymology. 1991;203:46-88). In an embodiment, the linker comprises an amino acid sequence of such length to separate the variable domains by about 3.5 nm.

[0243] In some embodiments, BA provided herein comprises a monovalent or “one-armed” antibody. The monovalent or "one-armed" antibodies as used herein refer to immunoglobulin proteins comprising a single variable domain. For example, the one-armed antibody may comprise a single variable domain within a Fab wherein the Fab is linked to at least one Fc fragment. In certain embodiments, the one-armed antibody comprises: (i) a heavy chain comprising a heavy chain constant region and a heavy chain variable region, (ii) a light chain comprising a light chain constant region and a light chain variable region, and (iii) a polypeptide comprising a Fc fragment or a truncated heavy chain. In certain embodiments, the Fc fragment or a truncated heavy chain comprised in the separate polypeptide is a "dummy Fc," which refers to an Fc fragment that is not linked to an antigen binding domain. The one-armed antibodies of the present disclosure may comprise any of the HCVR / ECVR pairs or CDR amino acid sequences. One-armed antibodies comprising a full-length heavy chain, a full-length light chain and an additional Fc domain polypeptide can be constructed using standard methodologies (see, e.g., W02010151792, which is incorporated herein by reference in its entirety), wherein the heavy chain constant region differs from the Fc domain polypeptide by at least two amino acids (e.g., H95R and Y96F according to the IMGT exon numbering system; or H435R and Y436F according to the EU numbering system). Such modifications are useful in purification of the monovalent antibodies (see W02010151792).

[0244] An antigen-binding fragment of an antibody will, in an embodiment, comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR, which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH - VH, VH - VL or VL - VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0245] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-67313810218250298.00092511911WO01 binding fragment of an antibody described herein include: (i) VH -CHI; (ii) VH -CH2; (iii) VH - CH3; (iv) VH-CH1-CH2; (V) VH -CH1-CH2-CH3; (vi) VH -CH2-CH3; (vii) VH -CL; (viii) VL- CHI; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2- CH3; and (xiv) VL -CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody described herein may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and / or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)). The present disclosure includes an antigen-binding fragment of an antigen-binding protein such as an antibody set forth herein.

[0246] Antigen-binding proteins (e.g., antibodies and antigen-binding fragments) may be monospecific or multi-specific (e.g., bispecific). Multispecific antigen-binding proteins are discussed further herein. The present disclosure includes monospecific as well as multispecific (e.g., bispecific) antigen-binding fragments comprising one or more variable domains from an antigen-binding protein that is specifically set forth herein.

[0247] The term “specifically binds” or “binds specifically” refers to those antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof) having a binding affinity to an antigen, expressed as KD, of at least about 10’9M (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 nM), as measured by real-time, label free bio-layer interferometry assay, for example, at 25°C or 37°C, e.g., an Octet® HTX biosensor, or by surface plasmon resonance, e.g., BIACORE™, or by solution-affinity ELISA.

[0248] " Isolated" antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof), polypeptides, polynucleotides and vectors, are at least partially free of other biological molecules from the cells or cell culture from which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antigenbinding protein may further be at least partially free of expression system components such as68313810218250298.00092511911WO01 biological molecules from a host cell or of the growth medium thereof. Generally, the term "isolated" is not intended to refer to a complete absence of such biological molecules (e.g., minor or insignificant amounts of impurity may remain) or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antigen-binding proteins (e.g., antibodies or antigen-binding fragments).

[0249] The term "human” antigen-binding protein, such as an antibody or antigen-binding fragment, as used herein, includes antibodies and fragments having variable and constant regions derived from human germline immunoglobulin sequences whether in a human cell or grafted into a non-human cell, e.g., a mouse cell. See e.g., US8502018, US6596541 or US5789215. The antibodies provided herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.However, the term "human antibody", as used herein, is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in a non-human mammal or in cells of a non-human mammal. The term is not intended to include natural antibodies directly isolated from a human subject. The present disclosure includes human antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof described herein).

[0250] As used herein, a "chimeric antibody" is an antibody having the variable domain from a first antibody and the constant domain from a second antibody, where the first and second antibodies are from different species, (see e.g., US4816567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855). The present disclosure includes chimeric antibodies comprising the variable domains which are set forth herein and a non-human constant domain.

[0251] The term “recombinant” antibodies or antigen-binding fragments thereof (which may be tethered to a payload), refers to such molecules created, expressed, isolated or obtained by technologies or methods known in the art as recombinant DNA technology which include, e.g., DNA splicing and transgenic expression. The term includes antibodies expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice), or a cell (e.g., CHO cells) such as a cellular expression system or isolated from a recombinant combinatorial human antibody library. The present disclosure includes recombinant antigen-binding proteins, such as antibodies and antigen-binding fragments as set forth herein.69313810218250298.00092511911WO01

[0252] An antigen-binding fragment of an antibody will, in an embodiment, comprise less than a full antibody but still binds specifically to antigen, including at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one (e.g., 3) CDR(s), which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH - VH, VH - VL or VL - VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH and / or VL domain which are bound non-covalently.

[0253] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigenbinding fragment of an antibody described herein include: (i) VH -CHI; (ii) VH -CH2; (iii) VH - CH3; (iv) VH-CH1-CH2; (V) VH -CH1-CH2-CH3; (vi) VH -CH2-CH3; (vii) VH -CL; (viii) VL - CHI; (ix) VL -CH2; (x) VL -CH3; (xi) VL -CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL -CH2- CH3; and (xiv) VL -CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody described herein may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and / or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)). The present disclosure includes an antigen-binding fragment of an antigen-binding protein such as an antibody set forth herein.

[0254] Antigen-binding proteins (e.g., antibodies and antigen-binding fragments) may be monospecific or multi-specific (e.g., bispecific). Multispecific antigen-binding proteins are discussed further herein. The present disclosure includes monospecific as well as multispecific (e.g., bispecific) antigen-binding fragments comprising one or more variable domains from an antigen-binding protein that is specifically set forth herein.70313810218250298.00092511911WO01

[0255] Antibodies and antigen-binding fragments described herein comprise immunoglobulin chains including the amino acid sequences specifically set forth herein (and variants thereof) as well as cellular and in vitro post-translational modifications to the antibody or fragment. For example, the present disclosure includes antibodies and antigen-binding fragments thereof comprising heavy and / or light chain amino acid sequences set forth herein as well as antibodies and fragments wherein one or more asparagine, serine and / or threonine residues is glycosylated, one or more asparagine residues is deamidated, one or more residues (e.g., Met, Trp and / or His) is oxidized, the N-terminal glutamine is pyroglutamate (pyroE) and / or the C-terminal lysine or other amino acid is missing.

[0256] In an embodiment, an antigen-binding protein, e.g., antibody or antigen-binding fragment, comprises a light chain constant domain, e.g., of the type kappa or lambda. In an embodiment, a VH as set forth herein is linked to a human heavy chain constant domain (e.g., IgG) and a VL as set forth herein is linked to a human light chain constant domain (e.g., kappa). The present disclosure includes antigen-binding proteins comprising the variable domains set forth herein, which are linked to a heavy and / or light chain constant domain, e.g., as set forth herein.

[0257] In some embodiments, BA provided herein comprises a humanized antibody or antigen binding fragment thereof, murine antibody or antigen binding fragment thereof, chimeric antibody or antigen binding fragment thereof, monoclonal antibody or antigen binding fragment thereof (e.g., monovalent Fab', divalent Fab2, F(ab)'3 fragments, single-chain variable fragment (scFv), bis-scFv, (scFv)2, diabody, bivalent antibody, one-armed antibody, minibody, nanobody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or antigen binding fragment thereof, bispecific antibody or biding fragment thereof, (e.g., bisscFv, or a bi-specific T-cell engager (BiTE)), trispecific antibody (e.g., F(ab)'3 fragments or a triabody), or a chemically modified derivative thereof. In some embodiments, BA provided herein can be bivalent. In some embodiments, BA provided herein can be monovalent (e.g., one-arm antibody).

[0258] The term “humanized antibody,” as used herein, includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences, or otherwise modified to increase their similarity to antibody variants produced naturally in humans.71313810218250298.00092511911WO01

[0259] In some cases, BA provided herein is an antibody which comprises one or more mutations in a framework region, e.g., in the CHI domain, CH2 domain, CH3 domain, hinge region, or a combination thereof. In some embodiments, the one or more mutations are to stabilize the antibody and / or to increase half-life. In some embodiments, the one or more mutations are to modulate Fc receptor interactions, to reduce or eliminate Fc effector functions such as FcyR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complementdependent cytotoxicity (CDC). In additional embodiments, the one or more mutations are to modulate glycosylation.

[0260] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of an antibody described herein (e.g., in a CH2 domain (residues 231-340 of human IgGl) and / or CH3 domain (residues 341-447 of human IgGl) and / or the hinge region, with numbering according to the Kabat numbering system (e.g., the EU index in Kabat)) to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and / or antigen-dependent cellular cytotoxicity. In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CHI domain) such that the number of cysteine residues in the hinge region are altered (e.g., increased or decreased) as described in, e.g., U.S. Patent No. 5,677,425. The number of cysteine residues in the hinge region of the CHI domain can be altered to, e.g., facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody or to facilitate linker conjugation.

[0261] In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into an IgG constant domain, or FcRn-binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to alter (e.g., decrease or increase) half- life of the antibody in vivo. See, e.g., PCT Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Pat. Nos. 5,869,046, 6,121,022, 6,277,375 and 6,165,745 for examples of mutations that will alter (e.g., decrease or increase) the half-life of an antibody in vivo. In some embodiments, the Fc region comprises a mutation at residue position L234, L235, or a combination thereof. In some embodiments, the mutations comprise L234 and L235. In some embodiments, the mutations comprise L234A and L235A.

[0262] The antibodies and antigen-binding fragments described herein may be modified after translation, e.g., glycosylated.72313810218250298.00092511911WO01

[0263] For example, antibodies and antigen-binding fragments described herein may be glycosylated (e.g., N-glycosylated and / or O-glycosylated) or aglycosylated. Typically, antibodies and antigen-binding fragments are glycosylated at the conserved residue N297 of the IgG Fc domain. Some antibodies and fragments include one or more additional glycosylation sites in a variable region. In an embodiment, the glycosylation site is in the following context: FN297S or YN297S.

[0264] In an embodiment, said glycosylation is any one or more of three different N-glycan types: high mannose, complex and / or hybrid that are found on IgGs with their respective linkage. Complex and hybrid types exist with core fucosylation, addition of a fucose residue to the innermost N-acetylglucosamine, and without core fucosylation.

[0265] In some cases, the antigen-binding protein is an aglycosylated antibody, i.e., an antibody that does not comprise a glycosylation sequence that might interfere with a transglutamination reaction, for instance an antibody that does not have a saccharide group at N180 and / or N297 on one or more heavy chains. In particular embodiments, an antibody heavy chain has an N 180 mutation. In other words, the antibody is mutated to no longer have an asparagine residue at position 180 according to the EU numbering system as disclosed by Kabat et al. In particular embodiments, an antibody heavy chain has an N180Q mutation. In particular embodiments, an antibody heavy chain has an N297 mutation. In particular embodiments, an antibody heavy chain has an N297Q or an N297D mutation. Antibodies comprising such abovedescribed mutations can be prepared by site-directed mutagenesis to remove or disable a glycosylation sequence or by site-directed mutagenesis to insert a glutamine residue at site apart from any interfering glycosylation site or any other interfering structure. Such antibodies also can be isolated from natural or artificial sources. Aglycosylated antibodies also include antibodies comprising a T299 or S298P or other mutations, or combinations of mutations that result in a lack of glycosylation.

[0266] In some cases, the antigen-binding protein is a deglycosylated antibody, i.e., an antibody in which a saccharide group at is removed to facilitate transglutaminase-mediated conjugation. Saccharides include, but are not limited to, N-linked oligosaccharides. In some embodiments, deglycosylation is performed at residue N180. In some embodiments, deglycosylation is performed at residue N297. In some embodiments, removal of saccharide groups is accomplished enzymatically, included but not limited to via PNGase.73313810218250298.00092511911WO01

[0267] In an embodiment, an antibody or fragment described herein is afucosylated.

[0268] The antibodies and antigen-binding fragments described herein may also be post- translationally modified in other ways including, for example: Glu or Gin cyclization at N- terminus; Loss of positive N-terminal charge; Lys variants at C-terminus; Deamidation (Asn to Asp); Isomerization (Asp to isoAsp); Deamidation (Gin to Glu); Oxidation (Cys, His, Met, Tyr, Trp); and / or Disulfide bond heterogeneity (Shuffling, thioether and trisulfide formation).

[0269] In some embodiments, an antibody disclosed herein comprises Q295 which can be native to the antibody heavy chain sequence. In some embodiments, an antibody heavy chain disclosed herein may comprise Q295. In some embodiments, an antibody heavy chain disclosed herein may comprise Q295 and an amino acid substitution N297D.

[0270] According to certain embodiments of the present disclosure, the antibodies and antigen-binding fragments are provided comprising an Fc domain comprising one or more mutations which enhance or diminish antibody binding to the FcRn receptor, e.g., at acidic pH as compared to neutral pH. For example, the present disclosure includes antibodies comprising a mutation in the CH2 or a CH3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in serum half-life of the antibody when administered to an animal.

[0271] Non-limiting examples of such Fc modifications include, e.g., a modification at position:• 250 (e.g., E or Q);• 250 and 428 (e.g., L or F);• 252 (e.g., L / Y / F / W or T),• 254 (e.g., S or T), and / or• 256 (e.g., S / R / Q / E / D or T); and / or a modification at position:• 428 and / or 433 (e.g., H / L / R / S / P / Q or K), and / or• 434 (e.g., A, W, H, F or Y); and / or a modification at position:• 250 and / or 428; and / or a modification at position:74313810218250298.00092511911WO01• 307 or 308 (e.g., 308F, V308F), and / or• 434.

[0272] In an embodiment, the modification comprises:• a 428L (e.g., M428L) and 434S (e.g., N434S) modification;• a 428L, 2591 (e.g., V259I), and 308F (e.g., V308F) modification;• a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification;• a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification;• a 250Q and 428L modification (e.g., T250Q and M428L); and / or• a 307 and / or 308 modification (e.g., 308F or 308P).

[0273] For example, the present disclosure includes antibodies comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of:• 250Q and 248L (e.g., T250Q and M248L);• 252Y, 254T and 256E (e.g., M252Y, S254T and T256E);• 2571 and 3111 (e.g., P257I and Q311I);• 2571 and 434H (e.g., P257I and N434H);• 376V and 434H (e.g., D376V and N434H);• 307A, 380A and 434A (e.g., T307A, E380A and N434A);• 428L and 434S (e.g., M428L and N434S); and• 433K and 434F (e.g., H433K and N434F).

[0274] In yet another embodiment, the modification comprises a 265 A (e.g., D265A) and / or a 297A (e.g., N297A) modification.

[0275] In an embodiment, the heavy chain constant domain is gamma4 comprising an S228P and / or S108P mutation. See Angal et al., “A Single Amino Acid Substitution Abolishes the Heterogeneity of Chimeric Mouse / Human (IgG4) Antibody,” Mol Immunol. 30(1): 105-108 (1993).

[0276] All possible combinations of the foregoing Fc domain mutations, and other mutations within the antibody variable domains disclosed herein, are contemplated within the scope of the present disclosure.

[0277] The antibodies described herein may comprise a modified Fc domain having reduced effector function. As used herein, a "modified Fc domain having reduced effector function" means any Fc portion of an immunoglobulin that has been modified, mutated, truncated, etc.,75313810218250298.00092511911WO01 relative to a wild-type, naturally occurring Fc domain such that a molecule comprising the modified Fc exhibits a reduction in the severity or extent of at least one effect selected from the group consisting of cell killing (e.g., ADCC and / or CDC), complement activation, phagocytosis and opsonization, relative to a comparator molecule comprising the wild-type, naturally occurring version of the Fc portion. In certain embodiments, a "modified Fc domain having reduced effector function" is an Fc domain with reduced or attenuated binding to an Fc receptor (e.g., FcyR).

[0278] In certain embodiments, the modified Fc domain is a variant IgGl Fc or a variant IgG4 Fc comprising a substitution in the hinge region. For example, a modified Fc for use in the context of the present disclosure may comprise a variant IgGl Fc wherein at least one amino acid of the IgGl Fc hinge region is replaced with the corresponding amino acid from the IgG2 Fc hinge region. Alternatively, a modified Fc for use in the context of the present disclosure may comprise a variant IgG4 Fc wherein at least one amino acid of the IgG4 Fc hinge region is replaced with the corresponding amino acid from the IgG2 Fc hinge region. Non-limiting, exemplary modified Fc regions that can be used in the context of the present disclosure are set forth in US Patent Application Publication No. 2014 / 0243504, the disclosure of which is hereby incorporated by reference in its entirety, as well as any functionally equivalent variants of the modified Fc regions set forth therein.

[0279] Also provided herein are antigen-binding proteins, antibodies or antigen-binding fragments, comprising a HCVR set forth herein and a chimeric heavy chain constant (CH) region, wherein the chimeric CH region comprises segments derived from the CH regions of more than one immunoglobulin isotype. For example, the antibodies of the disclosure may comprise a chimeric CH region comprising part or all of a CH2 domain derived from a human IgGl, human IgG2 or human IgG4 molecule, combined with part or all of a CH3 domain derived from a human IgGl, human IgG2 or human IgG4 molecule. According to certain embodiments, the antibodies provided herein comprise a chimeric CH region having a chimeric hinge region. For example, a chimeric hinge may comprise an “upper hinge” amino acid sequence (amino acid residues from positions 216 to 227 according to EU numbering) derived from a human IgGl, a human IgG2 or a human IgG4 hinge region, combined with a “lower hinge” sequence (amino acid residues from positions 228 to 236 according to EU numbering) derived from a human IgGl, a human IgG2 or a human IgG4 hinge region. According to certain embodiments, the76313810218250298.00092511911WO01 chimeric hinge region comprises amino acid residues derived from a human IgGl or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. An antibody comprising a chimeric CH region as described herein may, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. See, e.g., W02014 / 022540.

[0280] Other modified Fc domains and Fc modifications that can be used in the context of the present disclosure include any of the modifications as set forth in US2014 / 0171623; US 8,697,396; US2014 / 0134162; WO2014 / 043361, the disclosures of which are hereby incorporated by reference in their entireties. Methods of constructing antibodies or other antigen-binding fusion proteins comprising a modified Fc domain as described herein are known in the art.

[0281] In some embodiments, the antibodies and antigen-binding fragments described herein comprise an Fc domain comprising one or more mutations in the CH2 and / or CH3 regions.

[0282] In some embodiments, the CH3 region comprises one or more mutations, or a combination thereof, selected from the following: position 384 is Leu, Tyr, Met, or Vai; position 386 is Leu, Thr, His, or Pro; position 387 is Vai, Pro, or an acidic amino acid; position 388 is Trp; position 389 is Vai, Ser, or Ala; position 413 is Glu, Ala, Ser, Leu, Thr, or Pro; position 416 is Thr or an acidic amino acid; and position 421 is Trp, Tyr, His, or Phe, according to EU numbering. In an embodiment, the CH3 region comprises one or more amino acid mutations, or a combination thereof, selected from the following: a) position 380 is Trp, Leu, or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Vai, or Asn; position 390 is Ser or Asn; position 413 is Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe.

[0283] In some embodiments, the CH3 region comprises one or more mutations, or a combination thereof, selected from the following: a) Phe at position 382, Tyr at position 383, Asp at position 384, Asp at position 385, Ser at position 386, Lys at position 387, Leu at position 388, Thr at position 389, Pro at position 419, Arg at position 420, Gly at position 421, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440, Gly at position 442, and Glu at position 443; b) Phe at position 382, Tyr at position 383, Gly at position 384, N at position 385, Ala at position 386, Lys at position 387, Thr at position 389, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440; c) Phe at position 382, Tyr at position 383, Glu at position 384, Ala at position 385, Lys at77313810218250298.00092511911WO01 position 387, Leu at position 388, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440; d) Phe at position 382, Glu at position 384, Ser at position 386, Lys at position 387, Thr at position 389, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440; e) Phe at position 382, Gly at position 384, Ala at position 385, Lys at position 387, Ser at position 389, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440; f) Phe at position 382, Gly at position 384, Ala at position 385, Lys at position 387, Leu at position 388, Thr at position 389, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440; wherein the positions are determined according to EU numbering.

[0284] Additional mutations in CH2 and / or CH3 regions that can introduce non-native binding sites into the antigen-binding proteins descried herein include those described in US Patent Application Publication Nos. 2020 / 0223935, 2020 / 0369746, 2021 / 0130485, 2022 / 0017634; and PCT Application Publications Nos. WO2023 / 279099, WO2023 / 114499 and WO2023 / 114510, which are incorporated herein by reference in their entireties.

[0285] In some embodiments, the antibody is an anti-PSMA, anti-PRLR, anti-MUC16, anti- HER2, Her2, or anti-Her2, anti-EGFRvIII, anti-FelDl, anti-FGFR2, FGFR2, or anti-STEAP2 antibody. In some embodiments, the antibody is an anti-PRER or anti HER2 antibody. In some embodiments, the antibody, or antigen-binding fragment thereof, is anti-STEAP2. In some embodiments, the antibody, or antigen-binding fragment thereof, is anti-PRER. In some embodiments, the antibody, or antigen-binding fragment thereof, is aHer2. In some embodiments, the antibody, or antigen-binding fragment thereof, is aFelDl. In some embodiments, the antibody, or antigen-binding fragment thereof, is aFGFR2.

[0286] The antibody can have binding specificity for any antigen deemed suitable to those of skill in the art. In certain embodiments, the antigen is a transmembrane molecule (e.g., receptor). In one embodiment, the antigen is expressed on a tumor. In some embodiments, the binding agents interact with or bind to tumor antigens, including antigens specific for a type of tumor or antigens that are shared, overexpressed, or modified on a particular type of tumor. In one embodiment, the antigen is expressed on solid tumors. Exemplary antigens include, but are not limited to, lipoproteins; alpha 1 -antitrypsin; a cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; protein A or D; fibroblast growth factor receptor 2 (FGFR2), EpCAM, GD3, FelDl, FLT3, PSMA,78313810218250298.00092511911WO01PSCA, MUC1, MUC16, STEAP, STEAP2, CEA, TENB2, EphA receptors, EphB receptors, folate receptor, FOLRI, mesothelin, cripto, alpha vbeta6, VEGFR, EGFR, transferrin receptor, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5; CD proteins such as CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80, CD81, CD103, CD105, CD134, CD137, CD138, CD152, or an antibody which binds to one or more tumor-associated antigens or cell-surface receptors disclosed in U.S. Publication No. 2008 / 0171040 or U.S. Publication No. 2008 / 0305044 each incorporated in their entirety by reference; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); T-cell receptors; surface membrane proteins; integrins, such as CDl la, CDl lb, CDl lc, CD18, ICAM, VLA-4, and VCAM; a tumor associated antigen such as AFP, ALK, B7H4, BAGE proteins, P-catenin, brc-abl, BRCA1, BORIS, CA9 (carbonic anhydrase IX), caspase-8, CD123, CDK4, CLEC12A, c-kit, cMET, cyclin-Bl, CYP1B1, EGFRvIII, endoglin, EphA2, ErbB2 / Her2, ErbB3 / Her3, ErbB4 / Her4, ETV6-AML, Fra- 1, FOLRI, GAGE proteins, GD2, GloboH, glypican-3, GM3, gplOO, Her2, HEA / B-raf, HEA / EBNA1, HEA / k-ras, HLA / MAGE-A3, hTERT, IGF1R, LGR5, LMP2, MAGE proteins, MART-1, ML-IAP, CA-125, MUM1, NA17, NGEP, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, 0X40, pl5, p53, PAP, PAX3, PAX5, PCTA-1, PDGFR-a, PDGFR-0, PDGF-A, PDGF-B, PDGF- C, PDGF-D, PLAC1, PRLR, PRAME, PSGR, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, Steap-1, Steap-2, STn, survivin, TAG-72, TGF-0, TMPRSS2, Tn, TNFRSF17, TRP-1, TRP-2, tyrosinase, uroplakin-3, and fragments of any of the above-listed polypeptides; cell-surface expressed antigens; c-MET; molecules such as class A scavenger receptors including scavenger receptor A (SR-A), and other membrane proteins such as B7 family-related members including V-set and Ig domain-containing 4 (VSIG4), Colony stimulating factor 1 receptor (CSF1R), asialoglycoprotein receptor (ASGPR), and Amyloid beta precursor-like protein 2 (APLP-2); macrophage receptor with collagenous structure (MARCO), scavenger receptor with C-type lectin (SRCL), and scavenger receptor A-5 (SCARA5), COLEC12, class B macrophage scavenger receptors including CD36, LIMPII, SRBI, SRBII, class D scavenger receptor CD68, lysosomal membrane glycoprotein (LAMP), class E scavenger receptor including lectin-like oxidized low density lipoprotein receptor 1 LOX-1 and Dectin- 1, class F scavenger receptors including scavenger receptor expressed by endothelial cells-I (SREC-I) and SREC-II as well as multiple epidermal growth factor (EGF)-like domains (MEGF)10, class G scavenger receptor79313810218250298.000925 11911WO01CXC chemokine ligand 16 (CXCL16), class H scavenger receptors including Fasciclin, EGF-like, lamin type EGF-like and link domain-containing scavenger receptor- 1 (FEEL-1) and -2 (FEEL- 2), class I scavenger receptor CD163, and class J scavenger receptor receptor for advanced glycation end products (RAGE), other C-type lectin superfamily members including DEC205, CD206, Dectin-2, Mincle, DC-SIGN, and DNGR-1, and other membrane proteins such as B7 family-related member including V-set and Ig domain-containing 4 (VSIG4); AXL, BAFFR, BCR-list components, BDCA2, BDCA4, BTLA, BTNL2, BTNL3, BTNL8, BTNL9, C10orf54, CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CD168, CD177, CD209, CD209L, CD226, CD248, CD27, CD274, CD276, CD300A, CD45, CD46, CD47, CD48, CD62E, CD69, CD74, CD79a, CD79b, CD86, CD90.2, CD96, CLEC12B, CLEC7A, CLEC9A, CR1, CR3, CRTAM, CXCR1 / 2, CXCR4, CXCR5, DDR1, DDR2, DEC-205, DLL4, DR6, FAP, FCamR, FCMR, FcR's, Fire, GITR, HHLA2, HLA class II, HVEM, ICOSLG, IFNLR1, IL10R1, IL10R2, IL12R, IL13RA1, IL13RA2, IL15R, IL17RA, IL17RB, IL17RC, IL17RE, IL20R1, IL20R2, IL21R, IL22R1, IL22RA, IL23R, IL27R, IL29R, IL2Rg, IL31R, IL36R, IL3RA, IL4R, IL6R, IL5R, IL7R, IL9R, LAG3, LIFR, MAG / Siglec-4, MMR, MSR1, NCR3LG1, NKG2D, NKp30, NKp46, PDCD1, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGIRR, Siglec-1, Siglec-10, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, SIRPA, TACI, TCR-list components / assoc, PTCRA, TCRb, CD3z, TEK, TGFBR1, TGFBR2, TGFBR3, TfRl, TIGIT, TLR2, TLR4, TNF-a, TROY, TSLPR, TYRO, VLDLR, and VTCNL In some embodiments, the binding agent is adalimumab or infliximab. In some embodiments, the binding agent is alemtuzumab, muromonab, rituximab, tosituzumab, or agonistic antibodies (where immune stimulation might be part of the intended mechanism of action). In some embodiments, the antigen is PRLR or HER2. In some embodiments, the antigen is STEAP2. In some embodiments the antigen is human STEAP2. In some embodiments, the antigen is Her2. In some embodiments, the antigen is FelDl. In some embodiments, the antigen is FGFR2. In some examples, the MAGE proteins are selected from MAGE-1, -2, -3, -4, -6, and -12. In some examples, the GAGE proteins are selected from GAGE- 1 and GAGE-2.

[0287] Exemplary antigens also include, but are not limited to, BCMA, SLAMF7, GPNMB, MSR1, and UPK3A. Exemplary antigens also include, but are not limited to, MUC16, STEAP2, and HER2.80313810218250298.000925 11911WO01

[0288] In some embodiments, the antigens include MUC16. In some embodiments, the antigens include STEAP2. In some embodiments, the antigens include PSMA. In some embodiments, the antigens include MSR1. In some embodiments, the antigens include HER2. In some embodiments, the antigen is prolactin receptor (PRLR) or prostate-specific membrane antigen (PSMA). In some embodiments, the antigen is MUC16. In some embodiments, the antigen is HER2. In some embodiments, the antigen is STEAP2. In some embodiments, the antigen is MSR1.

[0289] In particular embodiments, the antibody or fragment is modified bonded to a primary amine compound. This can facilitate conjugation to a linker payload described herein. In certain embodiments, the primary amine compound is bonded to a glutamine side chain of the antibody or fragment. In certain embodiments, the glutamine residue is Q295. In certain embodiments, the glutamine residue is Q297. In certain embodiments, the glutamine residue is within a QTAG, for instance at a C-terminus.Mil. Primary Amine Compounds

[0290] In certain embodiments, primary amine compounds useful for the transglutaminase- mediated coupling of an antigen-binding protein (an antibody or an antigen binding fragment thereof) comprising a glutamine can be any primary amine compound deemed useful by the practitioner of ordinary skill. Generally, the primary amine compound has the formula H2N-R, where R can be any group compatible with the antibody and reaction conditions. In certain embodiments, R is alkyl, substituted alkyl, heteroalkyl, or substituted heteroalkyl. In particular embodiments, the primary amine compound comprises a tetrazine reactive group. In particular embodiments, the primary amine compound comprises a cyclooctyne reactive group or a transcyclooctene reactive group.

[0291] In certain embodiments, the primary amine compound is according to the formula H2N- LL-X, where LL is a divalent spacer and X is a tetrazine group. In certain embodiments, the primary amine compound is according to H2N-(CH2)n-X; where n is an integer selected from one to twelve; and X is selected from the group consisting of81313810218250298.00092511911WO01resulting modified glutamine residue

[0293] In some embodiments, the primary amine compound

[0294] The resulting modified glutamine residue

[0295] Exemplary conditions for the above reactions are provided in the Examples below.IX. Methods of Preparation

[0296] The compounds provided herein can be prepared, isolated, or obtained by any method apparent to those of skill in the art. Exemplary methods of preparation are described in detail in the Examples below and shown in FIGS. 1, 2A-2B, 3A-3B, 4, 5, and 6.

[0297] In one embodiment, provided is a method of making a conjugate comprising the steps of (a) treating a binding agent with a primary amine compound in the presence of microbial313810218250298.00092511911WO01 transglutaminase or sortase to provide a modified binding agent; (b) treating the modified binding agent with a linker-payload described herein; and (c) purifying the resulting conjugate.

[0298] In some embodiments, provided is a method of making a conjugate comprising the steps of (a) treating an antigen-bidning protein withorthe presence of microbial transglutaminase or sortase to provide a transglutaminase-modified or sortase-modified binding agent; (b) treating the transglutaminase- modified or sortase-modified binding agent with the linker payload described herein; and (c) purifying the conjugate.

[0299] The antigen-binding protein (BA) (such as an antibody) can be treated with the primary amine compounds described herein, under standard conjugation conditions (see, e.g., Doronina et al. Nature Biotechnology 2003, 21, 8, which is incorporated herein by reference in its entirety) to produce the antigen-binding proteins modified with the reactive group.

[0300] In some embodiments, an aglycosylated antigen-binding protein is reacted or treated with a primary amine compound to produce a glutaminyl-modified antigen-binding protein (such as a glutaminyl-modified antibody). In some embodiments, a deglycosylated antigen-binding protein is reacted or treated with a primary amine compound to produce a glutaminyl-modified antigen-binding protein (such as a glutaminyl-modified antibody).

[0301] In certain embodiments, the transglutaminase is an enzyme that catalyzes the formation of an isopeptide bond between a free amine group on the primary amine compound and the acyl group on the side chain of a glutamine residue present in the antigen-binding protein. Transglutaminase is also known as protein-glutamine-y-glutamyltransferase. In particular embodiments, the transglutaminase is classified as EC 2.3.2.13. The transglutaminase can be from any source deemed suitable. In certain embodiments, the transglutaminase is microbial. Useful transglutaminases have been isolated from Streptomyces mobaraense, Streptomyces cinnamoneum, Streptomyces griseo-carneum, Streptomyces lavendulae, and Bacillus subtilis. Non-microbial transglutaminases, including mammalian transglutaminases, can also be used. In83313810218250298.00092511911WO01 certain embodiments, the transglutaminase can be produced by any technique or obtained from any source deemed suitable by the practitioner of skill. In particular embodiments, the transglutaminase is obtained from a commercial source.

[0302] Then the antigen-binding proteins modified with the reactive group can be conjugated with linker-payload compounds via inverse electron demand Diels-Alder reactions. In some embodiments of the Diels-Alder reactions, the linker-payload includes a reactive group, for example an alkyne, that is capable of undergoing a regioisomeric Diels-Alder reaction with a tetrazine present on the antigen-binding protein. Such suitable reactive groups are described above. The antibody includes one or more dienophile or diene compounds.X. Pharmaceutical Compositions and Methods of Treatment

[0303] Provided herein are methods of treating and preventing diseases, conditions, or disorders comprising administering a therapeutically or prophylactically effective amount or one or more of the compounds disclosed herein, for example, one or more of the compounds of a formula provided herein. Diseases, disorders, and / or conditions include, but are not limited to, those associated with the antigens described herein.

[0304] The compounds described herein can be administered alone or together with one or more additional therapeutic agents. The one or more additional therapeutic agents can be administered just prior to, concurrent with, or shortly after the administration of the compounds described herein.

[0305] The present disclosure also includes pharmaceutical compositions comprising any of the compounds described herein in combination with one or more additional therapeutic agents, and methods of treatment comprising administering such combinations to subjects in need thereof.

[0306] The present disclosure also includes a composition or kit comprising the compound or pharmaceutical composition described herein in association with a further therapeutic agent.

[0307] Suitable additional therapeutic agents include, but are not limited to, a glucocorticoid, steroid, LXR modulator, an inflammatory therapeutic agent, an autoimmune therapeutic agent, a hormone, a biologic, or a monoclonal antibody. Suitable therapeutic agents also include, but are not limited to any pharmaceutically acceptable salts, acids, or derivatives of a compound set forth herein. The compounds described herein can also be administered and / or co-formulated in combination with antivirals, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotectants, metal chelators, IFN-gamma, and / or NSAIDs.84313810218250298.00092511911WO01

[0308] In some embodiments of the methods described herein, multiple doses of a compound described herein (or a pharmaceutical composition comprising a combination of a compound described herein and any of the additional therapeutic agents mentioned herein) may be administered to a subject over a defined time course. The methods according to this embodiment of the disclosure comprise sequentially administering to a subject multiple doses of a compound described herein. As used herein, “sequentially administering” means that each dose of the compound is administered to the subject at a different point in time, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure includes methods which comprise sequentially administering to the patient a single initial dose of a compound described herein, followed by one or more secondary doses of the compound, and optionally followed by one or more tertiary doses of the compound.

[0309] The terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of the compounds described herein. Thus, the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”); the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses can all include the same amount of a compound described herein, but generally can differ from one another in terms of frequency of administration. In certain embodiments, the amount of the compound included in the initial, secondary, and / or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., two, three, four, or five) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”).

[0310] In certain exemplary embodiments of the present disclosure, each secondary and / or tertiary dose is administered one to twenty-six (e.g., 1,3, 31 / 2, 4, 41 / 2, 5, 51 / 2, 6, 6I / 2, 7,71 / 2, 8, 8I / 2, 9, 91 / 2, 10, IOI / 2, 11, I II / 2, 12, 121 / 2, 13, 131 / 2, 14, 141 / 2, 15, 151 / 2, 16, 161 / 2, 17, 171 / 2, 18, I8I / 2, 19, 191 / 2, 20, 201 / 2, 21, 211 / 2, 22, 221 / 2, 23, 231 / 2, 24, 241 / 2, 25, 251 / 2, 26, 261 / 2, or more) weeks after the immediately preceding dose. The phrase “the immediately preceding dose,” as used herein means, in a sequence of multiple administrations, the dose the compound which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.85313810218250298.00092511911WO01

[0311] The methods according to this embodiment of the disclosure may comprise administering to a patient any number of secondary and / or tertiary doses of the compound. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, or more) tertiary doses are administered to the patient. The administration regimen may be carried out indefinitely over the lifetime of a particular subject, or until such treatment is no longer therapeutically needed or advantageous.

[0312] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient one to two weeks or one to two months after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient two to twelve weeks after the immediately preceding dose. In certain embodiments of the disclosure, the frequency at which the secondary and / or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.

[0313] The present disclosure includes administration regimens in which two to six loading doses are administered to a patient at a first frequency (e.g., once a week, once every two weeks, once every three weeks, once a month, once every two months, etc.), followed by administration of two or more maintenance doses to the patient on a less frequent basis. For example, according to this embodiment of the disclosure, if the loading doses are administered at a frequency of once a month, then the maintenance doses may be administered to the patient once every six weeks, once every two months, once every three months, etc.

[0314] The present disclosure includes pharmaceutical compositions of the compounds and / or conjugates described herein, for example, compositions comprising a compound described herein, or a pharmaceutically acceptable salt thereof, or regioisomer thereof, and a pharmaceutically acceptable carrier. The pharmaceutical compositions can further include a diluent, and / or an excipient. Examples of suitable carriers, diluents and excipients include, but are not limited to,86313810218250298.00092511911WO01 buffers for maintenance of proper composition pH (e.g., citrate buffers, succinate buffers, acetate buffers, phosphate buffers, lactate buffers, oxalate buffers, and the like), carrier proteins (e.g., human serum albumin), saline, polyols (e.g., trehalose, sucrose, xylitol, sorbitol, and the like), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxolate, and the like), antimicrobials, and antioxidants. In one embodiment, provided is a pharmaceutical composition including the compound, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0315] In some examples, set forth herein is a method of treating a disease, disorder or condition including administering to a patient having said disorder a therapeutically effective amount of a compound described herein, or a pharmaceutical composition thereof.

[0316] In some embodiments, the disease, disorder, or condition is an allergic state, including, but not limited to, asthma, atopic dermatitis, contact dermatitis, drug hypersensitivity reactions, anaphylactic rhinitis, perennial or seasonal allergic rhinitis, and serum sickness; dermatologic diseases and conditions including, but not limited to, skin itching, seborrheic dermatitis, neurodermatitis, bullous dermatitis herpetiformis, exfoliative erythroderma, mycosis fungoides, pemphigus, and severe erythema multiforme (Stevens-Johnson syndrome); endocrine disorders including, but not limited to, primary or secondary adrenocortical insufficiency, congenital adrenal hyperplasia, hypercalcemia associated with cancer, and nonsuppurative thyroiditis; gastrointestinal diseases; hematologic disorders including, but not limited to, acquired (autoimmune) hemolytic anemia, congenital (erythroid) hypoplastic anemia (Diamond-Blackfan anemia), idiopathic thrombocytopenic purpura in adults, pure red cell aplasia, and secondary thrombocytopenia; trichinosis; tuberculous meningitis with subarachnoid block or impending block; neoplastic diseases including, but not limited to, leukemias and lymphomas; nervous system disorders including, but not limited to, acute exacerbations of multiple sclerosis, cerebral edema associated with primary or metastatic brain tumor, craniotomy, or head injury; ophthalmic diseases including, but not limited to, sympathetic ophthalmia, temporal arteritis, uveitis, and ocular inflammatory conditions unresponsive to topical corticosteroids; renal diseases including, but not limited to, for inducing a diuresis or remission of proteinuria in idiopathic nephrotic syndrome or that due to lupus erythematosus; respiratory diseases including, but not limited to, berylliosis, fulminating or disseminated pulmonary tuberculosis when used concurrently with appropriate antituberculous chemotherapy, idiopathic eosinophilic pneumonias, symptomatic sarcoidosis; and Rheumatic disorders including, but not limited to, use as adjunctive therapy for short-term87313810218250298.00092511911WO01 administration (to tide the patient over an acute episode or exacerbation) in acute gouty arthritis, acute rheumatic carditis, ankylosing spondylitis, psoriaticarthritis, rheumatoid arthritis, including juvenile rheumatoid arthritis, and for use in dermatomyositis, polymyositis, and systemic lupus erythematosus.

[0317] In some examples, set forth herein is a method for treating a disease, disorder, or condition selected from an autoimmune disease, an allergy, arthritis, asthma, a breathing disorder, a blood disorder, a cancer, a collagen disease, a connective tissue disorder, a dermatological disease, an eye disease, an endocrine problem, an immunological disease, an inflammatory disease, an intestinal disorder, a gastrointestinal disease, a neurological disorder, an organ transplant condition, a rheumatoid disorder, a skin disorder, a swelling condition, a wound healing condition, and combinations thereof, comprising administering a payload or conjugate thereof described herein.

[0318] In some examples, the autoimmune disorder is selected from multiple sclerosis, autoimmune hepatitis, shingles, systemic lupus erythematosus (i.e., lupus), myasthenia gravis, Duchenne muscular dystrophy, and sarcoidosis. In some examples, the breathing disorder is selected from asthma, chronic respiratory disease, chronic obstructive pulmonary disease, bronchial inflammation, and acute bronchitis. In some examples, the cancer is selected from leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, Hodgkin’s lymphoma, Non-Hodgkin’ s lymphoma (NHL), and multiple myeloma. In some examples, the collagen disease is systemic lupus erythematosus. In some examples, the eye disease is keratitis. In some examples, the endocrine problem is selected from Addison's Disease, adrenal insufficiency, adrenal cortical dysfunction, adrenocortical, and congenital adrenal hyperplasia. In some examples, the inflammatory disease is selected from inflammation after cataract surgery, joint inflammation, immune inflammation, tendon inflammation, bursitis, epicondylitis, Crohn's disease, inflammatory bowels disease, lipid pneumonitis thyroiditis, urticaria (hives), pericarditis, nephrotic syndrome, and uveitis. In some examples, the intestinal disorder is selected from ulcerative colitis, Crohn’s disease, and inflammatory bowel disease. In some examples, the rheumatoid disorder is selected from rheumatoid arthritis, polymyalgia rheumatic, psoriatic arthritis, ankylosing spondylitis, and systemic lupus erythematosus. In some examples, the skin disorder is selected from psoriasis, eczema, and poison ivy. In some examples, the neurological disorder is chronic inflammatory demyelinating polyradiculoneuropathy.88313810218250298.00092511911WO01

[0319] In some embodiments, the compounds described herein are administered to a patient to treat an acute inflammatory event including, but not limited to, shock, brain edema, and graft-vs- host disease. In some embodiments, the compounds described herein are administered to treat lympholytic effects including, but not limited to, those associated with hematological malignancies, for example, leukemias, lymphomas, and myelomas.

[0320] In some examples, set forth herein is a method for reducing inflammation in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or conjugate thereof described herein. In some examples, set forth herein is a method for modulating the immune system in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or conjugate thereof described herein. In some examples, set forth herein is a method for modulating cortisol levels in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or conjugate thereof described herein. In some examples, set forth herein is a method of reducing lymphocyte migration in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or conjugate thereof described herein. In some examples, set forth herein is a method of treating hypercalcemia due to cancer, Meniere's disease, a migraine headache, a cluster headache, a severe aphthous ulcer, laryngitis, severe tuberculosis, a Herxheimer reaction to syphilis, a decompensated heart failure, allergic rhinitis or nasal polyps, comprising administering to a subject in need thereof a payload or conjugate thereof described herein. In some examples, the compounds disclosed herein can be used for treating inflammatory bowel disease, Crohn's disease, or ulcerative colitis. In some examples, the disease, disorder, or condition is a chronic inflammatory condition including, but not limited to, asthma, skin infections, and ocular infections. In some examples, compounds described herein are used for immunosuppression in patients undergoing organ transplantation.

[0321] In some examples, set forth herein is a method of treating a disease, disorder or condition selected from the group consisting of an immunological disease, autoimmune disease, inflammation, asthma, or an inflammatory bowel disorder, Crohn's disease, and ulcerative colitis.

[0322] In some examples, set forth herein is a method of treating a disease, disorder or condition by targeting an antigen, for example, cell-surface expressing antigen, to which compound delivery can achieve a therapeutic benefit comprising administering the conjugates89313810218250298.00092511911WO01 described herein. In some embodiments, the antigen is AXL, BAFFR, BCMA, BCR-list components, BDCA2, BDCA4, BTLA, BTNL2, BTNL3, BTNL8, BTNL9, C10orf54, CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CDl lc, CD137, CD138, CD14, CD168, CD177, CD19, CD20, CD209, CD209L, CD22, CD226, CD248, CD25, CD27, CD274, CD276, CD28, CD30, CD300A, CD33, CD37, CD38, CD4, CD40, CD44, CD45, CD47, CD46, CD48, CD5, CD52, CD55, CD56, CD59, CD62E, CD68, CD69, CD70, CD74, CD79a, CD79b, CD8, CD80, CD86, CD90.2, CD96, CLEC12A, CLEC12B, CLEC7A, CLEC9A, CR1, CR3, CRTAM, CSF1R, CTLA4, CXCR1 / 2, CXCR4, CXCR5, DDR1, DDR2, DEC-205, DLL4, DR6, FAP, FCamR, FCMR, FcR's, Fire, GITR, HHLA2, HLA class II, HVEM, ICOSLG, IFNLR1, IL10R1, IL10R2, IL12R, IL13RA1, IL13RA2, IL15R, IL17RA, IL17RB, IL17RC, IL17RE, IL20R1, IL20R2, IL21R, IL22R1, IL22RA, IL23R, IL27R, IL29R, IL2Rg, IL31R, IL36R, IL3RA, IL4R, IL6R, IL5R, IL7R, IL9R, Integrins, LAG3, LIFR, MAG / Siglec-4, MMR, MSR1, NCR3LG1, NKG2D, NKp30, NKp46, PDCD1, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGIRR, Siglec-1, Siglec-10, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, SIRPA, SLAMF7, TACI, TCR-list components / assoc, PTCRA, TCRb, CD3z, CD3, TEK, TGFBR1, TGFBR2, TGFBR3, TfRl, TIGIT, TLR2, TLR4, TROY, TSLPR, TYRO, VLDLR, VSIG4, or VTCN1. In some embodiments, the antigen is IL2R-y.

[0323] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.90313810218250298.000925 11911WO01EXAMPLES

[0324] The following examples illustrate specific aspects of the instant description. The examples should not be construed as limiting, as the examples merely provide specific understanding and practice of the embodiments and their various aspects.Example 1. Conjugation ChemistryConjugation of the Linker to the Antigen-Binding Protein

[0325] A tetrazine linker (100 times molar excess) was added to a binding protein, such as TfRl targeted monoclonal single arm antibody. mTG (1.5 units of mTG per mg of antibody) was then added and the mixture was incubated at room temperature on inversion machine to allow the transglutaminase enzymatic reaction to bind the tetrazine amine group to the glutamine on the Q295 region.

[0326] The crude mixture was purified using size exchange chromatography (SEC) in a 16 / 600 size column at a 2ml per minute rate to separate antibody linker conjugate from aggregate antibody, mTG, and excess linker. The UV vis attachment was used to confirm conjugation (280 should be higher than 260) (FIG. 7). This process can take up to 6 hours. UV vis spectra was used to select fractions with primarily conjugate.Conjugation of the Antigen-Binding Protein-Linker to the Linker-Polynucleotide

[0327] 1.2 -1.3 molar excess of the siRNA with the Cyclooctyne (ALO) handle attached was added to an antibody linker conjugate and the mixture was incubated overnight at room temperature to allow the diene(tetrazine) to bond to the dienophile (ALO) through IEDDA click chemistry.

[0328] The crude mixture was filtered using SEC in a 16 / 600 column at 1 ml per minute to separate Antibody Oligonucleotide Conjugate (AOC) from free siRNA and unconjugated antibody linker. 260 UV should be slightly higher than 280 UV for conjugates. UV vis method was used to confirm OAR 1 and OAR 2, OAR 1 should have a 260 / 280 value of 1.38 while OAR 2 should be 1.70. SEC was used to further check for aggregation of proteins which should elute first. Final conjugate samples were run through HIC to confirm OAR measurements from UV vis.91313810218250298.00092511911WO01Example 2. Conjugation ChemistryConjugation of the Linker to the Antigen-Binding Protein

[0329] Deglycosylated antibody with Q tags at the c-terminal was concentrated to -10-20 mg / mL in PBS using a 30K Amicon spin column. 30 equivalents of mTG linker per equivalent of antibody were added along with 1 Unit of Zidira mTG per mg of antibody (FIG. 13). The reaction was incubated at 37°C for 2 hours, and progression of the reaction was monitored using Q-ToF MS. The final product was purified using AKTA and SEC methods and concentrated using a 10K Amicon spin column, avoiding aggregation fractions.Conjugation of the Linker to siRNA

[0330] The desalted and dried oligonucleotide (oligo) was concentrated to 50 mg / mL using 90% DMSO in water. The linker (NHS ALO) was prepared by concentrating it to 50 mg / mL in DMSO. The linker solution (4 equivalents) was mixed with the oligo solution and 10 equivalents of TEA were added to adjust the pH to 11, enabling the reaction. The mixture was reacted at 40°C and 1000 rpm for 20-60 minutes (e.g., 20 minutes for Maleimide / ALO). The reaction was then immediately placed on ice. A sample for a mass spectrometry analysis was prepared by diluting the reaction lOOx (2 pL sample into 198 pL water). The reaction was analyzed using a Cl 8 RP column. If the reaction was incomplete, more linker was added and the pH was adjusted with TEA as needed.

[0331] Once the reaction was complete, the sample was transferred to a 15 mL tube, ACN (15 mL) and PBS (400 pL) were added, and the reaction mixture was vortexed until the oligo precipitated. The mixture was centrifuged and the supernatant was decanted. The pellet was allowed to dry upside down on a paper towel for 5 minutes. The oligo was re-solubilized in minimal water (-100 pL per 20 mg of conjugate) and the precipitation process was repeated a second time. Finally, the linker-conjugated oligo was dissolved in PBS or water and its concentration was measured using UV spectroscopy (lOOx dilution).Conjugation of the Antibody with Tetrazine Linker to siRNA with ALO Handle

[0332] The antibody was concentrated to -10 mg / mL using a 10K Amicon spin column (Amicon Ultra-15, UFC901096). ALO siRNA (1.5 equivalents) was added of to the solution and the reaction was incubated at 37 °C and 300 rpm on a thermoshaker overnight. The reaction (FIG. 14) was monitored using Agilent analytical SEC, ensuring that the absorbance at 260 nm was higher than at 280 nm, which indicated the presence of OAR1. If the main peak or back shoulder92313810218250298.000925 11911WO01 showed higher absorbance at 280 nm than 260 nm, OARO was present in the reaction. Upon completion, AKTA AEX purification was performed ensuring that the sample loop was displaced with mobile phase A instead of PBS after a 0.5N NaOH loop rinse for optimal AEX purification.Example 3. Biological Activity of the siRNA ConjugatesLipofectamine RNAiMAX, 0.3uL / well, 96 hours Incubation, 3T3-hTfRs

[0333] 3T3 cells expressing the human transferrin receptor (hTfR) were transfected using lipofection with: GAPDH control siRNA (siGAPDH), unconjugated B2M siRNA, B2M siRNA conjugated to an isotype antibody (anti-Fel Dl-hIgGl-b2m siRNA), or B2M siRNA conjugated to a TfRl antibody (anti-TFRl-hIgG3-b2m siRNA). Antibody-siRNA conjugates were prepared according to ALO-tetrazine reaction as described above. The transfection was performed at varying siRNA concentrations over a 96-hour period. Experimental results demonstrated that groups containing B2M siRNA, whether unconjugated or conjugated to the antibody, induced a measurable reduction in relative mRNA concentration (%) (FIG. 8, Table 1). In contrast, the control GAPDH siRNA group did not exhibit significant changes in relative mRNA levels (FIG. 8, data normalized to GAPDH control), serving as a baseline control for the study.Table 1. Lipofectamine Mediated Transfection of Conjugates or siRNASelf Delivery, 96 hours Incubation, 3T3-hTfRs Cells

[0334] 3T3 cells expressing human transferrin receptor (hTfR) were incubated with GAPDH control siRNA, unconjugated B2M siRNA, B2M siRNA conjugated to an isotype antibody (anti- Fel Dl-hIgGl-b2m siRNA), or B2M siRNA conjugated to a TfRl antibody (anti-TFRl-hIgG3- b2m siRNA) at varying siRNA concentrations for 96 hours. Antibody-siRNA conjugates were93313810218250298.000925 11911WO01 prepared according to ALO-tetrazine reaction as described above. Results demonstrated that the B2M siRNA conjugated to the TfRl antibody facilitated receptor-mediated delivery (FIG. 9, Table 2), leading to a dose dependent reduction in relative % mRNA concentration (normalized to GAPDH control). In contrast, both the unconjugated B2M siRNA and anti-Fel Dl-hIgGl-b2m siRNA exhibited no dose dependent reduction (FIG. 9, Table 2).Table 2. Knockdown of SOD1 in 3T3 hTfRl Cells Through Receptor-Mediated Uptake Using Conjugates

[0335] N2A cells were transfected using lipofection with four distinct siRNA groups: GAPDH control siRNA (siGAPDH), unconjugated SOD1 siRNA with ALO linker, SOD1 siRNA conjugated to a single arm TfRl antibody (Anti-TFRl-SODl-siRNA), and SOD1 siRNA conjugated to an isotype antibody (Anti-Fel DI -SOD 1 -siRNA). The transfection was performed at varying siRNA concentrations over a 96-hour period. Antibody-siRNA conjugates were prepared according to ALO-tetrazine reaction as described above. Experimental results demonstrated that groups transfected with SOD1 siRNA, whether unconjugated or conjugated to the TfRl or isotype antibody, exhibited a measurable reduction in target mRNA expression. In contrast, the GAPDH controlgroup did not induce significant changes in mRNA levels, serving as a negative control for the study (FIG. 10, Table 3).94313810218250298.00092511911WO01Table 3. Knockdown of SOD1 in 3T3 hTfRl Cells Using Conjugates Facilitated by Lipofectamine-Mediated Delivery96hr Incubation; 3T3-hTfR Cells

[0336] 3T3-hTfR cells were incubated with naked SOD1 siRNA with ALO linker, SOD1 conjugated to a TfRl antibody (Anti-TFRl -SOD 1 -siRNA, prepared with tetrazine-ALO reaction), or SOD1 siRNA conjugated to an isotype control antibody (Anti-Fel DI -SOD 1 -siRNA, prepared with tetrazine-ALO reaction). Results demonstrated that the SOD1 siRNA conjugated to the TfRl antibody facilitated receptor-mediated delivery, leading to a dose dependent reduction in relative % mRNA concentration. In contrast, both the unconjugated SOD1 siRNA and SOD1 conjugated to an isotype antibody showed no significant effect on the relative % mRNA concentration (FIG. 11, Table 4).Table 4. Knockdown of SOD1 in 3T3 hTfRl Cells Through Receptor-Mediated Uptake Using ConjugatesExample 4. Stability of the siRNA Conjugates

[0337] A 2.77 mg / ml sample of anti-TfrRl -tetrazine- ALO-MSTN siRNA was incubated in 300 pl PBS at 37°C on a thermoshaker set at 400 rpm. Samples at a concentration of 0.5 mg / ml were collected on days 1, 2, 3, and 7, and analyzed for DAR 1 percentage using analytical AEX95313810218250298.000925 11911WO01 and SEC. FIG. 12 shows minimal decrease in DAR 1 percentage over time, suggesting limited or no breakup of the conjugate.

[0338] As various changes can be made in the above-described subject matter without departing from the scope and spirit of the present disclosure, it is intended that all subject matter contained in the above description, or defined in the appended claims, be interpreted as descriptive and illustrative of the present disclosure. Many modifications and variations of the present disclosure are possible in light of the above teachings. Accordingly, the present description is intended to embrace all such alternatives, modifications, and variances which fall within the scope of the appended claims.

[0339] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.96313810218

Claims

250298.00092511911WO01WHAT IS CLAIMED IS:

1. A compound of the following formula:or a pharmaceutically acceptable salt thereof, wherein BA is an antigen-binding protein;RG' comprises a reactive group residue following reaction of a first reactive group RG1 with a second reactive group RG2;RG1 comprises a reactive group chosen fromRG2 comprises a reactive group chosen from; wherein each R1is independently hydrogen or C1-C10 alkyl;T is a polynucleotide; and k is 1 or 2.

2. The compound of claim 1 , wherein each RG' comprises313810218250298.00092511911WO013. The compound of claim 1, wherein each RG' is chosen from4. The compound of claim 1 , wherein each RG' comprises5. The compound of claim 1, wherein each RG' is chosen from6. A compound according to Formula (I):acceptable salt thereof, or regioisomer thereof, wherein each L is independently a linker.313810218250298.00092511911WO017. The compound of any of the previous claims according to Formula (la) or (lb):acceptable salt thereof, or regioisomer thereof, wherein each L is independently a linker.

8. A compound according to Formula (II):acceptable salt thereof, or regioisomer thereof, wherein L is a linker.The compound of any of the previous claims according to Formula (Ila) or (lib):313810218250298.00092511911WO01(lib), or a pharmaceutically acceptable salt thereof, or regioisomer thereof, wherein each L is independently a linker.

10. A compound according to Formula (III):acceptable salt thereof, or regioisomer thereof, wherein each L is independently a linker.

11. The compound of any of the previous claims according to Formula (Illa) or (Illb):acceptable salt thereof, or regioisomer thereof, wherein each L is independently a linker.100313810218250298.00092511911WO0112. The compound of any of the previous claims according to Formula (IV):pharmaceutically acceptable salt thereof, or regioisomer thereof, wherein L is a linker.

13. The compound of any of the previous claims according to Formula (IVa) or (IVb):acceptable salt thereof, or regioisomer thereof, wherein L is a linker.

14. The compound of any of the previous claims, wherein L is -N(R2)-Ci-Cioalkylene or -N(R2)-Ci-Cioalkylene-P03S-; and R2is hydrogen or Ci-Cio alkyl.

15. The compound of claim 2, wherein R1is methyl and R2is hydrogen.

16. The compound of any of the previous claims, wherein the reaction of RG1 and RG2 comprises an inverse electron demand Diels-Alder reaction.

17. The compound of claim 16, wherein RG' comprises a Diels-Alder adduct.313810218250298.00092511911WO0118. The compound of any of the previous claims selected from the group consisting ofor pharmaceutically acceptable salt and / or regioisomer thereof.

19. The compound of any of the previous claims, wherein BA comprises an antibody or an antigen binding fragment thereof.

20. The compound of any of the previous claims, wherein BA comprises an antigen binding fragment chosen from Fab fragments; F(ab')2 fragments; Fd fragments; Fv fragments; single-chain Fv (scFv) molecules; and dAb fragments.

21. The compound of any of the previous claims, wherein BA binds specifically to human transferrin receptor or a variant or an antigenic fragment thereof.102313810218250298.00092511911WO0122. The compound of any of the previous claims, wherein BA comprises one or two RG2 groups.

23. The compound of any of the previous claims, wherein BA comprises one or two residues according to the formula24. The compound of any of the previous claims, wherein BA comprises one or two residues according to the formula25. The compound of claim 23 or 24, wherein one of the residues is at position 295 or 297 of BA, numbered according to the EU index of Kabat.

26. The compound of claim 23 or 24, wherein one of the residues is at the C-terminus ofBA.

27. The compound of claim 23 or 24, wherein one of the modified glutamine residues is represented by Q* in the sequence LLQ*GA at the C-terminus of BA.

28. A linker-payload compound of the following formula:RG1 — L— T or a pharmaceutically acceptable salt thereof, whereinL is a linker covalently bound to RG1 and to T ;RG1 comprises a reactive group chosen from313810218250298.00092511911WO01 each R1is independently hydrogen or C1-C10 alkyl; andT is a polynucleotide.

29. The linker payload compound of claim 28, wherein RG1 comprises30. The linker payload compound of claim 28, wherein RG1 comprises31. The linker payload compound of claim 28, wherein RG1 comprises32. The linker payload compound of claim 28, according to the formulapharmaceutically acceptable salt thereof.

33. The linker payload compound of claim 28, according to the formulapharmaceutically acceptable salt thereof.

34. The linker payload compound of claim 28, according to the formula, or a pharmaceutically acceptable salt thereof.

35. The linker payload compound of any of the previous claims wherein L is -N(R2)-CI-CIO alkylene or -N(R2)-CI-CIO alkylene-PChS-; and R2is hydrogen or Ci- Cio alkyl.

36. The linker payload compound of claim 35, wherein R2is hydrogen.104313810218250298.00092511911WO0137. The linker payload compound of claim 28, chosen fromor a pharmaceutically acceptable salt thereof.

38. The compound of any of the previous claims, wherein the polynucleotide is an interfering nucleic acid molecule, a guide RNA, a ribozyme, an aptamer, a gapmer, a mixmer, a multimer, or an mRNA.

39. The compound of any of the previous claims, wherein the polynucleotide is an siRNA, an shRNA, a miRNA, or an antisense oligonucleotide.

40. The compound of any of the previous claims, wherein the polynucleotide is an siRNA.

41. The compound of any of the previous claims, wherein the polynucleotide is an antisense oligonucleotide.

42. The compound of any of the previous claims, wherein the polynucleotide comprises one or more modified nucleotides.

43. A pharmaceutical composition comprising the compound of the previous claims and a pharmaceutically acceptable carrier.

44. A composition or kit comprising the compound or pharmaceutical composition of any of the previous claims in association with a further therapeutic agent.105313810218250298.00092511911WO0145. A method of making a conjugate comprising the steps of(a) treating an antigen-bidning proteinthe presence of microbial transglutaminase or sortase to provide a transglutaminase-modified or sortase-modified binding agent;(b) treating the transglutaminase-modified or sortase-modified binding agent with the linker pay load of any of the previous claims; and(c) purifying the conjugate.313810218

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