Targeted therapeutics with an electrophilic group for covalent attachment and methods of use and manufacture thereof

Abstract

This disclosure pertains to programmable peptide modalities and the use thereof for specific targets. Further provided herein are conjugates comprising therapeutic moieties, wherein the therapeutic moiety can comprise enzymes, peptides, toxins, cytokines, or nucleic acids. Further provided herein are methods of treating disease with a therapeutic moiety.

Description

TARGETED THERAPEUTICS WITH AN ELECTROPHILIC GROUP FOR COVALENT ATTACHMENT AND METHODS OF USE AND MANUFACTURE THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and the benefit of U. S. Provisional Application No. 63 / 715,276 filed on November 1, 2024 which is incorporated by reference herein in its entirety for all purposes.BACKGROUND

[0002] Protein-protein interactions (PPIs) play central roles in cellular processes, including gene regulation, metabolism, cell signaling, and cell recognition (Stelzl U, et al., (2005). Cell, 122(6):957-968; Rual JF, et al., (2005). Nature, 437(7062): 1173-1178). Protein-protein interactions (PPIs) are intriguing targets in drug discovery and development. Peptides are well suited to target PPIs, which typically present with large surface areas lacking distinct features and deep binding pockets (Grob NM, et al., (2024). ACS Chem Biol, 19:101-109). To improve binding interactions with these topologies and advance the development of PPI-focused therapeutics, potential ligands can be equipped with electrophilic groups to enable binding through covalent mechanisms of action. Peptides are involved in modulating an estimated 15-40% of PPIs and constitute suitable candidates to address challenging PPI topologies (Cunningham A, et al., (2017). Curr Opin Struct Biol, 44:59-66; Neduva V, et al., (2005). PLoS Biol, 3(12):e405). Peptides mimic the structural features of protein domains and can occupy larger binding surfaces than typical small molecule drugs.

[0003] Peptides mediate biological signaling pathways with remarkable potency, selectivity, and low immunogenicity in humans (Fosgerau K, et al., (2015). Drug Discovery Today, 20(1): 122-128). An important challenge in the development of peptide drugs is their short circulation time resulting from rapid enzymatic degradation and renal clearance. (Diao L, et al., (2013). Clin Pharmacokinet, 52(10), 855-868). Stability issues have been addressed via strategies such as cyclization, incorporation of D- and noncanonical amino acids, and backbone modifications (Evans BJ, et al., (2020). Clin Pharmacokinet, 52(10): 855-868). The combination of 20 canonical amino acids and various noncanonical amino acids allows high-diversity peptide binders that are synthetically accessible by solid-phase peptide synthesis. Furthermore, peptide side chains arealso structurally tunable with new chemical moieties to improve peptide stability and cellular permeability (Zhang P, et al., (2024). J Am Chem Soc, 146:15627-15639). Methods to evade renal clearance have emerged, but extensive modifications may obstruct target binding by the introduction of steric hindrance (Muttenthaler M, et al., (2021). Nat Rev Drug Discovery, 20(4):309-325). What is needed, therefore, is an established approach to modulate pharmacokinetic and pharmacodynamic profiles of small-molecule drugs using the exploitation of covalent binding to the target of interest.SUMMARY

[0004] The present disclosure concerns a programmable controlled delivery and release peptide with the versatility to address diverse disease indications through the conjugation of various payloads.

[0005] In one aspect, the present disclosure is directed to a compound of Formulae (I’):R'— T-L-Tx(r).or a pharmaceutically acceptable salt thereof,wherein:R’ comprises a monovalent reactive group;T is a targeting moiety capable of specifically interacting with a target;Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

[0006] In another aspect, the present disclosure is directed to a compound of Formula (IF) T-R"-L— Tx(ir)or a pharmaceutically acceptable salt thereof,wherein:R” comprises a divalent reactive group;T is a targeting moiety capable of specifically interacting with a target;Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

[0007] In another aspect, the present disclosure is directed to a compound of Formula (III’)R'-T-R"-L— Tx(III )>or a pharmaceutically acceptable salt thereof,wherein:R’ comprises a monovalent reactive group;R” comprises a divalent reactive group;T is a targeting moiety capable of specifically interacting with a target;Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

[0008] In another aspect, the present disclosure is directed to a compound of Formula (IV’)or a pharmaceutically acceptable salt thereof,wherein:R” comprises a divalent reactive group;T is a targeting moiety capable of specifically interacting with a target;Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

[0009] In another aspect, the present disclosure is directed to a compound of Formula (V’)R' — T — L — R'— Tx(V )or a pharmaceutically acceptable salt thereof,wherein:R’ comprises a monovalent reactive group;R” comprises a divalent reactive group;T is a targeting moiety capable of specifically interacting with a target;Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

[0010] In another aspect, the present disclosure is directed to a compound of Formula (VI’)or a pharmaceutically acceptable salt thereof,wherein:R’ comprises a monovalent reactive group;T is a targeting moiety capable of specifically interacting with a target;Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

[0011] In another aspect, the present disclosure is directed to a compound compound of Formulaor a pharmaceutically acceptable salt thereof,wherein:R’ comprises a monovalent reactive group;R” comprises a divalent reactive group;T is a targeting moiety capable of specifically interacting with a target;Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

[0012] In another aspect, the present disclosure is directed to a pharmaceutical composition comprising a compound of the present disclosure (e.g., Formulae I, II, I’, IF, III’, IV’, V’, VI’, or VII’) and a pharmaceutically acceptable carrier.

[0013] In another aspect, the present disclosure is directed to a method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1-32 or a pharmaceutical composition according to claim 33.

[0014] Provided herein are pharmaceutical compositions comprising an effective amount of a targeted therapy comprising:a) [T]- a targeting moiety comprising a first polypeptide comprising a first polypeptide sequence capable of selectively interacting with a second polypeptide comprising a second polypeptide sequence;b) [R]- a reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, cysteine or tyrosine present in the second polypeptide sequence; and c) [Tx]- a therapeutic moiety.

[0015] Also provided are pharmaceutical compositions comprising an effective amount of a targeted therapy comprising:a) [T]- a targeting moiety comprising a first polypeptide comprising a first polypeptide sequence capable of selectively interacting with a cell surface-associated entity;b) [R]- a reactive moiety comprising an entity capable of covalent linkage to a reactive atom present in the cell surface-associated entity; andc) [Tx]- a therapeutic moiety.

[0016] Also provided are pharmaceutical compositions comprising an effective amount of a targeted therapy comprising:a) [T]- a targeting moiety comprising a first polypeptide comprising a first polypeptide sequence capable of selectively interacting with a second polypeptide comprising a second polypeptide sequence;b) [R]- a plurality of reactive moieties R1 to Rn (e.g., R’ and / or R”) independently comprising an entity capable of covalent linkage to a lysine, serine, cysteine, histidine, or tyrosine present in the second polypeptide sequence; andc) [Tx]- a therapeutic moiety.

[0017] Also provided are pharmaceutical compositions comprising an effective amount of a therapy comprising:a) [R1 ]- a first reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, or cysteine or tyrosine present in a targeting moiety comprising a first polypeptide comprising a first polypeptide sequence, wherein the targeting moiety is capable of selectively interacting with a second polypeptide comprising a second polypeptide sequence;b) [R2]- a second reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, cysteine or tyrosine present in the second polypeptide sequence; andc) [Tx]- a therapeutic moiety.

[0018] Also provided are pharmaceutical compositions comprising an effective amount of a targeted therapy comprising:a) [R1 ]- a first reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, or cysteine or tyrosine present in a first polypeptide comprising a first polypeptide sequence comprising a target cell surface molecule (e.g., a cell surface receptor);b) [Tx]- a therapeutic moiety; andc) [R2]- a second reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, or cysteine or tyrosine present in a targeting moiety comprising a second polypeptide sequence, wherein the targeting moiety is capable of selectively interacting with the first polypeptide.

[0019] Also provided are methods of treating a human subject suffering from a disease, disorder or condition, comprising administering to the human subject a pharmaceutical composition comprising an effective amount of a targeted therapy comprising:a) [T]- a targeting moiety comprising a first polypeptide comprising a first polypeptide sequence capable of selectively interacting with a second polypeptide comprising a second polypeptide sequence;b) [R]- a reactive moiety comprising an entity capable of covalent linkage to a cysteine or tyrosine present in the second polypeptide sequence; andc) [Tx]- a therapeutic moiety.

[0020] Also provided are methods of decorating the extracellular surface of a target cell population, comprising administering to the human subject a pharmaceutical composition comprising an effective amount of a targeted therapy comprising:a) [T]- a targeting moiety comprising a first polypeptide comprising a first polypeptide sequence capable of selectively interacting with a second polypeptide comprising a second polypeptide sequence present on the surface of a target cell population;b) [R]- a reactive moiety comprising an entity capable of covalent linkage to a cysteine or tyrosine present in the second polypeptide sequence; andc. [CPM]- a cell painting moiety;wherein the CPM decorates the extracellular surface of the target cell population.

[0021] Also provided are complexes comprising a human material conjugated to a composition comprising:a) [T]- a targeting moiety comprising a first polypeptide comprising a first polypeptide sequence capable of selectively interacting with a second polypeptide comprising a second polypeptide sequence;b) [R]- a reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, or cysteine or tyrosine present in the second polypeptide sequence; and c) [Tx]- a therapeutic moiety.

[0022] Also provided are complexes comprising a human material conjugated to a composition comprising:a) [R]- a reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, or cysteine or tyrosine present in a second polypeptide comprising a second polypeptide sequence; andb) [Tx]- a therapeutic moiety.

[0023] Also provided are compositions comprising:a) [M]- a modulating moiety comprising a first polypeptide comprising a first polypeptide sequence capable of selectively modulating a second polypeptide comprising a second polypeptide sequence;b) [R]- a reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, or cysteine or tyrosine present in the second polypeptide sequence.

[0024] Also provided are compositions comprising:a) [M]- a modulating moiety comprising a first polypeptide comprising a first polypeptide sequence capable of selectively modulating a second polypeptide comprising a second polypeptide sequence; andb) [R]- a plurality of reactive moieties R1 to Rn independently comprising an entity capable of covalent linkage to a lysine, serine, histidine, or cysteine or tyrosine present in the second polypeptide sequence.

[0025] Also provided are compositions comprising:a) [M]- a modulatory moiety comprising a first polypeptide comprising a first polypeptide sequence, wherein the modulating moiety is capable of selectively modulating a second polypeptide comprising a second polypeptide sequence;b) [R1 ]- a first reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, or cysteine or tyrosine present in the modulating moiety; and c) [R2]- a second reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, or cysteine or tyrosine present in the second polypeptide sequence.

[0026] Also provided are compositions comprising:a) [M]- a modulating moiety comprising a first polypeptide comprising a first polypeptide sequence, wherein the modulating moiety is capable of selectively modulating a second polypeptide comprising a second polypeptide sequenceb) [Rl]- a first reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, cysteine or tyrosine present in a modulating moiety; andc) [R2]- a second reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, or cysteine or tyrosine present in the second polypeptide sequence.

[0027] Also provided are conjugates represented by Formula (I) or (II):[T]-[R]-[L2]-[TX] (I)([R]-[Li ])m— [T]— ([L2]— [Tx])n(II)wherein:each [R] is independently a reactive moiety;[T] is a targeting moiety, wherein the targeting moiety is capable of interacting with a target; each [Tx] is independently a therapeutic moiety;each [Li] is independently a bond or a linker;each [L2] is independently a bond or a linker; andand m and n are each independently an integer from 0 to 5.

[0028] Also provided are pharmaceutical compositions comprising a conjugate described above and a pharmaceutically acceptable carrier.

[0029] Also provided are methods of treating a human subject suffering from a disease, disorder or condition, comprising administering to the human subject a pharmaceutical composition described above.

[0030] Also provided are methods of delivering a therapeutic agent to a target cell, comprising administering a conjugate described above.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0032] FIG. 1 is a schematic illustrating the peptide construct including the targeting moiety, reactive moiety, and the therapeutic moiety, which may be active or inactive.

[0033] FIG. 2 illustrates the variable positioning of the reactive moiety connected to the targeting moiety. The reactive moiety can be moved to different sites on the targeting moiety to position the reactive moiety within the binding site of the target.

[0034] FIG.3 illustrates the activated peptide construct binding to its target and the schematic upon activation, showing that all components of the therapeutic stay attached to the target upon binding and covalent modification.

[0035] FIG. 4 illustrates the activated peptide construct binding to its target and the schematic upon activation, showing that the targeting moiety is released and the therapeutic moiety stays attached to the target upon binding and covalent modification.

[0036] FIG. 5 illustrates the inactivated peptide construct binding to its target and the schematic upon activation, showing that the targeting moiety is released and the therapeutic moiety is activated and stays attached to the target upon binding and covalent modification.

[0037] FIG. 6 illustrates the activated peptide construct binding to its target and the schematic upon activation, showing the activated therapeutic moiety being released upon binding and covalent modification, as the targeting moiety stays attached to the target.

[0038] FIG. 7 illustrates the inactivated peptide construct binding to its target and the schematic upon activation, showing the inactivated therapeutic moiety is activated upon release, or subsequently, like a prodrug, and the targeting moiety stays attached to the target.

[0039] FIG. 8 illustrates a peptide construct including a targeting moiety, reactive moiety, and a therapeutic moiety, where the targeting moiety and reactive moiety are separated by an additional linker, for example, LAand / or LB, and where Tx is activated or inactivated.

[0040] FIG. 9 illustrates that a reactive moiety (e.g., R”) and therapeutic moiety (Tx) can each be independently moved to different sites on the targeting moiety to position the reactive moiety within a binding site and to place the therapeutic moiety.

[0041] FIG. 10 illustrates that a reactive moiety (e.g., R’) and therapeutic moiety (Tx) can each be independently moved to different sites on the targeting moiety to position the reactive moiety within the binding site and to place the therapeutic moiety.

[0042] FTG. 11 illustrates how PSMA-binding selectivity is retained in the presence of a linker, reactive moiety, and / or therapeutic component.DETAILED DESCRIPTIONSDefinitions

[0043] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N. Y. 1994), provides one skilled in the art with a general guide to many of the terms used in the present application.

[0044] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present disclosure. Indeed, the present disclosure is in no way limited to the methods and materials described. For purposes of the present disclosure, the following terms are defined.

[0045] The term “active” as used herein means biologically, therapeutically or pharmacologically active.

[0046] The terms “electrophilic group,” “electrophile,” and the like as used herein refers to an atom or group of atoms that can accept an electron pair to form a covalent bond. The electrophilic group used herein includes but is not limited to halide, carbonyl, and epoxide-containing compounds. Common electrophiles include halides such as thiophosgene, glycerin dichlorohydrin, phthaloyl chloride, succinyl chloride, chloroacetyl chloride, chlorosuccinyl chloride, etc.; ketones such as chloroacetone, bromoacetone, etc.; aldehydes such as glyoxal, etc.; isocyanates such as hexamethylene diisocyanate, tolylene diisocyanate, meta-xylylene diisocyanate, cyclohexylmethane-4,4-diisocyanate, and derivatives of these compounds.

[0047] The terms “nucleophilic group,” “nucleophile,” and the like as used herein refers to an atom or group of atoms that have an electron pair capable of forming a covalent bond. Groups of this type may be ionizable groups that react as anionic groups. The “nucleophilic group” used herein includes but is not limited to hydroxyl, carboxyl, primary amines, secondary amines, tertiary amines, indole nitrogen (e.g., from tryptophan), imidazole nitrogen (e.g., from histidine), thiols, thioethers (e.g., from methionine).

[0048] An “adjuvant,” as the term is used herein, is a compound that has little or no therapeutic value on its own, but increases the effectiveness of a therapeutic agent. Exemplary adjuvants include radiosensitizers, transfection-enhancing agents (such as chloroquine and analogs thereof), chemotactic agents and chemoattractants, peptides that modulate cell adhesion and / or cell mobility, cell permeabilizing agents, inhibitors of multidrug resistance and / or efflux pumps, etc.

[0049] The term “nucleophilic substitution” is meant a reaction between a nucleophile and an electrophile, in which a covalent bond is formed between the nucleophile and the electrophile and a bond is broken between the electrophile and a leaving group. Thus, a leaving group that had been bound to the electrophile is replaced by the nucleophile.

[0050] The term “conjugate addition” means a reaction between a nucleophile and a conjugated unsaturated group or conjugated unsaturated bond. For example, a nucleophile can react with an a, P unsaturated aldehyde or ketone, resulting in the formation of a covalent bond between the nucleophile and the P carbon and a bond between a hydrogen atom and the a carbon. In this reaction, a bond between the a and P carbons is also converted from a double to a single bond.

[0051] The terms “protein,” “peptide,” and “polypeptide” can be used interchangeably and in their broadest sense can refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits can be linked by peptide bonds. Alternatively, the subunits can be linked by other bonds, e.g., ester, ether, etc. A protein or peptide can contain at least two amino acids and no limitation can be placed on the maximum number of amino acids which can comprise a protein’s or peptide's sequence. As used herein the term “amino acid” can refer to natural amino acids, unnatural amino acids, or synthetic amino acids, including both the D and L optical isomers, amino acid analogs, and peptidomimetics.

[0052] “Cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and / or causes the destruction of cells. This term encompasses radioisotopes (eg,211At,1311,1251,90Y,186Re,188Re,153Sm,212Bi,32P,60Co, and177Lu) and chemotherapies. It is also intended to include toxins, which are, for example, small molecule toxins of bacterial origin, fungal origin, plant origin, or animal origin or enzymatically active toxins, including synthetic analogs or derivatives thereof.

[0053] As used herein, the term “targeting ligand” or “targeting moiety” refers to any material or substance which may promote targeting of receptors, cells, and / or tissues in vivo or in vitro with the compositions of the present disclosure. The targeting moiety may be synthetic, semisynthetic, or naturally occurring. Materials or substances which may serve as targeting ligands include, but are not limited to, proteins, including antibodies, antibody fragments, nanobodies, hormones, hormone analogues, glycoproteins and lectins, peptides, polypeptides, amino acids, sugars, saccharides, including monosaccharides and polysaccharides, carbohydrates, small molecules, vitamins, steroids, steroid analogs, hormones, cofactors, bioactive agents, and genetic material, including nucleosides, nucleotides, nucleotide acid constructs, and polynucleotides.

[0054] As used herein, the term “therapeutic moiety” refers to any material or substance which may promote a biological response due to binding. The therapeutic moiety may be selected from any one of the following enzymes, peptides, toxins, warheads, cytokines, or nucleic acids.

[0055] As used herein, the term “reactive moiety” refers to an entity capable of covalent linkage to a reactive atom present in a cell surface-associated entity, and / or capable of covalent linkage to a nucleophilic residue of a biological target, e.g., lysine, serine, histidine, cysteine, or tyrosine present in the polypeptide sequence of the target.

[0056] The term “in vivo” refers to an event that takes place in a subject’s body.

[0057] The term “half-life extending moiety” refers to any material or substance which may lead to an increase in half-life (serum half-life and / or therapeutic half-life) and / or increased absorption of the compositions disclosed herein (e.g., a peptide), compared to a reference such as an unconjugated form of a peptide or a wild-type peptide. In some embodiments, modifications described herein that improve or alter biological or biophysical properties (e.g., half-life extending) may be achieved when, to a pharmaceutically acceptable moiety, domain, or molecule, directly or via linkers (e.g. peptide components or PEG), is covalently linked (“conjugated” or “fused”) to a peptide. Half-life extending moieties described herein may be non-proteinaceous, such as a fatty acid or derivative thereof, a water-soluble polymer such as polyethylene glycol (PEG) or discontinuous PEG, hydroxyethyl starch (HES), lipid, branched or non-branched acyl groups, branched or unbranched C8-C30 acyl groups, branched or unbranched alkyl groups, and branched or unbranched C8-C30 alkyl groups. Half-life extending moieties described herein may also be proteinaceous, such as serum albumin, transferrin,Adnectin (e.g., albumin-binding or pharmacokinetic extension (PKE) Adnectin), Fc domain, and unstructured polypeptides such as XTEN and PAS polypeptides (e.g. a conformationally disordered polypeptide sequence consisting of the amino acids Pro, Ala, and / or Ser), and fragments of any of the foregoing.

[0058] A “therapeutically effective amount” of a compound, with respect to a method of treatment, refers to an amount of the compound(s) in a preparation which, when administered as part of a desired dosage regimen (to a mammal, preferably a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated or the cosmetic purpose, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment.

[0059] The term “linker” or “spacer” may be any component that connects the targeting moiety to the therapeutic moiety. Exemplary linkers include small organic compounds, water-soluble polymers of various lengths such as polyethylene glycol) or polydextran, and peptide-based linkers such as peptides of up to 50, 40, 30, 25, 20, 15, 10 or up to 6 amino acids in length.

[0060] The term “free radical polymerization” is meant the cross-linking of monomers that is initiated by a radical. The radical reacts with a monomer, producing a radical that can react with another monomer.

[0061] The term “solubility” refers to the amount of a substance that can be dissolved in another substance, for example, the amount of an unmodified or derived peptide that can be dissolved in an aqueous solution. An exemplary method for measuring the solubility of unmodified or derived peptides is the plug flow solubility test. Relative solubility can be determined with a comparison compound, for example, to identify polypeptides with increased solubility.

[0062] The term “biological activity” or “bioactivity” refers to any function in a biological system, pathway, molecule, or interaction with organisms including, but not limited to, viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans.“Function” refers to the ability of a molecule to affect any physical or biochemical property. For example, concerning the peptide disclosed herein, the biological activity includes any of the functions performed by the peptide.

[0063] The term “in vivo proteolysis” refers to the cleavage of a polypeptide that can occur by proteases occurring in an organism when introduced into a living system (e.g., when injected into an organism). Proteolysis can potentially affect the biological activity or half-life of the polypeptide. For example, wild-type peptides can undergo cleavage, producing a truncated, inactive polypeptide. An exemplary method for measuring in vivo proteolysis of peptides is the Meso Scale Discovery (MSD)-based electrochemiluminescence immunosorbent assay (ECLIA). The relative amount of in vivo proteolysis can be determined relative to a comparative compound, for example, to identify polypeptides with reduced in vivo proteolysis.

[0064] The term “agonist,” as used herein, is meant to refer to an agent that mimics or up-regulates (e.g., potentiates or supplements) the bioactivity of a protein of interest, or an agent that facilitates or promotes (e.g., potentiates or supplements) an interaction among polypeptides or between a polypeptide and another molecule (e.g., a steroid, hormone, nucleic acids, small molecules etc.). An agonist can be a wild-type protein or derivative thereof having at least one bioactivity of the wild-type protein. An agonist can also be a small molecule that up-regulates the expression of a gene or which increases at least one bioactivity of a protein. An agonist can also be a protein or small molecule which increases the interaction of a polypeptide of interest with another molecule, e.g., a target peptide or nucleic acid.

[0065] The term “antagonist,” as used herein, is meant to refer to an agent that down-regulates (e g., suppresses or inhibits) the bioactivity of a protein of interest, or an agent that inhibits / suppresses or reduces (e.g., destabilizes or decreases) interaction among polypeptides or other molecules (e.g., steroids, hormones, nucleic acids, etc.). An antagonist can also be a compound that down-regulates the expression of a gene of interest or which reduces the amount of the wild-type protein present. An antagonist can also be a protein or small molecule which decreases or inhibits the interaction of a polypeptide of interest with another molecule, e.g., a target peptide or nucleic acid.

[0066] As used herein, the term “warhead” refers to an atom or group of atoms that has a reactive functionality to engage in a covalent interaction, e.g., with a protein, enzyme, or receptor, in either a reversible or an irreversible manner. In some embodiments, a warhead may be an electrophilic group, which can form a covalent bond with a nucleophilic group of a protein, enzyme, or receptor, in either a reversible or an irreversible manner.

[0067] As used herein, the term “alkyl,” used alone or as part of another group, refers, in one embodiment, to a “Ci to Cis alkyl” and denotes linear and branched, saturated or unsaturated (e.g., alkenyl, alkynyl) groups, the latter only when the number of carbon atoms in the alkyl chain is greater than or equal to two, and can contain mixed structures. Non-limiting examples are alkyl groups having from 1 to 6 carbon atoms (Ci to G> alkyls), or alkyl groups having from 1 to 4 carbon atoms (Ci to C4 alkyls). Examples of saturated alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, amyl, tertamyl and hexyl. Examples of alkenyl groups include, but are not limited to, vinyl, allyl, butenyl and the like. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl and the like. Similarly, the term “Ci to Cis alkylene” denotes a bivalent radical of 1 to 18 carbons. The alkyl group can be unsubstituted, or substituted with one or more substituents selected from the group consisting of halogen, hydroxy, alkoxy, aryloxy, alkylaryloxy, heteroaryloxy, oxo, cycloalkyl, phenyl, heteroaryls, heterocyclyl, naphthyl, amino, alkylamino, arylamino, heteroarylamino, dialkylamino, diarylamino, alkylarylamino, alkylheteroarylamino, arylheteroarylamino, acyl, acyloxy, nitro, carboxy, carbamoyl, carboxamide, cyano, sulfonyl, sulfonylamino, sulfinyl, sulfinylamino, thiol, alkylthio, arylthio, or alkylsulfonyl groups. Any substituents can be unsubstituted or further substituted with any one of these aforementioned substituents.

[0068] The term “acyl” is art-recognized and refers to a group represented by the general formula hydrocarbyl-C(O) —, preferably alkyl-C(O) —.

[0069] The term “acyloxy” is art-recognized and refers to a group represented by the general formula hydrocarbyl-C(O)O —, preferably alkyl-C(O)O —.

[0070] The term “alkoxyalkyl” refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl -O-alkyl.

[0071] The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described below, but that contain at least one double or triple bond, respectively.

[0072] The term “alkoxy” refers to an alkyl group having an oxygen attached thereto.Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like.

[0073] The term “alkylamino,” as used herein, refers to an amino group substituted with at least one alkyl group.

[0074] The term “alkylcycloalkyl” refers to groups, which contain cycloalkyl as well as alkyl, alkenyl or alkynyl groups according to the above definition, e.g. alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyl, and alkynylcycloalkyl groups. Preferentially, a alkylcycloalkyl group is composed of a cycloalkyl group, comprising one or more rings, comprising three to ten, preferentially three, four, five, six or seven carbon-atoms and one or two alkyl, alkenyl, or alkynyl groups with one or two to six carbon atoms.

[0075] The term “alkylthio,” as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkyl-S —.

[0076] The term “amidine” denotes the group — C(NH) — NHR, wherein R is H or alkyl or aralkyl. A preferred amidine is the group — C(NH) — NH2.

[0077] The term “aralkyl,” as used herein, refers to an alkyl group substituted with an aryl group.

[0078] The term “aryl” used herein alone or as part of another group denotes an aromatic ring system having from 6-14 ring carbon atoms. The aryl ring can be a monocyclic, bicyclic, tricyclic, and the like. Non-limiting examples of aryl groups are phenyl, naphthyl including 1 -naphthyl and 2-naphthyl, and the like. The aryl group can be unsubtituted or substituted through available carbon atoms with one or more groups such as halogen, alkyl, aryl, hydroxy, alkoxy, aryloxy, alkylaryloxy, heteroaryl oxy, oxo, cycloalkyl, phenyl, heteroaryls, heterocyclyl, naphthyl, amino, alkylamino, arylamino, heteroaryl ami no, dialkylamino, diarylamino, alkylarylamino, alkylheteroarylamino, arylheteroarylamino, acyl, acyloxy, nitro, carboxy, carbamoyl, carboxamide, cyano, sulfonyl, sulfonylamino, sulfinyl, sulfmylamino, thiol, alkylthio, arylthio, alkylsulfonyl -OCN, -SCN, - N=C=O, -NCS, -NO, -N3, -OP(=O)(OR*)2, -P(=O)(OR*)2, -P(=O)(O-)2, -P(=O)(OH)2, - P(O)(OR*)(O-), -C(=O)R*, -C(=O)X, -C(S)R*, -C(S)OR*, -C(O)SR*, — C(S)SR*, -C(S)NR*2 or -C(=NR*)NR*2 groups, where each R* is independently H, alkyl, aryl, arylalkyl, a heterocycle, or a protecting group or prodrug moiety groups. Any substituents can be unsubstituted or further substituted with any one of these aforementioned substituents.

[0079] The term “carbonate” is art-recognized and refers to a group — OCO2-.

[0080] The term “carboxy,” as used herein, refers to a group represented by the formula CO2H.

[0081] The terms “carbocycle,” “carbocyclyl,” and “carbocyclic,” as used herein, refer to a non-aromatic saturated or unsaturated ring in which each atom of the ring is carbon. Preferably, a carbocycle ring contains from 3 to 10 atoms, more preferably from 5 to 7 atoms.

[0082] The term “carbonyl” is art-recognized and includes such moieties as may be represented by the general formula:wherein X is a bond or represents an oxygen or a sulfur, and Rn represents a hydrogen, an alkyl, an alkenyl, — (CH2)m— Rs or a pharmaceutically acceptable salt, Rn represents a hydrogen, an alkyl, an alkenyl or — (CH2)m— Rs, where m and Rs are as defined above. Where X is an oxygen and Rn or Rn is not hydrogen, the formula represents an “ester.” Where X is an oxygen, and Rn is as defined above, the moiety is referred to herein as a “carboxyl” group, and particularly when Rn is a hydrogen, the formula represents a “carboxylic acid.” Where X is an oxygen, and Rn is hydrogen, the formula represents a “formate.” In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a “thiocarbonyl” group. Where X is a sulfur and Rn or Rn is not hydrogen, the formula represents a “thioester.” Where X is a sulfur and Rn is hydrogen, the formula represents a “thiocarboxylic acid.” Where X is a sulfur and Rn is hydrogen, the formula represents a “thioformate.” On the other hand, where X is a bond, and Rn is not hydrogen, the above formula represents a “ketone” group. Where X is a bond, and Rn is hydrogen, the above formula represents an “aldehyde” group.

[0083] The term “cycloalkyl” refers to a saturated or partially unsaturated (e.g., cycloalkenyl) cyclic group, comprising one or several rings, preferentially one or two, containing three to fourteen ring carbon atoms, preferentially three to ten, preferentially three, four, five, six or seven ring carbon atoms. Furthermore the term cycloalkyl refers to a group where one or more hydrogen atoms are replaced by F, Cl, Br, I, OH, =0, SH, =S, NH2, =NH, or NO2, or cyclic ketones, for example cyclohexanone, 2-cyclohexenone or cyclopentanone. Examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentenyl, spiro[4,5]-decanyl, norbomyl,cyclohexyl, cyclopentenyl, cyclohexadienyl, decalinyl, cubanyl, bicyclo[4.3.0]nonyl, tetralin, cyclopentylcyclohexyl, fluor-cyclohexyl or the cyclohex-2-enyl group.

[0084] The term “ether,” as used herein, refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-0 —. Ethers may be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include “alkoxyalkyl” groups, which may be represented by the general formula alkyl -O-alkyl.

[0085] The terms “halo” and “halogen” as used herein means halogen and includes chloro, fluoro, bromo, and iodo.

[0086] The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to an alkyl group substituted with a heteroaryl group.

[0087] The terms “hetaralkenyl” and “heteroaralkenyl”, as used herein, refers to an alkenyl group substituted with a heteroaryl group.

[0088] The term “heteroalkyl” refers to a alkyl, alkenyl or alkynyl group, where several, preferentially one, two or three carbon atoms are replaced by a O, N, P, B, Se, Si, or S atom, preferentially O, S, N. The term heteroalkyl also includes a carboxylic acid or a thereof derived group, for example acyl (alkyl-CO), acylalkyl, alkoxycarbonyl, acyloxy, acyloxyalkyl, carboxyalkylamid or alkoxycarbonyloxy.

[0089] The term “heteroalkylcycloalkyl” refers to alkylcycloalkyl groups, according to the above definition, wherein one or several, preferentially one, two or three carbon atoms are replaced by O, N, Si, Se, P or S, preferentially O, S, N. In certain instances a heteroakylcycloalkyl group comprises one or two ring systems with three to ten, preferentially three, four, five, six or seven ring atoms and one or two alkyl, alkenyl, alkynyl or heteroalkyl groups with one or two to six carbon atoms. Examples of such a group are alkylheterocycloalkyl, alkylheterocycloalkenyl, alkenyl-heterocycloalkyl, alkynylheterocycloalkyl, heteroalkylcycloalkyl, heteroalkylheterocycloalkyl and heteroalkylheterocycloalkenyl, wherein the cyclic group is saturated or partially (e.g., twofold or threefold) unsaturated.

[0090] The terms “heteroaryl” and “hetaryl” include substituted or unsubstituted aromatic ring structures, such as 5- to 14-membered rings, preferably 5- to 7-membered rings, more preferably5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. Non-limiting examples of suitable heteroatoms which can be included in the aromatic ring include oxygen, sulfur, phosphate, and nitrogen. A “heteroaryl” may be a monocyclic ring system. The term “heteroaryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Non-limiting examples of heteroaryl rings include pyridinyl, pyrrolyl, oxazolyl, indolyl, isoindolyl, purinyl, furanyl, thienyl, benzofuranyl, thiophenyl, benzothiophenyl, carbazolyl, imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, quinolyl, isoquinolyl, pyridazyl, pyrimidyl, pyrazyl, etc. The heteroaryl group can be unsubstituted or substituted through available carbon atoms with one or more groups, such as halogen, alkyl, aryl, hydroxy, alkoxy, aryloxy, alkylaryloxy, heteroaryloxy, oxo, cycloalkyl, phenyl, heteroaryls, heterocyclyl, naphthyl, amino, amido, alkylamino, arylamino, heteroarylamino, dialkylamino, diarylamino, alkylarylamino, alkylheteroarylamino, arylheteroarylamino, acyl, acyloxy, nitro, carboxy, carbamoyl, carboxamide, cyano, sulfonyl, sulfonylamino, sulfinyl, sulfmylamino, thiol, alkylthio, arylthio, alkylsulfonyl, -OCN, -SCN, -N=C=O, -NCS, -NO, -N3, - OP(=O)(OR*)2, -P(=O)(OR*)2, -P(=O)(O-)2, -P(=O)(OH)2, -P(O)(OR*)(O-), -C(=O)R*, - C(=O)X, -C(S)R*, -C(S)OR*, -C(O)SR*, — C(S)SR*, -C(S)NR*2 or -C(=NR*)NR*2groups, where each R* is independently H, alkyl, aryl, arylalkyl, a heterocycle, or a protecting group or prodrug moiety. Any substituents can be unsubstituted or further substituted with any one of these aforementioned substituents.

[0091] The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0092] The term “heterocycloalkyl” refers to the above definition of cycloalkyl, wherein one or more, preferentially one, two or three ring carbon atoms are replaced by a O, N, Si, Se, P, or S, preferentially O, S, N. Preferentially, a heterocycloalkyl group is composed of one or two rings comprising three to ten, preferentially three, four, five, six or seven ring atoms. Moreover, the term heterocycloalkyl refers to groups where one or more hydrogen atoms are replaced by F, Cl, Br, I, OH, =0, SH, =S, NH2, NO2. Examples of heterocycloalkyl are piperidyl, morpholinyl, urotropinyl, pyrrolidinyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrofuryl,oxacyclopropyl, azacyclopropyl or 2-pyrazolinyl groups as well as lactams, lactones, cyclic imides and cyclic anhydrides.

[0093] The terms “heterocyclyl,” “heterocycle,” and “heterocyclic” refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heterocyclyl” and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyl s. Heterocyclyl groups include, for example, imidazolidinone, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0094] The term “hydrocarbyl,” as used herein, refers to a group that is bonded through a carbon atom that optionally has a =0 or =S substituent and typically has at least one carbonhydrogen bond and a primarily carbon backbone, but may optionally include functional groups with heteroatoms interrupting the carbon backbone. Examples of such functional groups with interrupting heteroatoms include amino, amide, carbonate, carbamate, ether (e.g., polyethylene glycol), ester, thioester, thiourea, and urea groups. For illustrative purposes, additional examples of hydrocarbyl groups include methyl, ethoxy ethyl, 2-pyridyl, trifluoromethyl, and acetyl, but not, for example, ethoxy (which is linked through oxygen, not carbon). Additional hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof. Hydrocarbyl also includes corresponding divalent species (i.e., hydrocarbylene), such as alkylene, arylene, etc.

[0095] The term “hydroxyalkyl,” as used herein, refers to an alkyl group substituted with a hydroxy group.

[0096] The term “lower” when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer non-hydrogen atoms in the substituent, preferably six or fewer. A “lower alkyl,” for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, loweralkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

[0097] The terms “polycyclyl,” “polycycle,” and “polycyclic” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are “fused rings.” Each of the rings of the polycycle may be substituted or unsubstituted. In certain embodiments, each ring of the poly cycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7.

[0098] The terms “biocompatible polymer” and “biocompatibility” when used in relation to polymers are art-recognized. For example, biocompatible polymers include polymers that are neither themselves toxic to the host (e.g., an animal or human), nor degrade (if the polymer degrades) at a rate that produces monomeric or oligomeric subunits or other byproducts at toxic concentrations in the host. In certain embodiments, biodegradation generally involves degradation of the polymer in an organism, e.g., into its monomeric subunits, which may be known to be effectively non-toxic. Intermediate oligomeric products resulting from such degradation may have different toxicological properties, however, or biodegradation may involve oxidation or other biochemical reactions that generate molecules other than monomeric subunits of the polymer. Consequently, in certain embodiments, toxicology of a biodegradable polymer intended for in vivo use, such as implantation or injection into a patient, may be determined after one or more toxicity analyses. It is not necessary that any subject composition have a purity of 100% to be deemed biocompatible. Hence, a subject composition may comprise 99%, 98%, 97%, 96%, 95%, 90% 85%, 80%, 75% or even less of biocompatible polymers, e.g., including polymers and other materials and excipients described herein, and still be biocompatible.

[0099] The term “biodegradable” is art-recognized, and includes polymers, compositions and formulations, such as those described herein, that are intended to degrade during use.Biodegradable polymers typically differ from non-biodegradable polymers in that the former may be degraded during use. In certain embodiments, such use involves in vivo use, such as in vivo therapy, and in other certain embodiments, such use involves in vitro use. In general, degradation attributable to biodegradability involves the degradation of a biodegradable polymer into its component subunits, or digestion, e.g., by a biochemical process, of the polymer intosmaller, non-polymeric subunits. In certain embodiments, two different types of biodegradation may generally be identified. For example, one type of biodegradation may involve cleavage of bonds (whether covalent or otherwise) in the polymer backbone. In such biodegradation, monomers and oligomers typically result, and even more typically, such biodegradation occurs by cleavage of a bond connecting one or more of subunits of a polymer. In contrast, another type of biodegradation may involve cleavage of a bond (whether covalent or otherwise) internal to sidechain or that connects a side chain to the polymer backbone. For example, a therapeutic agent or other chemical moiety attached as a side chain to the polymer backbone may be released by biodegradation. In certain embodiments, one or the other or both general types of biodegradation may occur during use of a polymer.

[0100] As used herein, the term “biodegradation” encompasses both general types of biodegradation. The degradation rate of a biodegradable polymer often depends in part on a variety of factors, including the chemical identity of the linkage responsible for any degradation, the molecular weight, crystallinity, biostability, and degree of cross-linking of such polymer, the physical characteristics (e.g., shape and size) of an implant, and the mode and location of administration. For example, the greater the molecular weight, the higher the degree of crystallinity, and / or the greater the biostability, the biodegradation of any biodegradable polymer is usually slower. The term “biodegradable” is intended to cover materials and processes also termed “bioerodible”.

[0101] The phrase “controlled release” or “sustained release” refers to the use of systems that allow for the controlled or tunable delivery of one or more of the present compounds or compositions over time. For example, in certain instances, the present compounds or compositions are used in conjunction with a controlled release system that delivers an effective amount (such as an approximately continuous amount, an increasing amount, or a decreasing amount) of the compound(s) over a certain period of time, for example, over a period of at least about 4, 8, 12, 24, 48, or 72 hours, over a period of at least about 1, 2, 3, 4, or 5 days, over a period of at least about 1, 2, or 3 weeks, or over a period of at least about 1, 2, 3, 4, 5, or 6 months. Such controlled release systems may be used in conjunction with medical devices, such as stents and catheters, to provide medical devices which offer controlled release of the present compounds and / or compositions. By way of example, some suitable controlled release systems include hydrogels, polymers, meshes, and others demonstrated in the art.

[0102] An “effective amount” of a subject compound, with respect to the subject method of treatment, refers to an amount of the therapeutic in a preparation which, when applied as part of a desired dosage regimen provides a benefit according to clinically acceptable standards for the treatment or prophylaxis of a particular disorder.

[0103] As used herein, the term “low aqueous solubility” refers to water insoluble compounds having poor solubility in water, that is <5 mg / ml at physiological pH (6.5-7.4). Preferably, their water solubility is <1 mg / ml, more preferably <0.1 mg / ml. It is desirable that the drug is stable in water as a dispersion; otherwise a lyophilized or spray -dried solid form may be desirable.

[0104] As used herein, “polymerizations” include radical, anionic, and cationic mechanisms, as well as reactions of bifunctional molecules (analogous to the formation of nylon, e.g., reacting molecules each of which bears two or more different reactive moi eties that react with each other (but, preferably, are disfavored from reacting intramolecularly by steric, conformational, or other constraints), or reacting two or more different compounds, each compound bearing two or more reactive moieties that react only with reactive moieties of different compounds (i.e., intermolecularly)), as well as metal -catalyzed polymerizations such as olefin metathesis, and other polymerization reactions known to those of skill in the art.

[0105] As used herein, the term “precursor” to a targeting ligand refers to any material or substance which may be converted to a targeting ligand. Such conversion may involve, for example, anchoring a precursor to a targeting ligand. Exemplary targeting precursor moieties include maleimide groups, disulfide groups, such as ortho-pyridyl disulfide, vinylsulfone groups, azide groups, and a-iodo acetyl groups. The attachment of the targeting ligand or precursor thereof to the polymer may be accomplished in various ways including, but not limited to chelation, covalent attachment, or formation of host-guest complexes. In certain embodiments, an optional linker group may be present between the targeting ligand or precursor thereof and the polymer, wherein the linker group is attached to the polymer via chelation, covalent attachment or form host guest complexes. For example, the one terminal end of a linker group may be attached to the targeting ligand while the other may be attached to an adamantane group, or other such hydrophobic moiety, which forms a host-guest complex with a cyclodextrin moiety. Thus the targeting ligand may be attached to a grafted cyclodextrin moiety, to a cyclodextrin moiety within the polymeric chain, or to the polymeric chain itself. The number of targeting ligands perpolymeric chain may vary according to various factors including but not limited to the identity of the therapeutic agent, nature of the disease, type of polymer chain. Structures of possible linker groups are the same as linker groups defined elsewhere in this application.

[0106] The term “purification step” refers to a step that increases the purity of a product. The product can be separated from some of the other components of a mixture such as starting materials, accelerators, side-products, and solvents. Products can be purified base on their characteristics — such as size, shape, charge, hydrophobicity, solubility, or boiling point — using standard techniques.

[0107] The term “small molecule” refers to a compound having a molecular weight less than about 2500 amu, preferably less than about 2000 amu, even more preferably less than about 1500 amu, still more preferably less than about 1000 amu, or most preferably less than about 750 amu.

[0108] The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents may be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents may include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain maythemselves be substituted, if appropriate. Unless specifically indicated as un substituted, all occurrences of moieties bearing one or more C — H bonds may be either unsubstituted or substituted as defined herein. By way of example, a reference to an “alkyl” or “aryl” group will be understood to include unsubstituted or substituted variants thereof.

[0109] It is not intended that the administration of the biomaterials, precursor components, or compounds of the present disclosure be limited to a particular mode of administration, dosage, or frequency of dosing; the present mode contemplates all modes of administration, including oral, intravenous, intramuscular, subcutaneous, parenteral, or any other route sufficient to provide a dose adequate to prevent or treat a disease, disorder, or infection. One or more of the biomaterials, precursor components, or compounds may be administered to a mammal in a single dose or multiple doses, possibly in the presence of pharmaceutical stabilizing compounds. When multiple doses are administered, the doses can be separated from one another by, for example, one week to one month. It is to be understood that for any particular subject, specific dosage regimes should be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions.Conjugates

[0110] Provided herein are conjugates comprising (i) a first targeting moiety, (ii) a reactive moiety, and (iii) a therapeutic moiety, wherein the targeting moiety comprises at least one first polypeptide sequence whereby the first targeting moiety is capable of interacting with a first target at the site of a second polypeptide sequence. In some embodiments, the reactive moiety comprises an entity capable of covalent linkage to a lysine, serine, histidine, or cysteine or tyrosine present in the second polypeptide sequence. In some embodiments, the linkage is reversible. In other embodiments, the linkage is non-reversible. In some embodiments, the therapeutic moiety comprises an antibody, antibody fragment, or antigen-binding domain. In some embodiments, the therapeutic moiety comprises a single-domain antibody (sdAb). In some embodiments, the reactive moiety is comprised within or within proximity of a region of the first targeting moiety that interfaces with the target. In some embodiments, the targeting moiety, the reactive moiety, and the therapeutic moiety are linked to form a fusion protein. In some embodiments, the targeting moiety, the reactive moiety, and the therapeutic moiety are linked bychemical conjugation. In some embodiments, the targeting moiety, and the therapeutic moiety are linked by a linker. In some embodiments, the target is a cell surface molecule.

[0111] In some embodiments, the conjugate is represented by Formula (I) or (II):[T]-[R]-[L2]-[TX] (I)([R]-[Li])m-[T]-([L2]-[Tx])n(II)wherein:each [R] is independently a reactive moiety;[T] is a first targeting moiety;each [Tx] is independently a therapeutic moiety;each [Li] is independently a bond or a linker;each [L2] is independently a bond or a linker; andand m and n are each independently an integer from 0 to 5.

[0112] In some embodiments, TM1 comprises a first polypeptide sequence. In some embodiments, TM1 is capable of interacting with a first target comprising a second polypeptide sequence. In some embodiments, TM1 interacts with the first target at the second polypeptide sequence. In some embodiments, RM comprises an entity capable of covalent linkage to a lysine, serine, histidine, cysteine, or tyrosine present in the second polypeptide sequence.

[0113] In some embodiments of the present disclosure, provided herein are compounds (i.e., conjugates) comprising a reactive group, a targeting moiety, a linker, and a optionally present linker.

[0114] In some embodiments, provided herein is a compound of Formulae (F):R'— T-L-Tx(r).or a pharmaceutically acceptable salt thereof,wherein:R’ comprises a monovalent reactive group;T is a targeting moiety capable of specifically interacting with a target;Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

[0115] In some embodiments of Formula (I’), Tx is absent. In some embodiments of Formula (I’), Tx is a therapeutic moiety. In some embodiments of Formula (F), Tx is a diagnostic moiety.

[0116] In some embodiments of Formula (F), L is absent. In some embodiments of Formula (F), L is a linker.

[0117] In some embodiments, a compound of Formula (F) can be represented by the structures of FIG. 10.

[0118] In some embodiments, provided herein is a compound of Formula (IF)T— R"— L— Tx(II )or a pharmaceutically acceptable salt thereof,wherein:R” comprises a divalent reactive group;T is a targeting moiety capable of specifically interacting with a target;Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

[0119] In some embodiments of Formula (IF), Tx is absent. In some embodiments of Formula (IF), Tx is a therapeutic moiety. In some embodiments of Formula (IF), Tx is a diagnostic moiety.

[0120] In some embodiments of Formula (IF), L is absent. In some embodiments of Formula (IF), L is a linker.

[0121] In some embodiments, a compound of Formula (IF) can be represented by the structures of FIGs. 1-8, and / or 9.

[0122] In some embodiments, provided herein is a compound of Formula (III’)R’-T-R’-L— Tx(nr)>or a pharmaceutically acceptable salt thereof,wherein:R’ comprises a monovalent reactive group;R” comprises a divalent reactive group;T is a targeting moiety capable of specifically interacting with a target;Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

[0123] In some embodiments of Formula (III’), Tx is absent. In some embodiments of Formula (Hl’), Tx is a therapeutic moiety. In some embodiments of Formula (HF), Tx is a diagnostic moiety.

[0124] In some embodiments of Formula (III’), L is absent. In some embodiments of Formula (III’), L is a linker.

[0125] In some embodiments, provided herein is a compound of Formula (IV’)or a pharmaceutically acceptable salt thereof,wherein:R” comprises a divalent reactive group;T is a targeting moiety capable of specifically interacting with a target;Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

[0126] In some embodiments of Formula (IV’), Tx is absent. In some embodiments of Formula (IV’), Tx is a therapeutic moiety. In some embodiments of Formula (IV’), Tx is a diagnostic moiety.

[0127] In some embodiments of Formula (IV’), L is absent. In some embodiments of Formula (IV’), L is a linker.

[0128] In some embodiments, provided herein is a compound of Formula (V’)R' — T— L — R"- Tx( )or a pharmaceutically acceptable salt thereof,wherein:R’ comprises a monovalent reactive group;R” comprises a divalent reactive group;T is a targeting moiety capable of specifically interacting with a target;Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

[0129] In some embodiments of Formula (V’), Tx is absent. In some embodiments of Formula (V’), Tx is a therapeutic moiety. In some embodiments of Formula (V’), Tx is a diagnostic moiety.

[0130] In some embodiments of Formula (V’), L is absent. In some embodiments of Formula (V’), L is a linker.

[0131] In some embodiments, provided herein is a compound of Formula (VF)or a pharmaceutically acceptable salt thereof,wherein:R’ comprises a monovalent reactive group;T is a targeting moiety capable of specifically interacting with a target;Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

[0132] In some embodiments of Formula (VI’), Tx is absent. In some embodiments of Formula (VI’), Tx is a therapeutic moiety. In some embodiments of Formula (VF), Tx is a diagnostic moiety.

[0133] In some embodiments of Formula (VF), L is absent. In some embodiments of Formula (VI’), L is a linker.

[0134] In some embodiments, provided herein is a compound of Formula (VIF)or a pharmaceutically acceptable salt thereof,wherein:R’ comprises a monovalent reactive group;R” comprises a divalent reactive group;T is a targeting moiety capable of specifically interacting with a target;Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

[0135] In some embodiments of Formula (VIF), Tx is absent. In some embodiments of Formula (VIF), Tx is a therapeutic moiety. In some embodiments of Formula (VIF), Tx is a diagnostic moiety.

[0136] In some embodiments of Formula (VII’), L is absent. In some embodiments of Formula (VIF), L is a linker.

[0137] In some embodiments of Formulae (!’), (Ill’), (V’), (VI’), or (VIF), R’ is represented by Formula (ILA):wherein:LAis a bond or chain of 1-60 carbon atoms in length,wherein one or more carbon atoms are optionally substituted with halogen, hydroxyl, cyano, CFH2, CF2H, CF3, alkoxy, C1-C5 alkyl, or -C(=O)-NH2; andwherein one or more carbon atoms of the backbone are optionally replaced by a divalent group independently selected from oxygen, alkylamino, carbonyl, 3-14 membered cycloalkylene, 3-8 membered monocyclic heterocyclic group, 5-14 membered bridged heterocyclic group, 5-14 membered spiro heterocyclic group, 5-14 membered heteroarylene, and 7-14 membered heteroaryLfused-heterocyclylene, and wherein each of the 3-14 membered cycloalkylene, 3-8 membered monocyclic heterocyclic group, 5-14 membered bridged heterocyclic group, 5-14 membered spiro heterocyclic group, 5-14 membered heteroarylene, and 7-14 membered heteroaryLfused-heterocyclylene is independently substituted with 0, 1, 2, 3, 4, 5, or 6 Ra;each Rais independently selected from alkylamino, halogen, hydroxyl, C1-C5 alkoxy, C1-C5 alkyl, C1-C5 alkylcarboxyl, C1-4 alkenyl, C3-5 cycloalkyl, C1-5haloalkyl, and an oxo group; andRwis an electrophilic functional group capable of forming a covalent bond with the target.Polymeric Conjugates

[0138] The polymeric conjugates of the present disclosure may be useful to improve solubility and / or stability of a therapeutic agent, reduce drug-drug interactions, reduce interactions with blood elements including plasma proteins, reduce or eliminate immunogenicity, increase protease resistance, protect the agent from metabolism, modulate drug-release kinetics, improve circulation time, improve drug half-life (e.g., in the serum, or in selected tissues, such as tumors), attenuate toxicity, improve efficacy, normalize drug metabolism across subjects of different species, ethnicities, and / or races, and / or provide for targeted delivery into specific cells or tissues. Poorly soluble and / or toxic compounds may benefit particularly from incorporation into polymeric compounds of the disclosure. In certain embodiments, the therapeutic agent is a small molecule, a macromolecule, an antibody, a peptide, a protein, an enzyme, a nucleic acid, or a polymer that has a therapeutic function.

[0139] In another aspect, the rate of release of any material from any polymer matrix of the present disclosure may be presented as the half-life of such material in the matrix.

[0140] In addition to the embodiment involving protocols for in vitro determination of release rates, in vivo protocols, whereby in certain instances release rates for polymeric systems may be determined in vivo, are also contemplated by the present disclosure. Other assays useful for determining the release of any material from the polymers of the present system are known in the art.Targeting Moieties

[0141] The present disclosure contemplates the use of both naturally occurring and non-naturally occurring amino acids and amino acid analogs in a targeting moiety.

[0142] The targeting moiety can be selected from various modular, programmable modalities. In some embodiments, the targeting moiety is a nanobody. In some embodiments, the targetingmoiety is a cyclic peptide. In some embodiments, the targeting moiety is a miniprotein. In other embodiments, the targeting moiety is a knottin.

[0143] In some embodiments, the targeting moiety comprises an antibody, antibody fragment, or an antigen-binding domain. In some embodiments, the targeting moiety comprises an antigen binding domain. In some embodiments, the targeting moiety comprises a CDR region and at least one Unnatural amino acid (“UAA”) residue, meaning an amino acid not found naturally in a human being but being made synthetically or naturally in a non-human organism. Exemplary UAAs include, but are not limited to, hydroxyproline (Hyp), beta-alanine, citrulline (Cit), ornithine (Om), norleucine (Nle), 3 -nitrotyrosine, nitroarginine, pyroglutamic acid (Pyr). In some embodiments, the UAA residue is within or in the proximity of the CDR region. In some embodiments, the UAA residue is comprised within the CDR region. In some embodiments, the targeting moiety comprises a single domain antibody (sdAb).

[0144] In some embodiments, a targeting moiety may comprise a peptidomimetic macrocycle. Any amino acid or amino acid analog is amenable to the synthetic methods employed for the synthesis of stable bis-sulfhydryl-containing peptidomimetic macrocycles can be used in the present disclosure. For example, cysteine or tyrosine is contemplated as a useful amino acid in the present disclosure. However, sulphur-containing amino acids other than cysteine or tyrosine that contain a different amino acid side chain are also useful. For example, cysteine contains one methylene unit between the a-carbon of the amino acid and the terminal — SH of the amino acid side chain. The present disclosure also contemplates the use of amino acids with multiple methylene units between the a-carbon and the terminal — SH. Non-limiting examples include a-methyl-L-homocysteine and a-m ethyl -D-homocysteine. In some embodiments, the amino acids and amino acid analogs are of the D-configuration. In other embodiments, they are of the L-configuration. In some embodiments, some of the amino acids and amino acid analogs contained in the peptidomimetic are of the D-configuration while some of the amino acids and amino acid analogs are of the L-configuration. In some embodiments, the amino acid analogs are a,a-di substituted, such as a-methyl-L-cysteine and a-methyl-D-cysteine.

[0145] Looped peptides, such as bicyclic peptides, may be incorporated together into conjugates of the present disclosure. For example, two looped peptides of the same specificity can be linked together, increasing the avidity of the derivative for its targets. In some embodiments, a pluralityof looped peptides are combined to form a multimer. For example, two different looped peptides are combined to create a bispecific molecule. Alternatively, three or more looped peptides, which may be the same or different, can be combined to form a multispecific molecule. In one embodiment, multivalent complexes may be constructed by linking together the looped peptides, which may be the same or different.

[0146] In certain embodiments, a bicyclic peptide is used as the targeting moiety. In other embodiments, the looped bicyclic peptide bind with high affinity to a target.

[0147] In terms of protease stability, bicyclic peptides should ideally demonstrate stability to plasma proteases, epithelial (“membrane-anchored”) proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, and the like. Protease stability should be maintained between different species such that the bicyclic peptide or a conjugate comprising the bicyclic peptide can be developed in animal models as well as administered with confidence to humans.

[0148] The desirable solubility profile is a function of the proportion of charged and hydrophilic versus hydrophobic residues and intra / inter-molecular H-bonding, which is important for formulation and absorption purposes.

[0149] Optimal plasma half-life in circulation depends upon the clinical indication and treatment regimen, and may be required to develop a bicyclic peptide for short exposure in an acute illness management setting, or develop a bicyclic peptide with enhanced retention in the circulation, and is therefore optimal for the management of more chronic disease states. Other factors driving the desirable plasma half-life are requirements of sustained exposure for maximal therapeutic efficiency versus the accompanying toxicology due to sustained exposure of the agent.

[0150] In some embodiments, the therapeutic moiety is an affibody. The affibody molecule constitutes a highly suitable carrier for directing molecules of interest (e.g., toxins, radioisotopes, therapeutic peptides) to, e.g., tumor cells due to specific target binding and lack of irrelevant interactions, such as the Fc receptor binding displayed by some antibodies.

[0151] Common advantages of affibody molecules over antibodies are better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs.

[0152] Affibodies are exemplified by, but not limited to, Anti-ErbB2 AFFIBODY® (also referred to as anti-HER2 AFFIBODY®), Anti-EGFR AFFIBODY®, Anti-TNFalpha AFFIBODY®, Anti-fibrinogen AFFIBODY®, Anti -transferrin AFFIBODY®, Anti-HSA AFFIBODY®, Anti-Insulin AFFIBODY®, Anti-IgG AFFIBODY®, Anti- IgM AFFIBODY®, Anti-IgA AFFIBODY®, and Anti-IgE AFFIBODY® (e.g., from Abeam, Cambridge, Mass.).

[0153] As disclosed in US9708374, affibodies tolerate modification and are independently folding when incorporated into fusion proteins. Head-to-tail fusions of Affibody molecules of the same specificity have proven to give avidity effects in target binding, and head-to-tail fusion of Affibody molecules of different specificities makes it possible to get bi-specific or multispecific affinity proteins. Fusions with other proteins can also be created (Ronnmark et al. (2002) “Construction and characterization of affibody-Fc chimeras produced in Escherichia coli" J. Immunol. Methods 261: 199-211. PMID 11861078; Ronnmark et al. (2003) “Affibody-beta-galactosidase immunoconjugates produced as soluble fusion proteins in the Escherichia coli cytosol,” J. Immunol. Methods 281: 149-160. PMID 14580889). A site for site-specific conjugation is facilitated by the introduction of a single cysteine or tyrosine at a desired position.

[0154] A number of different Affibody molecules have been produced by chemical synthesis. Since they do not contain cysteines or disulfide bridges, they fold spontaneously and reversibly into the correct three-dimensional structures when the protection groups are removed after synthesis (Nord et al. (2001) “Recombinant human factor Vlll-specific affinity ligands selected from phage-displayed combinatorial libraries of protein A,” Eur. J. Biochem. 268: 1-10. PMID 11488921; Engfeldt et al. (2005) “Chemical synthesis of triple-labeled three-helix bundle binding proteins for specific fluorescent detection of unlabeled protein,” Chem. BioChem. 6: 1043-1050. PMID 15880677).

[0155] In another aspect, a disclosed conjugate may comprise a bifunctional peptide, e.g., a stapled or stitched bifunctional peptide. In some embodiments, the therapeutic moiety comprises a bifunctional peptide, e.g., a stapled or stitched peptide. In some embodiments, a bifunctional peptide comprises 5-50 amino acids. As disclosed in US9458189, a bifunctional peptide typically comprises two peptide domains, a targeting domain that binds to a target, and an effector domain that recruits a protein or protein complex to the target and / or actson or modifies the activity of the target. One or both of the targeting domain and effector domain of the bifunctional peptide are stapled or stitched to stabilize the conformation of the peptide.. Typically, the two domains are covalently associated with one another through a linker; however, non-covalent associations may also be used. The linker may range in structure from simply a covalent bond to a bifunctional molecule to a polymeric linker. In essence, the bifunctional peptide works to bring two proteins or other biomolecules into close proximity to one another. Therefore, ligating a bifunctional stapled peptide to a protein allows for bringing three or more proteins into close proximity. Multimeric protein complexes may be formed using such bifunctional peptides multiple peptide domains.

[0156] In some embodiments, the therapeutic moiety comprises a stapled or stitched peptide. In some embodiments, the stapled or stitched peptide comprises 5-50 amino acids. In some embodiments, a stapled or stitched peptide may be formed by a peptide that comprises one or more unnatural amino acids with alkenyl side chains to form a staple or stitch. Given the stability of stapled / stitched peptides, they may be used as agents for recruiting proteins or other biomolecules to a particular protein, nucleic acid, other biomolecule, cell, or organelle (i.e., tethering two cellular components together or bringing them into close proximity).

[0157] In some embodiments, a stapled or stitched peptide comprises a helix. In some embodiments, a stapled or stitched peptide comprises an a-helix. Crosslinks can be incorporated across one (i, i+3 or i, i+4) and / or two turns (i, i+7) of a helix (e g., a-helix). In certain embodiments, the peptide only includes crosslinks across one turn of the helix. In certain embodiments, the peptide includes crosslinks across one and two turns of the helix. In certain embodiments, a staple is incorporated across one turn of a helix using (R)-2-amino-2-methylhept-6-enoic acid in both the i and i+4 positions. In other embodiments, a staple is incorporated across one turn of a helix using (S)-2-amino-2-methylhept-6-enoic acid in both the i and i+4 positions. In certain embodiments, a staple is incorporated across two turns of a helix using (R)-2-amino-2-methylhept-6-enoic acid in the i position and (S)-2-amino-2-methyldec-9-enoic acid in the i+7 position. In other embodiments, a staple is incorporated across two turns of a helix using (S)-2-amino-2-methylhept-6-enoic acid in the i position and (R)-2-amino-2-methyldec-9-enoic acid in the i+7 position. In certain other embodiments, a staple is incorporated across two turns of a helix using (R)-2-amino-2-methyldec-9-enoic acid in the i position and (S)-2-amino-2-methylhept-6-enoic acid in the i+7 position. In certain other embodiments, a staple isincorporated across two turns of a helix using (S)-2-amino-2-methyldec-9-enoic acid in the i position and (R)-2-amino-2-methylhept-6-enoic acid in the i+7 position.

[0158] In certain embodiments, the a,a-dialkenyl residue used in a stitched peptide is 2-amino-2-(pent-4-enyl)hept-6-enoic acid, also known as bispentenyl glycine. Bispentylglycine can be stitched with a-alkenyl residues of either R or S stereochemistry across either one or two turns of a helix. In certain embodiments, bispentenylglycine in the i position is stitched with 2-amino-2-methylhept-6-enoic acid in the i-4 position. In certain embodiments, bispentenyl glycine is stitched with 2-amino-2-methylhept-6-enoic acid in the i+4 position. In certain embodiments, bispentenyl glycine is stitched with 2-amino-2-methyldec-9-enoic acid in the i-7 position. In certain embodiments, bispentenyl glycine is stitched with 2-amino-2-methyldec-9-enoic acid in the i+7 position. In certain embodiments, two or more bispentyl glycine residues are incorporated to give three or more stitches.

[0159] It is important to note that when designing crosslinking residues into a helical portion of a peptide, the crosslinkers do not interfere with any important interactions that the peptide may make with other biomolecules (e.g., proteins, nucleic acids, lipids, sugars, etc.). The design requires a strategy based on any structural information available (e.g., crystal structure, mutagenesis studies, or homology models) to place the crosslinkers on the appropriate portion of the helix, out of the way of crucial interactions.

[0160] In some embodiments, the targeting moiety is a cell binding molecule. Cellbinding molecules include, but are not limited to, large molecular weight proteins, such as whole antibodies (polyclonal, monoclonal, dimeric, multimeric, multispecific (e.g., bispecific antibodies)), single chain antibodies, antibody fragments such as Fab, fab ', F (ab1) 2 Fv (Parham, J. Immunol.1983, 131:2895-2902), fragments produced from Fab expression libraries, antiidiotype antibodies (anti-Id), CDR's, and epitope-binding fragments of any of the above antibodies, which antibodies are immunospecific antigens bound to cancer cells, viral antigens, microbial antigens or proteins produced by the immune system that recognize, bind to specific antigens or produce desired biological activity (Miller et al, j. Immunol.2003, 170:4854-4861), interferons (e.g., types I, II, III); a polypeptide; lymphokines such as IL-2, IL-3, IL-4, IL-6, GM-CSF, interferon gamma (IFN-gamma); hormones such as insulin, TRH (thyroid stimulating hormone releasing hormone), MSH (melanocyte stimulating hormone), steroid hormones such asandrogens and estrogens; growth factors and colony stimulating factors such as Epidermal Growth Factor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF), transforming Growth Factors (TGF), such as TGF alpha, TGF beta, insulin and insulin-like growth factors (IGF-I and IGF-II), G-CSF, M-CSF and GM CSF (Burgess, immunol.Today, 1984, 5:155-158); vaccinia Virus Growth Factor (VGF); fibroblast Growth Factor (FGF); small molecular weight proteins, peptides and peptide hormones, such as bombesin, gastrin releasing peptide; platelet-derived growth factor; interleukins and cytokines such as interleukin-2 (IL-2), interleukin-6 (IL-6), leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor (GM-CSF); vitamins such as folic acid; apolipoproteins and glycoproteins, such as transferrin (O' keefa et al, j. biol. Chem.1985, 932-937); a sugar-binding protein or lipoprotein, such as lectin; a transport molecule for cell nutrition; and small molecule inhibitors such as Prostate Specific Membrane Antigen (PSMA) inhibitors and small molecule Tyrosine Kinase Inhibitors (TKI), non-peptide or any other cell -binding molecules or substances such as biopolymers (Dhar, et al, proc. Natl. Acad. Sci.2008, 105:17356-61); dendrimers (Lee, et al, nat. Biotechnol.2005,23:1517-26; Almutairi et al, proc. Natl. Acad. Sci.2009, 106:685-90); nanoparticles (Liong et al, ACS Nano, 2008, 19: 1309-12; Medarova et al, nat. Med.2007, 13:372-7; javier et al, bioconj. Chem.2008, 19:1309-12); liposomes (Medinai et al, curr. Phar. Res.2004, 10:2981-9); viral capsids (Flenniken et al, viruses nanotechnol 2009, 327:71-93). In general, monoclonal antibodies are preferred cell surface binding agents, if appropriate. Antibodies may be of murine, human, humanized, chimeric, or other origin species.

[0161] In some embodiments, the targeting moiety and / or therapeutic moiety of compounds and / or conjugates described herein comprise an amino acid sequence have 95%, 96%, 97%, 98%, or 99% sequence identity to any of the particular sequences or disclosed herein. In some embodiments, the targeting moiety and / or therapeutic moiety of compounds and / or conjugates described herein comprise an amino acid sequence having 1, 2, 3, 4, or 5 substitutions, insertions, or deletions relative thereto.Target

[0162] In some embodiments, the targeting moiety is capable of interacting with a target. In some embodiments, the targeting moiety binds selectively to a biological target. For example, the targeting moiety may bind with an affinity greater than about KD=1000 nM, suitably greater thanabout Kd=l OOnM, for example greater than about Kd=l OnM as determined by the method described herein. Typically, the target will be analogous to an epitope. One skilled in the art will appreciate that the choice of target molecule is large and varied. They may be for instance human or animal proteins, cytokines, cytokine receptors, enzymes co-factors for enzymes or DNA binding proteins. Suitable cytokines and growth factors include but are not limited to: ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor, ENA78, Eotaxin, Eotaxin-2, Exodus-2, FGF-acidic, FGF-basic, fibroblast growth factor-10 (30). FLT3 derivative, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-1, insulin, IFNy, IGF-I, IGF-II, IL-la, IL-1 (3, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-17a, IL-17c, IL-17d, IL-17e, IL-17f, IL-18 (IGIF), IL-21, IL-22, IL-23, IL-31, IL-32, IL-33, IL-34, Inhibin a, Inhibin P, IP- 10, keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian inhibitory substance, monocyte colony inhibitory factor, monocyte attractant protein (30 ibid), M-CSF, MDC (67 a. a.), MDC (69 a. a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a. a ), MDC (69 a. a ), MIG, MIP-la, MIP-ip, MIP-3a, MIP3 (3, MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1), NAP -2, Neurturin, Nerve growth factor, P-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDFla, SDFlp, SCF, SCGF, stem cell factor (SCF), TARC, TGF-a, TGF-, TGF-2, TGF-3, tumour necrosis factor (TNF), TNF-a, TNF-P, TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO / MGSA, GRO-a, GRO-a, HCC1, 1-309, HER 1, HER 2, HER 3 and HER 4. Cytokine receptors include receptors for the foregoing cytokines. Chemokine targets include CC chemokine derivatives CCL21 / 6Ckine, CCL12 / MCP-5, CCL6 / C10, CCL22 / MDC, CCL14 / HCC-1 / HCC-3, CCL3L1 / MIP-1 alpha Isoform LD78 beta, CCL23 / Ckbeta 8-1, CCL3 / MIP-1 alpha, CCL28, CCL4L1 / LAG-1, CCL27 / CTACK, CCL4 / MIP-1 beta, CCL24 / Eotaxin-2 / MPIF-2, CCL15 / MIP-1 delta, CCL26-like / Eotaxin-3-like, CCL9 / 10 / MIP-1 gamma, CCL26 / Eotaxin-3, CCL19 / MIP-3 beta, CCL11 / Eotaxin, CCL20 / MIP-3 alpha, CCL14a / HCC-l, CCL23 / MPIF-1, CCL14b / HCC-3, CCL18 / PARC, CCL16 / HCC-4, CCL5 / RANTES, CCL1 / I-309 / TCA-3, TAFA1 / FAM19A1, MCK-2, TAFA5 / FAM19A5, CCL2 / JE / MCP-1, TAFA3 / FAM19A3, CCL8 / MCP-2, TAFA4 / FAM19A4, CCL7 / MCP-3 / MARC, CCL17 / TARC, CCL13 / MCP-4 and CCL25 / TECK; chemokine receptors include CCR1, CCR7, CCR2, CCR8, CCR3, CCR9, CCR4, CCR10, CCR5, CCRL2 / LCCR / CRAM-A / B and CCR6; CXC chemokine derivatives includeCXCL13 / BLC / BCA-1, CXCL10 / IP-10 / CRG-2, CXCL14 / BRAK, LIX, CXCL16, CXCL 15 / Lungkine, CXCL5 / ENA-78, CXCL9 / MIG, CXCL6 / GCP-2, CXCL7 / NAP-2, CXCL1 / 2 / 3 / GRO, CXCL4 / PF4, CXCL1 / GRO alpha / KC / CINC-1, CXCL12 / SDF-1 alpha, CXCL2 / GRO beta / MIP-2 / CINC-3, CXCL12 / SDF-1 beta, CXCL3 / GRO gamma / CINC-2 / DCIP-1, CXCL12 / SDF-1, CXCL11 / I-TAC, CXCL7 / Thymus Chemokine-1 and CXCL8 / IL-8; CXC chemokine receptors include CXCR3, CXCR7 / RDC-1, CXCR4, CXCR1 / IL-8 RA, CXCR5, CXCR2 / IL-8 RB and CXCR6; TNF Superfamily derivatives include 4-1BB Derivative / TNFSF9, LIGHT / TNFSF14, APRIL / TNFSF13, Lymphotoxin, BAFF / BLyS / TNFSF13B, Lymphotoxin beta / TNFSF3, CD27 Derivative / TNFSF7, 0X40 Derivative / TNFSF4, CD30 Derivative / TNFSF8, TL1A / TNFSF15, CD40 Derivative / TNFSF5, TNF-alpha / TNFSFlA, EDA (pan), TNF-beta / TNFSFIB, EDA-Al / Ectodysplasin Al, TRAIL / TNFSF10, EDA-A2, TRANCE / TNFSF11, Fas Derivative / TNFSF6, TWEAK / TNFSF12 and GITR Derivative / TNFSF18; TNF Superfamily receptors include 4-1BB / TNFRSF9 / CD137, NGF R / TNFRSF16, BAFF R / TNFRSF13C, Osteoprotegerin / TNFRSFl IB, BCMA / TNFRSF17, OX40 / TNFRSF4, CD27 / TNFRSF7, RANK / TNFRSF11A, CD30 / TNFRSF8, RELT / TNFRSF19L, CD40 / TNFRSF5, TACVTNFRSF13B, DcR3 / TNFRSF6B, TNFRH3 / TNFRSF26, DcTRAIL R1 / TNFRSF23, TNF R1 / TNFRSF1 A, DcTRAIL R2 / TNFRSF22, TNF RIETNFRSF1B, DR3 / TNFRSF25, TRAIL R1 / TNFRSF10A, DR6 / TNFRSF21, TRAIL R2 / TNFRSF1 OB, EDAR, TRAIL R3 / TNFRSF10C, Fas / TNFRSF6 / CD95, TRAIL R4 / TNFRSF10D, GITR / TNFRSF18, TROY / TNFRSF19, HVEM / TNFRSF14, TWEAK R / TNFRSF12, Lymphotoxin beta R / TNFRSF3 and XEDAR; Toll-Like Receptors including TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8 and TLR-9; enzymes, including Cathepsin A, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin F, MMP 1, MMP2, MMP 3, MMP 7, MMP 8, MMP 9, MMP 10, MMP 11, MMP 12, MMP 13, MMP 14, MMP 15, MMP 16, MMP 17, MMP 19, MMP 20, MMP 21, MMP 23 A, MMP 23B, MMP 26, MMP 27, MMP 28, urokinase, kallikreins, including KLK1, KLK2, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLK10, KLK11, KLK12, KLK13, KLK14 and KLK15; components of the complement system; intracellular signaling molecules and transcription factors; p53; and MDM2. Targets may also be large plasma proteins, such as serum albumins, as set forth herein. It will be appreciated that this list is by no means exhaustive.

[0163] In some embodiments, the targeting moiety is capable of interacting with a target. In some embodiments, the targeting moiety, T is selected from PTH(l-34), PTHrP(l-36), abaloparatide, M-PTH(1-14), and LA-PTH.

[0164] In some embodiments, the target is overexpressed on tumor cells. In some embodiments, the target comprises surface markers or receptors. In some embodiments, the target is selected from one or more of PSMA, PTHR, PTH1R, PTH2R, PTH3R, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-1, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD 166, CLL-1, ADAM9, cKIT, CD36, CD73, ITGaVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, ROR1, TROP-2, CX3CR1, CDH17, NECTIN4, cMET, CD 19, CD20, CD22, CD30, CD33, CD 123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, tau, TREM2, TSLP, mesothelin, PCSK9, ANGPTL3, Lp(a), FGFRlc / p-Klotho, C5, Factor D, MASP-2, properdin, VEGF-A, Ang-2, ROCK, IL-2R, CTLA-4, CD28, CD40L, IL-4Ra, IL-5Ra, IL-10R, IgE, CRTH2, GLP-1, LAG-3, TIGIT, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, pCadherin, CEAC AM6, CD47, and EpCAM. In some embodiments, the target is selected from two or more of PSMA, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-1, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD 16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD166, CLL-1, ADAM9, cKIT, CD36, CD73, ITGaVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, IL-6R, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, ROR1, TROP-2, CX3CR1, CDH17, NECTIN4, cMET, CD19, CD20, CD22, CD30, CD33, CD123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, tau, TREM2, TSLP, mesothelin, PCSK9, ANGPTL3, Lp(a), FGFRlc / p-Klotho, C5, Factor D, MASP-2, properdin, VEGF-A, Ang-2, ROCK, IL-2R, CTLA-4, CD28, CD40L, IL-4Rot, IL-5Ra, IL-1 OR, IgE, CRTH2, GLP-1, LAG-3, TIGIT, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, pCadherin, CEACAM6, CD47, and EpCAM. In some embodiments, a conjugate describedherein brings a payload (e.g., the therapeutic moiety) to the tumor cell, when the conjugate is bound (e.g., via the targeting moiety) to the one or more targets on the tumor cell and the payload kills, inhibits or slows the growth of the tumor cell.

[0165] In some embodiments, the target is a membrane protein. In some embodiments, the target is a cell (e.g., beta cell) surface protein. In some embodiments, the target is a cell (e.g., beta cell) surface receptor. In some embodiments, the target is selected from the group consisting of PSMA, PTHR, PTH1R, PTH2R, PTH3R, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-1, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD 166, CLL-1, ADAM9, cKIT, CD36, CD73, ITGaVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, IL-6R, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, ROR1, TROP-2, CX3CR1, CDH17, NECTIN4, cMET, CD19, CD20, CD22, CD30, CD33, CD123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, tau, TREM2, TSLP, mesothelin, PCSK9, ANGPTL3, Lp(a), FGFRlc / p-Klotho, C5, Factor D, MASP-2, properdin, VEGF-A, Ang-2, ROCK, IL-2R, CTLA-4, CD28, CD40L, IL-4Ra, IL-5Ra, IL-10R, IgE, CRTH2, GLP-1, LAG-3, TIGIT, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, pCadherin, CEACAM6, CD47, DPP6, ZNT8, GPR44, PTPRN, GLP1R, and EpCAM. In some embodiments, the target is DPP6. Accordingly, the targeting moiety is a binder to DPP6. In some embodiments, the target is ZNT8. Accordingly, the targeting moiety is a binder to ZNT8 (e.g., as described in Kasinathan D, et al., (2024). Diabetes, 73 (5): 806-818). In some embodiments, the target is GPR44. Accordingly, the targeting moiety is a binder to GPR44 (e.g., CRTH2 / GPR44 (extracellular) blocking peptide (#BLP-PR062), Alomone Labs). In some embodiments, the target is PTPRN. Accordingly, the targeting moiety is a binder to PTPRN. In some embodiments, the target is GLP1R. Accordingly, the targeting moiety is a binder to GLP1R.

[0166] In some embodiments, the target is a serum protein. In some embodiments, the target is an extracellular membrane.

[0167] In some embodiments, T is or comprisesReactive Moieties (Also Referred to as “Reactides”)

[0168] As discussed in Grob et al., peptide drugs may particularly benefit from covalent binding to overcome their pharmacokinetic limitations. The crystal structure of a peptide-protein complex is used to determine chemically suitable and sterically exposed nucleophiles in the proximity of the binding site. Reactive moieties are subsequently incorporated into a peptide ligand at a spatially favorable position to enable covalent binding to the target protein. Many of the covalent, peptide-based inhibitors that emerged from structure-based design target Cys and Lys residues exposed in the binding pocket, whereas other nucleophilic residues have not been deeply explored (Grob NM, et al., (2024). ACS Chem Biol, 19:101-109).

[0169] As disclosed in Grob et al., sulfonyl fluorides are versatile, broadly reactive moieties well-suited to electrophile scanning. Sulfonyl fluorides have been reported to react with side chains of various amino acids including Cys, Lys, Tyr, His, Ser, and Thr by Sulfur(VI) Fluoride Exchange (SuFEx) and have found widespread use in bioconjugation and medicinal chemistry. Palladium oxidative addition complexes (Pd-OACs) are useful reagents for the functionalization of peptides by palladium-mediated Cys arylation, and have been employed for the introduction of electrophilic handles such as carbamates. Pd-OACs have advantages such as the rapid and selective functionalization of the low proteome abundance amino acid, Cys, resulting in theformation of a stable S-aryl bond. For example, a peptide sequence with single Cys substitutions can be prepared using automated fast-flow peptide synthesis to deprotect the reactive site and cleave the bonds from the solid support; the peptide is then subject to palladium-mediated cysteine arylation using a Pd-OA complex, yielding the complex’ covalently attachment to Cys (Grob NM, et al., (2024). ACS Chem Biol, 19:101-109). The introduction of a reactive moiety on peptides obtained by solid-phase synthesis permits the exploration of various electrophilic groups from a single peptide precursor. Electrophiles with broad reactivity such as sulfonyl fluoride would enable the evaluation of cross-linking reactivity with a wide range of potential nucleophilic residues.

[0170] In various embodiments, the reactive moiety comprises a warhead. Tn some embodiments, the warhead is an electrophile. In some embodiments, the electrophile is cationic. In some embodiments, the electrophile comprises an atom that carries a partial positive charge or does not have an octet of electrons. In some embodiments, the electrophile has an electrophilicity index of about 0.8 eV or higher, such as about 1.5 eV or higher. The electrophilicity index (co) is calculated as follows:co = p2 / q,wherein p represents the electronic chemical potential, and q is the chemical hardness of the molecule (Parr RG, et al., (1999). J Am Chem Soc, 121(9): 1922-1924). p and q may be approached in terms of the one-electron energies of the frontier molecular orbital HOMO andand q ~ EL- EH, respectively (Perez P, et al., (2007). Comput TheorChem, 19:139-201.

[0171] In some embodiments, a compound of the present disclosure of Formula (II- A) is represented by a compound of Formula (II- A- 1):whereinLA1and LA2are each independently a bond or chain of 1-30 carbon atoms in length, wherein one or more carbon atoms are optionally substituted with halogen, hydroxyl, cyano, oxo, CFH2, CF2H, CF3, alkoxy, C1-C5 alkyl, or -C(=O)-NH2; andwherein one or more carbon atoms of the backbone are optionally replaced by a divalent group independently selected from oxygen, alkylamino, carbonyl, 3-14 membered cycloalkylene, 3-8 membered monocyclic heterocyclic group, 5-14 membered bridged heterocyclic group, 5-14 membered spiro heterocyclic group, 5-14 membered heteroarylene, and 7-14 membered heteroaryl-fused-heterocyclylene, and wherein each of the 3-14 membered cycloalkylene, 3-8 membered monocyclic heterocyclic group, 5-14 membered bridged heterocyclic group, 5-14 membered spiro heterocyclic group, 5-14 membered heteroarylene, and 7-14 membered heteroaryl-fused-heterocyclylene is independently substituted with 0, 1, 2, 3, 4, 5, or 6 Ral;each Ralis independently selected from alkylamino, halogen, hydroxyl, C1-C5 alkoxy, C1-C5 alkyl, C1-C5 alkylcarboxyl, C1-4 alkenyl, C3-5 cycloalkyl, C1-5 haloalkyl, glycosyl, amino acid, and an oxo group;Ring A is a bond or optionally substituted bivalent 3- to 14-membered ring system; optionally substituted bivalent 3- to 10-membered monocyclic, bicyclic, bridged, or spirocyclic carbocyclic ring system; optionally substituted bivalent 3- to 10-membered monocyclic, bicyclic, bridged, or spirocyclic heterocyclic ring system; optionally substituted bivalent 5- to 10-membered heteroarylene; and optionally substituted bivalent Ce-14 arylene, wherein each of the 3- to 14-membered ring system; 3- to 10-membered monocyclic, bicyclic, bridged, or spirocyclic carbocyclic ring system; 3- to 10-membered monocyclic, bicyclic, bridged, or spirocyclic heterocyclic ring system; 5- to 10-membered heteroarylene; and Ce-14 arylene is optionally independently substituted with 0, 1, 2, 3, 4, 5, or 6 RA; andeach RAis independently selected from alkylamino, halogen, hydroxyl, C1-C5 alkoxy, C1-C5 alkyl, C 1-4 alkenyl, C3-5 cycloalkyl, C1-5 haloalkyl, glycosyl, amino acid, and an oxo group.

[0172] In some embodiments, LA1and LA2are each independently:wherein each nm is independently an integer selected from 0 to 15.

[0173] In some embodiments, Formula (II-A-1) is represented by:wherein nm is an integer selected from 0 to 15.

[0174] In some embodiments, Rwis represented by:wherein:X10is a bond or selected from -N(R10)-, -O-, -CH2-, -(CH2)2-, -(CH2)3- and optionally substituted 5- to 10-membered heteroaryl;X20is selected from -C(O)-, -S(O)2-, -S(O)(NR20)-, and optionally substituted 5- to 10- membered heteroaryl;X30is selected from -F, -Cl, and optionally substituted 5- to 10-membered heteroaryl; X40is selected from C, -CH=, -CF=, -CCH3=, -CCF3=, -C(CN)=, and -C(N(R40)2)=; X50is selected from =CH2, =CHCH3, =CHF, =CF2, =CHCF3;X60is selected -N(R10)2-, or -OCi-6alkyl;each of R10, R20, and R40are independently selected from hydrogen, C1-3 alkyl, optionally substituted C1-3 haloalkyl, optionally substituted C1-3 alkoxyl.

[0175] In some embodiments, Rwis represented by:

[0176] In some embodiments, Rwis represented by:

[0177] In some embodiments, Rwis represented by:

[0178] In some embodiments, Rwis represented by:wherein:X20is selected from C(O)-, -S(O)2-, -S(O)(NR20)-;X30is selected from -CH=, -CCH3=, -CF=, -CCF3=, -C(CN)=, and -C(N(R30)2)=; X40is selected from -CH=, -CCH3=, -CF=, -CCF3=, -C(CN)=, and -C(N(R30)2)=; X50is selected from -CH2-, -CHCH3-, -CHF-, -CF2-, and -CHCF3-;each R20is independently selected from hydrogen, optionally substituted Ci-3alkyl, optionally substituted Ci-3haloalkyl, optionally substituted Ci-3alkoxyl;each R30is independently selected from hydrogen, optionally substituted C1-3 alkyl, optionally substituted C1-3 haloalkyl, optionally substituted C1-3 alkoxyl; andp is 1 or 2.

[0179] In some embodiments, Rwis

[0180] In some embodiments, R is

[0181] In some embodiments, Rwis selected from:

[0182] In some embodiments, Rwis selected from:

[0183] In some embodiments, the electrophile may comprise one of the following structures:

[0184] In some embodiments, RwisIn some embodiments, Rwis,embodiments,some embodiments,In some embodiments,. In some embodiments, Rwis., jn someembodiments, Rw, . In some embodiments, Rwis, . In some embodiments, Rwisembodiments,In some embodiments, Rwis, some

[0185] In some embodiments, the reactive moiety is linked to the targeting moiety at a Cys or Lys residue present in the targeting moiety. In some embodiments, the reactive moiety is linked to an Asp or Glu residue, or a UAA. In some embodiments, the reactive moiety is linked to the N-terminus or C-terminus of a polypeptide sequence, or is linked to a post-translational modification (PTM) on the polypeptide.

[0186] In some embodiments, R” is represented by Formula (II-B):wherein:LAand LBare each independently a bond or chain of 1-60 carbon atoms in length, wherein one or more carbon atoms are optionally substituted with halogen, hydroxyl, cyano, CFH2, CF2H, CF3, alkoxy, C1-C5 alkyl, or -C(=O)-NH2; andwherein one or more carbon atoms of the backbone are optionally replaced by a divalent group independently selected from oxygen, alkylamino, carbonyl, 3-14 membered cycloalkylene, 3-8 membered monocyclic heterocyclic group, 5-14 membered bridged heterocyclic group, 5-14 membered spiro heterocyclic group, 5-14 membered heteroarylene, and 7-14 membered heteroaryl-fused-heterocyclylene, and wherein each of the 3-14 membered cycloalkylene, 3-8 membered monocyclic heterocyclic group, 5-14 membered bridged heterocyclic group, 5-14 membered spiro heterocyclic group, 5-14 membered heteroarylene, and 7-14 membered heteroaryl-fused-heterocyclylene is independently substituted with 0, 1, 2, 3, 4, 5, or 6 Ra;each Rais independently selected from alkylamino, halogen, hydroxyl, C1-C5 alkoxy, C1-C5 alkyl, C1-C5 alkylcarboxyl, C1-4 alkenyl, C3-5 cycloalkyl, C1-5haloalkyl, and an oxo group; andRwis an electrophilic functional group capable of forming a covalent bond with the target.

[0187] In some embodiments, the compound of Formula (II-B) comprises a Michael acceptor capable of Elcb elimination (retro-Michael) of -Tx or -L-Tx.

[0188] In some embodiments, Formula (II-B) is or comprises:wherein* represents the point of attachment to T; andnq is an integer from 0 to 15.

[0189] In some embodiments, Formula (someOembodiments, Formula (II-B) is. In some embodiments, Formula (II-B) isasome embodiments,embodiments, Formula (II-B) isIn some embodiments,Therapeutic Moieties

[0190] The therapeutic moiety can be selected from various modular, programmable modalities. In some embodiments, the therapeutic moiety is an enzyme. In some embodiments, the therapeutic moiety is an antibody. In some embodiments, the therapeutic moiety is a peptide. In some embodiments, the therapeutic moiety is a toxin. In some embodiments, the therapeutic moiety is a cytokine. In other embodiments, the therapeutic moiety is a nucleic acid. In some embodiments, the therapeutic moiety is a corticosteroid (e.g., fluticasone, dexamethasone, or an analog thereof).

[0191] In some embodiments, therapeutic moieties of the present disclosure include those having utility for anti cancer activity. In some embodiments, a therapeutic moiety has a cytotoxic effect or a cytostatic effect. In certain embodiments, e g., in any of the uses and methods of treating a disease or cancer provided herein, a therapeutic moiety may be an immune checkpoint inhibitor, which blocks the activity of an immune checkpoint protein. Non-limiting examples of immune checkpoint proteins include CTLA4, PD-1, LAG3, B7H3, B7H4, TIM3, CD25, CD137, 4- IBB, IDO, PD-1, and PD-L1. The immune checkpoint inhibitor can be an antibody, a fusion protein, an aptamer, a small molecule inhibitor, or an immune checkpoint protein-binding fragment thereof or other inhibitors that mitigate the immunosuppressive effect / environment of cancers, such as IDO inhibitors (IDOI). In any of the provided methods, theimmune checkpoint inhibitor can be an anti-immune checkpoint protein antibody or antigenbinding fragment thereof or a small molecule inhibitor. Such inhibitors are known to those of skill in the art.

[0192] In some embodiments, the therapeutic moiety is an enzyme is selected from one or more of iduronate-2-sulfatase, L-iduronidase, aspartylglucosaminidase, acid lipase, cysteine transporter, Lamp-2, a-galactosidase A, acid ceramidase, a-L-fucosidase, P-hexosaminidase A, GM2 -ganglioside activator (GM2A), a-D-mannosidase, -D-mannosidase, arylsulfatase A, saposin B, neuraminidase, a-N-acetylglucosaminidase phosphotransferase, phosphotransferase y-subunit, heparan-N-sulfatase, a-N-acetylglucosaminidase, acetylCoA: N-acetyltransferase, N-acetylglucosamine 6-sulfatase, galactose 6-sulfatase, P-galactosidase, N-acetylgalactosamine 4-sulfatase, hyaluronoglucosaminidase, sulfatases, palmitoyl protein thioesterase, tripeptidyl peptidase I, acid sphingomyelinase, cathepsin A, cathepsin K, a-galactosidase B, NPC1, NPC2, sialin, and sialic acid transporter, including active fragments and variants thereof.

[0193] In some embodiments, the therapeutic moiety is a small molecule inhibitor such as ibrutinib, dacomitinib, boceprevir, narlaprevir, nirmatrelvir, gefitinib, afatinib, osimertinib, clopidogrel, rivastigmine, zanubrutinib, acalabrutinib, SML-8-83-1, SML-10-70-1, sotorasib, adagrasib, GDC-6036, JNJ-74699157, PROTAC, remdesivir, and nirmatrelvir (Schaefer D, et al., (2023). Pharmaceuticals (Basel), 16(5):663).

[0194] In some embodiments, the therapeutic moiety is a phosphatase. General examples of phosphatases include acid phosphatases and alkaline phosphatases. Examples of acid phosphatases include prostatic acid phosphatase, lysosomal acid phosphatase, erythrocytic acid phosphatase, macrophage acid phosphatase, osteoclastic acid phosphates, and potato acid phosphatase. Examples of commonly employed alkaline phosphatases include shrimp alkaline phosphatase, calf-intestinal alkaline phosphatase, placental alkaline phosphatase, and secreted alkaline phosphatase (i.e., a C-terminal truncation of placental alkaline phosphatase).

[0195] In some embodiments, the therapeutic moiety is a lysosomal enzyme, wherein the lysosomal enzyme is selected from the group consisting of: acid-al,4-glucosidase; P-galactosidase; -hexosaminidase A; P-hexosaminidase B; a-galactosidase A; glucocerebrosidase; arylsulfatase A; galactosylceramidase; acid sphingomyelinase; acid ceramidase; acid lipase; a-L-iduronidase; iduronate sulfatase; heparan N-sulfatase; a-N-acetylglucosaminidase; acetyl-CoA-glucosaminide acetyltransferase; N-acetylglucosamine-6-sulfatase; galactosamine-6-sulfatase; P-galactosidase; aryl sulfatase B; 0-glueuronidase; a-mannosidase; P-mannosidase; a-L-fucosidase; N-aspartyl-P-glucosaminidase; a-neuraminidase; lysosomal protective protein; a-N-acetyl-galactosaminidase; N-acetylglucosamine-l-phosphotransferase; and palmitoyl-protein thioesterase.

[0196] In some embodiments, the therapeutic moiety is released from any one of Formulae (I), (II), (F), (IF), (III’), (IV’), (V’), (VI’), or (VI ) by the action of any one of Cathepsin A, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin F, acid-al,4-glucosidase; P-galactosidase; P-hexosaminidase A; P-hexosaminidase B; a-galactosidase A; glucocerebrosidase; arylsulfatase A; galactosylceramidase; acid sphingomyelinase; acid ceramidase; acid lipase; a-L-iduronidase; iduronate sulfatase; heparan N-sulfatase; a-N-acetylglucosaminidase; acetyl-CoA-glucosaminide acetyltransferase; N-acetylglucosamine-6-sulfatase; galactosamine-6-sulfatase; P-galactosidase; arylsulfatase B; P-glucuronidase; a-mannosidase; P-mannosidase; a-L-fucosidase; N-aspartyl-P-glucosaminidase; a-neuraminidase; lysosomal protective protein; a-N-acetyl-galactosaminidase; N-acetylglucosamine-l-phosphotransferase; and palmitoyl-protein thioesterase.

[0197] In some embodiments, the therapeutic moiety is a radionuclide metal or halogen selected from the group comprising:111In,99mTc,94mTc,67Ga66Ga,68Ga52Fe,69Er,72As,97Ru,203Pb,62Cu,64Cu,67Cu186Re,188Re,86Y,90Y,51Cr,52mMn,157Gd,177Lu,161Tb,169Y,175Yb,105Rh,166Dy,166Ho,153Sm,149Pm,151Pm,172Tm,121Sn,177mSn,213Bi,142Pr,143Pr,198Au,199Au,18F,123I,124I,131I,75Br,76Br,77Br, and82Br.

[0198] In some embodiments, the therapeutic moiety is an antibody (e.g., monoclonal antibody).

[0199] Antibodies used for the therapeutic moiety may be selected from various antibodies that bind specific targets and are well-known in the art. These include, but are not limited to anti-TNF antibody (U. S. Pat. No. 6,258,562), anti-IL-12 and / or anti-IL-12p40 antibody (U. S. Pat. No.6,914,128); anti-IL-18 antibody (U. S. Patent Publication No, 2005 / 0147610), anti-C5, anti-CBL, anti-CD147, anti-gpl20, anti-VLA-4, anti-CDl la, anti-CD18, anti-VEGF, anti-CD40L, anti-CD-40 (e.g., see PCT Publication No. WO 2007 / 124299) anti-Id, anti-lCAM-1, anti-CXCL13, anti-CD2, anti-EGFR, anti-TGF-beta 2, anti-HGF, anti-cMet, anti DLL-4, anti-NPRl, anti-PLGF, anti-ErbB3, anti -E- sei ectin, anti-Fact VII, anti-Her2 / neu, anti-F gp, anti-CDl 1 / 18, anti-CD14, anti-ICAM-3, anti-RON, anti-SOST, anti-CD-19, anti-CD80 (e.g., see PCT Publication No, WO2003 / 039486, anti-CD4, anti-CD3, anti-CD23, anti-beta2-integrin, anti-alpha4beta7, anti-CD52, anti-HLA DR, anti-CD22 (e.g., see U. S. Pat. No. 5,789,554), anti-CD20, anti-MIF, anti-CD64 (FcR), anti-TCR alpha beta, anti-CD2, anti-Hep B, anti-CA 125, anti-EpCAM, anti-gpl20, anti-CMV, anti-gpllbllla anti-IgE, anti-CD25, anti-CD33, anti-HLA, anti-IGFl,2, anti IGFR, anti-VNRintegrin, anti-IL-1 alpha, anti -IL- lb eta, anti-IL-1 receptor, anti-IL-2 receptor, anti-IL-4, anti-IL-4 receptor, anti-IL5, anti-IL-5 receptor, anti-IL-6, anti-IL-8, anti-IL-9, anti-IL-13, anti-IL-13 receptor, anti-IL-17, anti-IL-6R, anti-RANKL, anti-NGF, anti-DKK, anti-alphaVbeta3, anti-IL-17A, anti-IL-23pl9 and anti-IL-23 (see Presta, L. G. (2005) J. Allergy Clin. Immunol, 116: 731-6.

[0200] Antibodies used for the therapeutic moiety may also be selected fromvarious therapeutic antibodies approved for use, in clinical trials, or in development for clinical use. Such therapeutic antibodies include, but are not limited to, rituximab (Rituxan®, IDEC / Genentech / Roche) (see, for example, U. S. Pat. No. 5,736,137), a chimeric anti-CD20 antibody approved to treat Non-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20 currently being developed by Genmab, an anti-CD20 antibody described in U. S. Pat. No.5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM (Intracel), and PRO70769 (PCT Application No. PCT / US2003 / 040426), trastuzumab (Herceptin®, Genentech) (see, for example, U. S. Pat. No. 5,677,171), a humanized anti-Her2 / neu antibody approved to treat breast cancer; pertuzumab (rhuMab-2C4, Omnitarg®), currently being developed by Genentech; an anti-Her2 antibody (U. S. Pat. No. 4,753,894; cetuximab (Erbitux®, Imclone) (U. S. Pat. No. 4,943,533; PCT Publication No. WO 96 / 40210), a chimeric anti-EGFR antibody in clinical trials for a variety of cancers; ABX-EGF (U. S. Pat. No.6,235,883), currently being developed by Abgenix-Immunex-Amgen; HuMax-EGFr (U. S. Pat. No. 7,247,301), currently being developed by Genmab; 425, EMD55900, EMD62000, and EMD72000 (Merck KGaA) (U. S. Pat. No. 5,558,864; Murthy, et al. (1987) Arch. Biochem. Biophys. 252(2): 549-60; Rodeck, et al. (1987) J. Cell. Biochem. 35(4): 315-20; Kettleborough, et al. (1991) Protein Eng. 4(7): 773-83); ICR62 (Institute of Cancer Research) (PCT Publication No. WO 95 / 20045; Modjtahedi, et al. (1993) J. Cell. Biophys. 22(1-3): 129-46; Modjtahedi, et al. (1993) Br. J. Cancer 67(2): 247-53; Modjtahedi, et al. (1996) Br. J. Cancer 73(2): 228-35;Modjtahedi, et al. (2003) Int. J. Cancer 105(2): 273-80); TheraCIMhR3 (YM Biosciences, Canada and Centro de Immunologia Molecular, Cuba (U. S. Pat. No. 5,891,996; U. S. Pat. No.6,506,883; Mateo, et al. (1997) Immunotechnol. 3(1): 71-81); mAb-806 (Ludwig Institute for Cancer Research, Memorial Sloan-Kettering) (Jungbluth, et al. (2003) Proc. Natl. Acad. Sci. USA. 100(2): 639-44); KSB-102 (KS Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT Publication No. WO 01 / 62931A2); and SC100 (Scancell) (PCT Publication No. WO 01 / 88138); alemtuzumab (Campath®, Millenium), a humanized mAb currently approved for treatment of B-cell chronic lymphocytic leukemia; muromonab-CD3 (Orthoclone OKT3®), an anti-CD3 antibody developed by Ortho Biotech / Johnson & Johnson, ibritumomab tiuxetan (Zevalin®), an anti-CD20 antibody developed by IDEC / Schering AG, gemtuzumab ozogamicin (Mylotarg®), an anti-CD33 (p67 protein) antibody developed by Celltech / Wyeth, alefacept (Amevive®), an anti-LFA-3 Fc fusion developed by Biogen), abciximab (ReoPro®), developed by Centocor / Lilly, basiliximab (Simulect®), developed by Novartis, palivizumab (Synagis®), developed by Medimmune, infliximab (Remicade®), an anti-TNF alpha antibody developed by Centocor, adalimumab (Humira®), an anti-TNF alpha antibody developed by Abbott, Humicade®, an anti-TNF alpha antibody developed by Celltech, golimumab (CNTO-148), a fully human TNF antibody developed by Centocor, etanercept (Enbrel®), an p75 TNF receptor Fc fusion developed by Immunex / Amgen, lenercept, an p55TNF receptor Fc fusion previously developed by Roche, ABX-CBL, an anti-CD147 antibody being developed by Abgenix, ABX-IL8, an anti-IL8 antibody being developed by Abgenix, ABX-MA1, an anti- MUC18 antibodybeing developed by Abgenix, Pemtumomab (R1549, 90Y-muHMFGl), an anti-MUC1 in development by Antisoma, Therex (R1550), an anti-MUCl antibody being developed by Antisoma, AngioMab (AS1405), being developed by Antisoma, HuBC-1, being developed by Antisoma, Thioplatin (AS 1407) being developed by Antisoma, Antegren® (natalizumab), an anti-alpha-4-beta-l (VLA-4) and alpha-4-beta-7 antibody being developed by Biogen, VLA-1 mAb, an anti -VLA-1 integrin antibody being developed by Biogen, LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibody being developed by Biogen, CAT- 152, an anti-TGF-P2 antibody being developed by Cambridge Antibody Technology, ABT 874 (J695), an anti-IL-12 p40 antibody being developed by Abbott, CAT-192, an anti-TGFpi antibody being developed by Cambridge Antibody Technology and Genzyme, CAT-213, an anti- Eotaxinl antibody being developed by Cambridge Antibody Technology, LymphoStat-B® an anti-BLyS antibody being developed by Cambridge Antibody Technology and Human Genome Sciences Inc., TRAIL-R1 mAb, an anti-TRAIL-Rl antibody being developed byCambridge Antibody Technology and Human Genome Sciences, Inc., Avastin® bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody being developed by Genentech, an anti-HER receptor family antibody being developed by Genentech, Anti-Tissue Factor (ATF), an anti-Tissue Factor antibody being developed by Genentech, Xolair® (Omalizumab), an anti-IgE antibody being developed by Genentech, Raptiva® (Efalizumab), an anti- CD1 la antibody being developed by Genentech and Xoma, MLN-02 Antibody (formerly LDP-02), being developed by Genentech and Millenium Pharmaceuticals, HuMax CD4, an anti-CD4 antibody being developed by Genmab, HuMax-IL15, an anti-IL15 antibody being developed by Genmab and Amgen, HuMax-Inflam, being developed by Genmab and Medarex, HuMax-Cancer, an anti-Heparanase I antibody being developed by Genmab and Medarex and Oxford GlycoSciences, HuMax-Lymphoma, being developed by Genmab and Amgen, HuMax-TAC, being developed by Genmab, IDEC-131, and anti-CD40L antibody being developed by IDEC Pharmaceuticals, IDEC-151 (Clenoliximab), an anti-CD4 antibody being developed by IDEC Pharmaceuticals, IDEC-114, an anti-CD80 antibody being developed by IDEC Pharmaceuticals, IDEC- 152, an anti-CD23 being developed by IDEC Pharmaceuticals, antimacrophage migration factor (MIF) antibodies being developed by IDEC Pharmaceuticals, BEC2, an anti-idiotypic antibody being developed by Imclone, IMC-1C11, an anti- KDR antibody being developed by Imclone, DC101, an anti-flk-1 antibody being developed by Imclone, anti-VE cadherin antibodies being developed by Imclone, CEA-Cide® (labetuzumab), an anti-carcinoembryonic antigen (CEA) antibody being developed by Immunomedics, LymphoCide® (Epratuzumab), an anti-CD22 antibody being developed by Immunomedics, AFP-Cide, being developed by Immunomedics, MyelomaCide, being developed by Immunomedics, LkoCide, being developed by Immunomedics, ProstaCide, being developed by Immunomedics, MDX-010, an anti-CTLA4 antibody being developed by Medarex, MDX-060, an anti-CD30 antibody being developed by Medarex, MDX-070 being developed by Medarex, MDX-018 being developed by Medarex, Osidem® (IDM-1), and anti-Her2 antibody being developed by Medarex and Immuno-Designed Molecules, HuMax®-CD4, an anti-CD4 antibody being developed by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibody being developed by Medarex and Genmab, CNTO 148, an anti- TNFa antibody being developed by Medarex and Centocor / J& J, CNTO 1275, an anticytokine antibody being developed by Centocor / J& J, MORI 01 and MORI 02, anti -intercellularadhesion molecule- 1 (ICAM-1) (CD54) antibodies being developed by MorphoSys, MOR201, an anti-fibroblast growth factor receptor 3 (FGFR-3) antibody being developed by MorphoSys, Nuvion® (visilizumab), an anti-CD3 antibody being developed by Protein Design Labs, HuZAF®, an anti-gamma interferon antibody being developed by Protein Design Labs, Anti-a 5p 1 Integrin, being developed by Protein Design Labs, anti -IL- 12, being developed by Protein Design Labs, ING-1, an anti-Ep-CAM antibody being developed by Xoma, Xolair® (Omalizumab) a humanized anti-IgE antibody developed by Genentech and Novartis, and MLN01, an anti-Beta2 integrin antibody being developed by Xoma. In another embodiment, the therapeutics include KRN330 (Kirin); huA33 antibody (A33, Ludwig Institute for Cancer Research); CNTO 95 (alpha V integrins, Centocor); MEDI522 (alpha V03 integrin, Medimmune); volociximab (alpha Vpi integrin, Biogen / PDL); Human mAb 216 (B cell glycosolated epitope, NCI); BiTE MT103 (bispecific CD19*CD3, Medimmune); 4G7*H22 (Bispecific BcellxFcgammaRl, Medarex / Merck KGa); rM28 (Bispecific CD28xMAPG, EP Patent No. EP1444268); MDX447 (EMD 82633) (Bispecific CD64 EGFR, Medarex);Catumaxomab (removab) (Bispecific EpCAM*anti-CD3, Trion / Fres); Ertumaxomab (bispecific HER2 / CD3, Fresenius Biotech); oregovomab (OvaRex) (CA-125, ViRexx); Rencarex® (WX G250) (carbonic anhydrase IX, Wilex); CNTO 888 (CCL2, Centocor); TRC105 (CD 105 (endoglin), Tracon); BMS-663513 (CD137 agonist, Bristol Myers Squibb); MDX-1342 (CD19, Medarex); Siplizumab (MEDI-507) (CD2, Medimmune); Ofatumumab (Humax-CD20) (CD20, Genmab); Rituximab (Rituxan) (CD20, Genentech); veltuzumab (hA20) (CD20, Immunomedics); Epratuzumab (CD22, Amgen); lumiliximab (IDEC 152) (CD23, Biogen); muromonab-CD3 (CD3, Ortho); HuM291 (CD3 fc receptor, PDL Biopharma); HeFi-1, CD30, NCI); MDX-060 (CD30, Medarex); MDX-1401 (CD30, Medarex); SGN-30 (CD30, Seattle Genentics); SGN-33 (Lintuzumab) (CD33, Seattle Genentics); Zanolimumab (HuMax-CD4) (CD4, Genmab); HCD122 (CD40, Novartis); SGN-40 (CD40, Seattle Genentics); Campath1H (Alemtuzumab) (CD52, Genzyme); MDX-1411 (CD70, Medarex); hLLl (EPB-1) (CD74.38, Immunomedics); Galiximab (IDEC-144) (CD80, Biogen); MT293 (TRC093 / D93) (cleaved collagen, Tracon); HuLuc63 (CS1, PDL Pharma); ipilimumab (MDX-010) (CTLA4, Bristol Myers Squibb); Tremelimumab (Ticilimumab, CP-675,2) (CTLA4, Pfizer); HGS-ETR1 (Mapatumumab) (DR4 TRAIL-R1 agonist, Human Genome Science / Glaxo Smith Kline); AMG-655 (DR5, Amgen); Apomab (DR5, Genentech); CS-1008 (DR5, Daiichi Sankyo); HGS-ETR2(lexatumumab) (DR5 TRAIL-R2 agonist, HGS); Cetuximab (Erbitux) (EGFR, Imclone); IMC-11F8, (EGFR, Imclone); Nimotuzumab (EGFR, YM Bio); Panitumumab (Vectabix) (EGFR, Amgen); Zalutumumab (HuMaxEGFr) (EGFR, Genmab); CDX-110 (EGFRvIII, AVANT Immunotherapeutics); adecatumumab (MT201) (Epcam™, Merck); edrecolomab (Panorex, 17-1A) (Epcam™, Glaxo / Centocor); MORAb-003 (folate receptor a, Morphotech); KW-2871 (ganglioside GD3, Kyowa); MORAb-009 (GP-9, Morphotech); CDX-1307 (MDX-1307) (hCGb, Celldex); Trastuzumab (Herceptin) (HER2, Celldex); Pertuzumab (rhuMAb 2C4) (HER2 (DI), Genentech); apolizumab (HLA-DR beta chain, PDL Pharma); AMG-479 (IGF-1R, Amgen); anti-IGF-lR R1507 (IGF1-R, Roche); CP 751871 (IGF1-R, Pfizer); IMC-A12 (IGF1-R, Imclone); BIIB022 (IGF-1R, Biogen); Mik-beta-1 (IL-2Rb (CD122), Hoffman LaRoche); CNTO 328 (IL6, Centocor); Anti -KIR (1-7F9) (Killer cell Ig-like Receptor (KIR), Novo); Hu3S193 (Lewis (y), Wyeth, Ludwig Institute of Cancer Research); hCBE-11 (LTpR, Biogen);HuHMFGl (MUC1, Antisoma / NCl); RAV12 (N-linked carbohydrate epitope, Raven); CAL (parathyroid hormone-related protein (PTH-rP), University of California); CT-011 (PD1, CureTech); MDX-1106 (ono-4538) (PD1, Medarex / Ono); MAb CT-011 (PD1, Curetech); IMC-3G3 (PDGFRa, Imclone); bavituximab (phosphatidylserine, Peregrine); huJ591 (PSMA, Cornell Research Foundation); muJ591 (PSMA, Cornell Research Foundation); GC1008 (TGFb (pan) inhibitor (IgG4), Genzyme); Infliximab (Remicade) (TNFa, Centocor); A27.15 (transferrin receptor, Salk Institute, INSERN WO 2005 / 111082); E2.3 (transferrin receptor, Salk Institute); Bevacizumab (Avastin) (VEGF, Genentech); HuMV833 (VEGF, Tsukuba Research Lab, PCT Publication No. WO / 2000 / 034337, University of Texas); IMC-18F1 (VEGFR1, Imclone); IMC-1121 (VEGFR2, Imclone).

[0201] In some embodiments of the present disclosure, the therapeutic is or comprises Tx, wherein Tx is selected from:nts, Tx issome embodiments, Tx is,

[0203] Examples of antibodies (e.g., Tx of the present disclosure) include, but are not limited to, 3F8 (anti-GD2), Abagovomab (anti CA-125), Abciximab (anti CD41 (integrin alpha-IIb), Adalimumab (anti-TNF-a), Adecatumumab (antiEpCAM, CD326), Aduhelm (anti-amyloid-beta), Afelimomab (anti-TNF-a); Afutuzumab (anti-CD20), Alacizumab pegol (anti-VEGFR2), ALD518 (anti -IL-6), Alemtuzumab (Campath, MabCampath, anti- CD52), Altumomab (anti-CEA), Anatumomab ( anti-TAG-72), Anrukinzumab (IMA-638, anti-IL-13), Apolizumab (anti-HLA-DR), Arcitumomab (anti -CEA), Aselizumab (anti-L-selectin (CD62L), Atlizumab (tocilizumab, Actemra, RoActemra, anti-IL-6 receptor), Atorolimumab (anti-Rhesus factor), Bamlanivimab (anti-SARS-CoV-2; anti-RSV, or anti -influenza), Bapineuzumab (anti-beta amyloid), Basiliximab (Simulect, antiCD25 (a chain of IL-2 receptor), Bavituximab (antiphosphatidylserine), Bectumomab (LymphoScan, anti-CD22), Belimumab (Benlysta, LymphoStat-B, anti-BAFF), Benralizumab (anti-CD125), Bertilimumab (anti-CCLll (eotaxin-1)), Besilesomab (Scintimun, anti-CEA-related antigen), Bevacizumab (Avastin, anti-VEGF-A), Biciromab (FibriScint, anti-fibrin II beta chain), Bivatuzumab (antiCD44 v6), Blinatumomab (BiTE, anti-CD19), Brentuximab (cAClO, anti-CD3() TNFRSF8), Briakinumab (anti-IL-12, IL-23) Canakinumab (Haris, anti-IL-1), Cantuzumab (C242, antiCanAg), Capromab, Catumaxomab (Removab, anti-EpCAM, anti-CD3), CC49 (anti-TAG-72), Cedelizumab (anti-CD4), Certolizumab pegol (Cimzia anti-TNF-a), Cetuximab (Erbitux, IMCC225, anti-EGFR), Citatuzumab bogatox (anti-EpCAM), Cixutumumab (anti-lGF-1), Clenoliximab (anti-CD4),Clivatuzumab (anti-MUCl), Conatumumab (anti-TRAIL-R2), CR6261 (anti-influenza A hemagglutinin), Dacetuzumab (anti-CD40), Daclizumab (Zenapax, anti-CD25 (a chain of IL-2 receptor)), Daratumumab (anti-CD38 (cyclic ADP ribose hydrolase), Denosumab (Prolia, anti-RANKL), Detumomab (anti-B-lymphoma cell), Dorlimomab, Dorlixizumab, Dupixent (anti-IL-4Ra), Ecromeximab (anti-GD3 ganglioside), Eculizumab (Soliris, anti-C5), Ultomiris (anti-C5), Evkeeza (anti-ANGPTL3), Edobacomab (anti-endotoxin), Edrecolomab (Panorex, MAM7-1A, anti-EpCAM), Efalizumab (Raptiva, anti-LFA-1 (CD Ila), Efungumab (Mycograb, anti-Hsp90), Elotuzumab (anti-SLAMF7), Elsilimomab (anti-IL-6), Enlimomab pegol (anti-ICAM-1 (CD54)), Epitumomab (anti -epi sialin), Epratuzumab (anti-CD22), Erlizumab (anti-ITGB2 (CD 18)), Ertumaxomab (Rexomun, anti-HER2 / neu, CD3), Etaracizumab (Abegrin, anti-integrin avP3), Exbivirumab ( anti -hepatitis B surface antigen), Eylea (anti-VEGF), Fanolesomab (NeutroSpec, anti-CD15), Faralimomab (anti -interferon receptor), Farletuzumab (anti-folate receptor 1), Fasenra (anti-IL-5Ra), Felvizumab (antirespiratory syncytial virus), Fezakinumab (anti-IL-22), Figitumumab (anti-IGF-1 receptor), Fontolizumab (anti-fFN-y), Foravirumab (anti-rabies virus glycoprotein), Fresolimumab (antiTGF-0), Galiximab (anti-CD80), Gantenerumab (anti- beta amyloid), Gavilimomab, CD147 (basigin)), Gemtuzumab (anti-CD33), Girentuximab (anti-carbonic anhydrase 9), Glembatumumab (CR011, anti-GPNMB), Golimumab (Simponi, anti-TNF-ot), Gomiliximab (anti-CD23 (IgE receptor)), Ibalizumab (anti-CD4), Ibritumomab (anti-CD20), Igovomab (Indimacis-125, anti-CA-125), Imciromab (Myoscint, anti-cardiac myosin), Infliximab (Remicade, anti-TNF-q), Intetumumab (anti-CD51), Inolimomab (anti-CD25 (a chain of IL-2 receptor)), Inotuzumab (anti-CD22), Ipilimumab (anti-CD152), Iratumumab (anti- CD30 (TNFRSF8)), Keliximab (anti-CD4), Labetuzumab (CEA-Cide, anti-CEA), Lebrikizumab (antiIL-13), Lemalesomab (anti-NCA-90 (granulocyte antigen)), Lerdelimumab (anti-TGF beta 2), Leqembi (anti-amyloid-beta), Lexatumumab (anti-TRAIL-R2), Libivirumab (anti-hepatitis B surface antigen), Lintuzumab (anti-CD33), Lucatumumab (anti-CD40), Lumiliximab (anti-CD23 (IgE receptor), Mapatumumab (anti-TRAIL-Rl), Maslimomab (anti- T-cell receptor), Matuzumab (antiEGFR), Mepolizumab (Bosatria, anti-IL-5), Metelimumab (anti-TGF beta 1), Milatuzumab (anti-CD74), Minretumomab (anti-TAG-72), Mitumomab (BEC-2, anti-GD3 ganglioside), Morolimumab (anti-Rhesus factor), Motavizumab (Numax, anti-respiratory syncytial virus), Muromonab-CD3 (Orthoclone OKT3, anti-CD3), Nacolomab (anti-C242), Naptumomab (anti5T4), Natalizumab (Tysabri, anti-integrin a4). Nebacumab (anti-endotoxin),Necitumumab (antiEGFR), Nerelimomab (anti-TNF-a), Nimotuzumab (Theracim, Theraloc, anti-EGFR), Nofetumomab, Nulojix (anti-CTLA-4), Ocrelizumab (anti-CD20), Odulimomab (Afolimomab, anti-LFA-1 (GDIla)), Ofatumumab (Arzerra, anti-CD20), Olaratumab (anti-PDGF-R a), Omalizumab (Xolair, antilgE Fc region), Oportuzumab (anti-EpCAM), Oregovomab (OvaRex, anti-CA-125), Otelixizumab (anti-CD3), Pagibaximab (anti-lipoteichoic acid), Palivizumab (Synagis, Abbosynagis, anti-respiratory syncytial virus), Panitumumab (Vectibix, ABX-EGF, antiEGFR), Panobacumab (anti-Pseudomonas aeruginosa), Pascolizumab (anti-IL-4), Pemtumomab (Theragyn, anti-MUCl), Pertuzumab (Omnitarg, 2C4, anti-IIER2 / neu), Pexelizumab (anti-C5), Pintumomab (anti -adenocarcinoma antigen), Praluent (anti-PCSK9), Priliximab (anti-CD4), Pritumumab (antivimentin), PRO 140 (anti-CCR5), Racotumomab (1E10, anti-(N-glycolylneuraminic acid (NeuGc, NGNA)-gangliosides GM3)), Rafivirumab (anti-rabies virus glycoprotein), Ramucirumab (anti-VEGFR2), Ranibizumab (Lucentis, anti-VEGF-A), Raxibacumab (antianthrax toxin, protective antigen), Regavirumab (anti-cytomegalovirus glycoprotein B), Repatha (anti-PCSK9), Reslizumab (anti-IL-5), Rilotumumab (anti-HGF), Rituximab (Mab Thera, Rituxanmab, antiCD20), Robatumumab (anti-IGF-1 receptor), Rontalizumab (anti-IFN-a), Rovelizumab (LeukArrest, anti-CDll, CD 18), Ruplizumab (Antova, anti-CD154 (CD40L)), Satumomab (anti -TAG-72), Sevirumab (anti-cytomegalovirus), Sibrotuzumab (anti-FAP), Sifalimumab (anti-IFN-a), Siltuximab (anti-IL-6), Siplizumab (anti-CD2), (Smart) MI95 (anti-CD33), Solanezumab (anti-beta amyloid), Sonepcizumab (anti-sphingosine-1 -phosphate), Sontuzumab (anti-episialin), Sotrovimab (anti-SARS-CoV-2), Stamulumab (anti-myostatin), Sulesomab (LeukoScan, (anti-NCA-90 (granulocyte antigen), Tacatuzumab (anti-alpha-fetoprotein), Tadocizumab (anti-integrin αvβ3), Talizumab (anti-IgE), Tanezumab (anti-NGF), Taplitumomab (anti-CD19), Tefibazumab (Aurexis, (anti-clumping factor A), Telimomab, Tenatumomab (anti-tenascin C), Teneliximab (anti-CD40), Teplizumab (anti-CD3), Tezspire (anti-TSLP), TGN1412 (anti-CD28), Ticilimumab (Tremelimumab, (anti-CTLA-4), Tigatuzumab (anti-TRAlL-R2), TNX-650 (anti-IL-13), Tocilizumab (Atlizumab, Actemra, RoActemra, (anti -IL-6 receptor), Toralizumab (anti-CD154 (CD40L)), Tositumomab (anti-CD20), Trastuzumab (Herceptin, (anti-HER2 / neu), Tremelimumab (antiCTLA-4), Tucotuzumab celmoleukin (anti-EpCAM), Tuvirumab (anti -hepatitis B virus), Uplizna (anti-CD19), Urtoxazumab (anti-Escherichia coli), Ustekinumab (Stelara, anti-IL-12, IL-23), Vabysmo (anti-VEGF-A / Ang-2), Vapaliximab (anti-AOC3 (VAP-1)), Vedolizumab, (anti-integrin αvβ3), Veltuzumab (anti-CD20), Vepalimomab (anti-AOC3 (VAP-1), Visilizumab (Nuvion, anti-CD3), Vitaxin (anti-vascular integrin avb3), Volociximab (anti-integrin α4β1), Votumumab (HumaSPECT, anti-tumor antigen CTAA16.88), Zalutumumab (HuMax-EGFr, (anti-EGFR), Zanolimumab (HuMax-CD4, antiCD4), Ziralimumab (anti-CD147 (basigin)), Zolimomab (anti-CD5), Etanercept (Enbrel®), Alefacept (Amevive®), Abatacept (Orencia®), Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (Iron Regulatory Protein 2)], 14G2a (anti-GD2 ganglioside, from Nat. Cancer Inst, for melanoma and solid tumors), J591 (anti-PSMA, Weill Cornell Medical School for prostate cancers), 225.28S [anti-HMW-MAA (High molecular weight-melanoma-associated antigen), Sorin Radiofarmaci S. R. L. (Milan, Italy) for melanoma], COL-1 (anti-CEACAM3, CGM1, from Nat. Cancer Inst. USA for colorectal and gastric cancers), CYT-356 (Oncoltad®, for prostate cancers), HNK20 (OraVax Inc. for respiratory syncytial virus), ImmuRAIT (from Immunomedics for NHL), Lym-1 (anti-HLA-DR10, Peregrine Pharm. for Cancers), MAK-195F [anti-TNF (tumor necrosis factor; TNFA, TNF-alpha; TNFSF2), from Abbott / Knoll for Sepsis toxic shock], MEDI-500 [T10B9, anti-CD3, THaP (T cell receptor alpha / beta), complex, from Medlmmune Inc for Graft -versus-host disease], RING SCAN [ anti-TAG 72 (tumour associated glycoprotein 72), from Neoprobe Corp, for Breast, Colon and Rectal cancers], Avicidin (anti-EPCAM (epithelial cell adhesion molecule), anti-TACSTDl (Tumor-associated calcium signal transducer 1), antiGA733-2 (gastrointestinal tumor-associated protein 2), anti-EGP-2 (epithelial glycoprotein 2); anti-KSA; KS1 / 4 antigen; M4S; tumor antigen 17-1 A; CD326, from NeoRx Corp, for Colon, Ovarian, Prostate cancers and NHL]; LymphoCide (Immunomedics, NJ), Smart ID 10 (Protein Design Labs), Oncolym (Techniclone Inc, CA), Allomune (BioTransplant, CA), anti-VEGF (Genentech, CA); CEAcide (Immunomedics, NJ), IMC-1C11 (ImClone, NJ) and Cetuximab (ImClone, NJ).

[0204] Disclosed in patent application AU2021202647, are a list of therapeutic moiety combinations, and are hereby incorporated by reference.

[0205] In some embodiments, the therapeutic moiety comprises an imaging agent, a radioligand agent, or a cytotoxic agent. In some embodiments, the cytotoxic moiety comprises a small molecule drug, or a chemotherapeutic drug. In some embodiments, the radioligand agent comprises a radionuclide selected from the group consisting of153Sm,177Lu,90Y,131I,149Tb,211At,212Pb,212Bi,213Bi,223Ra,225Ac, and227Th. In some embodiments, the radioligand agent comprises a radionuclide selected from the group consisting of99mTc,131I,201Tl,111In, and67Ga.In some embodiments, the radioligand agent further comprises a chelator. In some embodiments, the chelator complexes with the radionuclide.Linker

[0206] In various embodiments, conjugates disclosed herein comprises one or more linkers. In some embodiments, the therapeutic moiety is attached to the targeting moiety via a linker. In some embodiments, the reactive moiety is attached to the targeting moiety via a linker. In some embodiments, the linker comprises a polymer. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is 0.01 kDa to 2.5 kDa in size. In some embodiments, the linker is linear, branched, multimeric, or dendrimeric.

[0207] In some embodiments, the linker is a bifunctional or multifunctional linker. In some embodiments, the linker comprises a bifunctional or multifunctional polymer. In some embodiments, the linker comprises a water-soluble polymer. In some embodiments, the water-soluble polymer is polyethylene glycol (PEG). In some embodiments, the PEG has a molecular weight between 0.1 kDa and 2.5k Da. In some embodiments, the PEG comprises 1-8 monomers.

[0208] It is known that extensive modifications to the peptide construct may obstruct target binding by the introduction of steric hindrance. A high degree of stability and minimal hydrolysis side reactions of the linker and the macrocyclic peptide are key for any practical cross-linking methodology. Compared to other cysteine-lysine cross-linking reagents such as N-hydroxysuccinimide(NHS)-maleimide, NHS-pyridyldithiol, or NHS-haloacetyl cross-linkers, the palladium oxidative complex shows potential advantages in high linker stability and minimal hydrolysis side reactions stability (Kubota K, et al., (2018). J Am Chem Soc., 140(8):3128-3133).

[0209] In some embodiment, the linker comprises a domain which improves pharmacodynamics.

[0210] In some embodiments, L comprises a chain of 1-60 carbon atoms in length, wherein one or more carbon atoms are optionally substituted with halogen, hydroxyl, cyano, CFH2, CF2H, CF3, alkoxy, C1-C5 alkyl, or -C(=O)-NH2; andwherein one or more carbon atoms of the backbone are optionally replaced by a divalent group independently selected from oxygen, alkylamino, carbonyl, 3-14 memberedcycloalkylene, 3-8 membered monocyclic heterocyclic group, 5-14 membered bridged heterocyclic group, 5-14 membered spiro heterocyclic group, 5-14 membered heteroarylene, and 7-14 membered heteroaryl-fused-heterocyclylene, and wherein each of the 3-14 membered cycloalkylene, 3-8 membered monocyclic heterocyclic group, 5-14 membered bridged heterocyclic group, 5-14 membered spiro heterocyclic group, 5-14 membered heteroarylene, and 7-14 membered heteroaryl-fused-heterocyclylene is independently substituted with 0, 1, 2, 3, 4, 5, or 6 RL; andeach RLis independently selected from alkylamino, halogen, hydroxyl, C1-C5 alkoxy, C1-C5 alkyl, C1-C5 alkylcarboxyl, C1-4 alkenyl, C3-5 cycloalkyl, C1-5 haloalkyl, and an oxo group.

[0211] In some embodiments, L is a chain of 1-60 carbon atoms in length, wherein one or more carbon atoms are optionally substituted with halogen, hydroxyl, cyano, CFH2, CF2H, CF3, alkoxy, C1-C5 alkyl, or -C(=O)-NH2; andwherein one or more carbon atoms of the backbone are optionally replaced by a divalent group independently selected from oxygen, alkylamino, carbonyl, 3-14 membered cycloalkylene, 3-8 membered monocyclic heterocyclic group, 5-14 membered bridged heterocyclic group, 5-14 membered spiro heterocyclic group, 5-14 membered heteroarylene, and 7-14 membered heteroaryl-fused-heterocyclylene, and wherein each of the 3-14 membered cycloalkylene, 3-8 membered monocyclic heterocyclic group, 5-14 membered bridged heterocyclic group, 5-14 membered spiro heterocyclic group, 5-14 membered heteroarylene, and 7-14 membered heteroaryl-fused-heterocyclylene is independently substituted with 0, 1, 2, 3, 4, 5, or 6 RL; andeach RLis independently selected from alkylamino, halogen, hydroxyl, C1-C5 alkoxy, C1-C5 alkyl, C1-C5 alkylcarboxyl, C1-4alkenyl, C3-5 cycloalkyl, C1-5haloalkyl, and an oxo group.

[0212] In some embodiments, L comprises a mercapto-succinimidyl adduct group, a hydrophilic PEG spacer, a valine-citrulline (Val-Cit) dipeptide, a valine-alanine (Val-Ala) dipeptide, and alanine-alanine (Ala-Ala) dipeptide, a carbohydrate-substituted aryl(e.g., phenolic glucuronide capable of immolative fragmentation via an ortho- or paraquinone methide), a peptide further substituted with a carbohydrate, a para-aminobenzyl (PAB) spacer, or a combination thereof.

[0213] In some embodiments, L is represented by:wherein:# represents the point of attachment to T;* represents the point of attachment to R’; and$ represents the point of attachment to Tx.

[0214] In some embodiments, L is represented by:

[0215] In some embodiments, -L-Tx is represented by:

[0216] In some embodiments, -L-Tx is represented by:

[0217] In some embodiments, -L-R”- is represented by:

[0218] In some embodiments, -L-R”-T is represented by:

[0219] In some embodiments, higher-order linkers comprise bifunctional linkers, such as homobifunctional linkers or heterobifunctional linkers. Exemplary homobifunctional linkers include, but are not limited to, Lomanf s reagent dithiobis (succinimidylpropionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl proprionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate(sulfo DST), ethylene glycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG), N, N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), l,4-di-3 '-(2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), such as e.g. l,5-difluoro-2,4-dinitrobenzene or l,3-difluoro-4,6-dinitrobenzene, 4, 4'-difluoro-3, 3 '-dinitrophenyl sulfone (DFDNPS), bis-[|3-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3 '-dimethylbenzidine, benzidine, a,a'-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N, N'-ethylene-bis(iodoacetamide), or N, N'-hexamethylene-bis(iodoacetamide).

[0220] In some embodiments, the bifunctional linker comprises a heterobifunctional linker. Exemplary heterobifunctional linker include, but are not limited to, amine-reactive and sulfhydryl cross-linkers such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble-long-chain N-succinimidyl 3 -(2 -pyridyldithio) propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[a-methyl-a-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-l -carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxy succinimide ester (MBs), m-maleimidobenzoyl-N-hydroxy sulfosuccinimide ester (sulfo-MBs), N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB), sulfosuccinimidyl(4-iodoacteyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(y-maleimidobutyryloxy)succinimide ester (GMBs), N-(y-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (slAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-l -carboxylate (sIAC), succinimidyl 6-((((4-iodoacetyl)amino)methyl)cyclohexane-l-carbonyl)amino) hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl -reactive and sulfhydryl-reactive cross-linkers such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-l-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactiveand photoreactive cross-linkers such as N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl- 1,3 '-dithiopropionate (sAsD), N-hydroxy succinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'- azido-2 '-nitrophenyl amino)hexanoate (s ANP AH), sulfosuccinimidyl-6-(4 '-azi do-2 '- nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2 -nitrobenzoyloxysuccinimide (ANB- NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl- 1,3 '-dithiopropionate (sAND), N- succinimidyl-4(4-azidophenyl) 1,3 '-dithiopropionate (s ADP), N-sulfosuccinimidyl(4- azidophenyl)- 1,3 '-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-l,3'- dithiopropi onate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3 -acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP- DTP), sulfhydryl-reactive and photoreactive crosslinkers such asl-(p-Azidosalicylamido)-4- (iodoacetamido)butane (AsIB), N-[4-(p-azidosalicylamido)butyl]-3 '-(2'- pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4- maleimide carbonyl -reactive and photoreactive cross-linkers such as p-azidobenzoyl hydrazide (ABH), carboxylate-reactive and photoreactive cross-linkers such as 4-(p- azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive cross-linkers such as p-azidophenyl glyoxal (APG).

[0221] Additional bifunctional linkers are well known in the art (see, for example, U. S. Pat. No.5,208,020; Isalm and Dent in Bioconjugation chapter 5, p 218-363, Groves Dictionaries Inc. New York, 1999), for example, N-succinimidyl-3-(2pyridyldithiojpropionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), Nsuccinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-3-(2-pyridyldithio)butyrate (SDPB), 2-iminothiolane, N-succinimidyl-4-(5-nitro-2-pyridyldithio) butyrate (SNPB), N-succinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate (SNPP), N-sulfosuccinimidyl4-(5-nitro-2-pyridyldithio) butyrate (SSNPB), N-succinimidyl-4-methyl-4-(5-nitro-2pyridyldithio)pentanoate (SMNP), N-sulfosuccinimidyl 4-(5-nitro-2-pyri dyl di thi o)-pentanoate (S SNPP), 4-succi ni mi dyl -oxy carbonyl -a-m ethyl -a-(2-py ri dyl di th i o) -toluene (SMPT), Nsulfosuccinimidyl-4-methyl-4-(5-nitro-2-pyridyldithio)pentanoate (SSMNP); N-succinimidyl4-methyl-4-(2-pyridyldithio)pentanoate (SMPDP), N-succinimidyl-4-(5-N, N-dimethylcarboxamido-2-pyridyldithio) butyrate (SCPB), N-sulfosuccinimidyl-4-(5-N, N-dimethylcarboxamido-2-pyridyldithio) butyrate (SSCPB), N-succinimidyl-4,4-dimethyl-4-(2pyridyldithio)pentanoate (SDMPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-l carboxylate (SMCC), N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), bismaleimidopolyethyleneglycol (BMPEG), BM(PEG)i~2o, N-(P-maleimidopropyloxy)succinimide ester (BMPS), iminothiolane (IT), dimethyl adipimidate HC1 or derivatives of imidoesters, active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bisdiazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro2,4-dinitrobenzene), gamma-maleimidobutyric acid N-succinimidyl ester (GMBS), Emaleimidocaproic acid N-hydroxysuccinimide ester (EMCS), 5-maleimidovaleric acid NHS, HBVS, N-succinimidyl -4-(N-maleimidomethyl)-cyclohexane-l-carboxy-(6-amidocaproate) (a long chain analog of SMCC (LC-SMCC)), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)-butyric acid hydrazide or HC1 salt (MPBH), Nsuccinimidyl 3-(bromoacetamido)propionate (SBAP), N-succinimidyl iodoacetate (SIA), kappa-maleimidoundecanoic acid N-succinimidyl ester (KMUA), N-succinimidyl 4-(pmaleimidophenyl)-butyrate (SMPB), succinimidyl-6-(beta-maleimidopropionamido)hexanoate (SMPH), succinimidyl-(4-vinylsulfonyl)benzoate (SVSB), dithiobismaleimidoethane (DTME), 1,4-bis-maleimidobutane (BMB), 1,4 bismaleimidyl-2,3dihydroxybutane (BMDB), bis-maleimidohexane (BMH), bis-maleimidoethane (BMOE), sulfosuccinimidyl 4-(N-maleimido-methyl)cyclohexane-l -carboxylate (sulfo-SMCC), sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate (sulfo-SIAB), m-maleimidobenzoyl-Nhydroxysulfosuccinimide ester (sulfo-MBS), N-(gamma-maleimidobutryloxy)sulfosuccinimdeester (sulfo-GMBS), N-(epsilon-maleimidocaproyloxy)sulfosuccimido ester (sulfoEMCS), N-(kappa-maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS), and sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB); or the commercially available linkers (such as from Thermo Scientific’s Pierce: Imidoester Crosslinkers: DMA (Dimethyl adipimidate*2 HC1), DMP (Dimethyl pimelimidate*2 HC1), DMS (Dimethyl Suberimidate»2 HC1), DTBP (Dimethyl 3,3'-dithiobispropionimidate»2 HC1); NHS-ester Crosslinkers- Amine Reactive: BS(PEG)s (Bis(succinimidyl) penta(ethylence glycol), BS(PEG)9 (Bis(succinimidyl)nona(ethylence glycol), BS3 (Bisfsulfosuccinimidyl] suberate), BSOCOES (Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone), DSG (Disuccinimidyl glutarate), DSP (Dithiobis[succinimidyl propionate]), DSS (Disuccinimidyl suberate), DST (Disuccinimidyl tartarate), DTSSP (3,3'-Dithiobis[sulfosuccinimidylpropionateJ), EGS (Ethylene glycol bisfsuccinimidylsuccinate]), Sulfo-EGS (Ethylene glycol bis[sulfosuccinimidylsuccinate]), TSAT (Tris-succinimidyl aminotriacetate), DFDNB (1,5 Difluoro-2,4-dinitrobenzene); Amine-to-Sulfhydryl Crosslinkers: Sulfo-SIAB (Sulfosuccinimidyl (4-iodoacetyl)aminobenzoate), SIAB (Succinimidyl (4iodoacetyl)aminobenzoate), SBAP(Succinimidyl 3-(bromoacetamido)propionate), SIA (Succinimidyl iodoacetate), Sulfo-SMCC (Sulfosuccinimidyl -4-(N-maleimidomethyl)cyclohexane-l -carboxylate), SM(PEG)n (NHS-PEG-Maleimide Crosslinkers: Succinimidyl([N-maleiniidopropionamido])-#ethyleneglycol)ester, #=1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 24), LC-SMCC (Succinimidyl 4-(N-maleimidom ethyl) cyclohexane- lcarboxy-(6-amidocaproate)), Sulfo-EMCS (N-epsilon-Maleimidocaproyloxysulfosuccinimide ester), EMCS (N-epsilon-Malemidocaproyl-oxysuccinimide ester), Sulfo-GMBS (N-gamma-Maleimidobutyryl-oxysulfosuccinimide ester), GMBS (N-gammaMaleimidobutyryl-oxysuccinimide ester), Sulfo-KMUS (N-kappa-Maleimidoundecanoyloxysulfosuccinimide ester), Sulfo-MBS (m-Maleimidobenzoyl-N-hydroxy sulfosuccinimide ester), MBS (m -Mai eimidobenzoyl-N-hydroxy succinimide ester), Sulfo-SMPB ((Sulfosuccinimidyl 4-(p-maleiimidophenyl)butyrate), SMPB (Succinimidyl 4-(pmaleimidophenyl)butyrate), AMAS N-(a-Maleimidoacetoxy) succinimide ester), BMPS ( Nbeta-Maleimidopropyl-oxysuccinimide ester), SMPH (Succinimidyl 6-[(betamaleimidopropionamido)hexanoate]), PEG12-SPDP (2-Pyridyldithioltetraoxaoctatriacontane-N-hydroxysuccinimide), PEG4-SPDP (2-Pyridyldithioltetraoxatetradecane-N-hydroxysuccinimide), Sulfo-LC-SPDP (Sulfosuccinimidyl 6-[3’-(2pyridyldithio)propionamido]hexanoate), LC-SPDP (Succinimidyl 6-[3-(2pyridyldithio)propionamido]hexanoate), SMPT (4-Succinimidyloxycarbonyl-alpha-methylalpha(2-pyridyldithio)toluene); Carboxyl-to-Amine Crosslinkers: DCC (Dicyclohexylcarbodiimide), EDC (l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide);Photoreactive Crosslinkers: ANB-NOS (N-5-Azido-2-nitrobenzoyloxysuccinimide), NHSDiazirine (SDA) Crosslinkers: SDA (NHS-Diazirine) (Succinimidyl 4,4'-azipentanoate), LCSDA (NHS-LC-Diazirine) (Succinimidyl 6-(4,4'-azipentanamido)hexanoate), SDAD(NHSSS-Diazirine) (Succinimidyl 2-([4,4'-azipentanamido]ethyl)-l,3'-dithiopropi onate), SulfoSDA (Sulfo-NHS-Diazirine) (Sulfosuccinimidyl 4,4'-azipentanoate), Sulfo-LC-SDA (SulfoNHS-LC-Diazirine) (Sulfosuccinimidyl 6-(4,4’-azipentanamido)hexanoate), Sulfo-SDAD (Sulfo-NHS-SS-Diazirine) (Sulfosuccinimidyl 2-([4,4'-azipentanamido]ethyl)-1,3'dithiopropionate), Sulfo-SANPAH (Sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate), SPB (Succinimidyl-[4-(psoralen-8-yloxy)]-butyrate); Sulfhydryl-to-Carbohydrate Crosslinkers: BMPH (N-beta-Maleimidopropionic acid hydrazide-TFA), EMCH (N-epsilonMaleimidocaproic acid hydrazide-TFA), KMUII (N-kappa-Maleimidoundecanoic acid hydrazide-TFA), MPBH (4-(4-N-Maleimidophenyl)butyric acid hydrazide-IICl), PDPH (3-(2Pyridyldithio)propionyl hydrazide); Sullhydryl-to-Hydroxyl Crosslinkers: PMPI (pMaleimidophenyl isocyanate); Sullhydryl-to-Sulfhydryl Crosslinkers: BM(PEG)2 (l,8Bismaleimido-diethyleneglycol), BM(PEG)3 (1,11 -Bismaleimido-tri ethyleneglycol), BMB (1,4-Bismaleimidobutane), BMDB (l,4-Bismaleimidyl-2,3-dihydroxybutane), BMH (Bismaleimidohexane), BMOE (Bismaleimidoethane), DIME (Dithiobismaleimido ethane), TMEA (Tris(2-maleimidoethyl)amine), and SVSB (succinimidyl-(4vinylsulfone)benzoate).

[0222] In some embodiments, the linker comprises one or more functional reactive groups for conjugating or binding a reactive moiety or therapeutic moiety to a targeting moiety. In some instances, a described moiety of the construct (e.g., a targeting moiety, reactive moiety, or therapeutic moiety) comprises a functional reactive group that reacts with a linker (optionally pre-attached to a therapeutic moiety, targeting moiety, or other portion of a conjugate) described herein.

[0223] In some embodiments, a linker comprises a functional reactive group that reacts with a natural amino acid in a targeting moiety or therapeutic moiety described herein. In some embodiments, the functional reactive group comprises a nucleophilic group that is reactive to an electrophilic group present on a targeting moiety. Exemplary electrophilic groups include carbonyl groups — such as aldehyde, ketone, carboxylic acid, ester, amide, enone, acyl halide or acid anhydride. In some embodiments, the functional reactive group is aldehyde. Exemplary nucleophilic groups include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In some embodiments, an unnatural amino acid incorporated into a conjugate described herein comprises an electrophilic group.

[0224] In some embodiments, the linker is a non-cleavable linker. Non-cleavable linkers are characterized by higher stability in circulation, and lower off-target activity due to reduced killing of antigen-negative cells. The resistance to extracellular cleavage may additionally increase the specificity of the drug release, and an increase in pharmacokinetic and pharmacodynamic properties.

[0225] In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker is a dipeptide linker. In some embodiments, the dipeptide linker is valinecitrulline (Val-Cit), phenylalanine-lysine (Phe-Lys), valine-alanine (Vai-Ala) and valinelysine (Val-Lys). In some embodiments, the dipeptide linker is valine-citrulline. The linker may comprise a cleavable sequence or a sequence that is recognized by a protease.

[0226] In some embodiments, the linker is pH cleavable. In some embodiments, the linker is hydrolysable. In some embodiments, the linker is protease-resistant. In some embodiments, the linker can have flexibility and a varied length.

[0227] In some embodiments, the cleavable linker is glucuronide. In some embodiments, the cleavable linker comprises a glucuronide. In some embodiments, the cleavable linker is an acid-cleavable linker. In some embodiments, the acid-cleavable linker is hydrazine. In some embodiments, the cleavable linker is reducible.

[0228] Appropriate physiologically cleavable linkages include but are not limited to ester, carbonate ester, carbamate, sulfate, phosphate, acyloxy alkyl ether, acetal, and ketal. Such conjugates should possess a physiologically cleavable bond that is stable upon storage and upon administration. For instance, a targeting domain or modified targeting domain linked to a polymer should maintain its integrity upon manufacturing of the final pharmaceutical composition, upon dissolution in an appropriate delivery vehicle, if employed, and upon administration irrespective of route.

[0229] In various embodiments, the linker is a polypeptide linker. In certain embodiments, the targeting moiety comprises a polypeptide linker, which may be connected (e.g., at the C-terminal) to a therapeutic moiety. The linker may be expressed via recombinant technology and may be encoded using a nucleic acid sequence in conjunction with the expression of the targeting moiety. The linker may link the targeting moiety to form a fusion protein. The presence of a linker in the conjugates may allow the targeting moiety to function properly without stericinterference from the other targeting domain. The linker may link a targeting domain to a tag, such as an expression tag or purification tag. The linker may be a flexible linker. The flexibility of the linker may allow the targeting domains to adopt independent conformations with minimal interference from the other targeting domain. In some embodiments, the linker is a peptide linker comprising, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids. In some instances, the peptide linker comprises at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, or less amino acids. In additional cases, the peptide linker comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In additional cases, the polypeptide linker comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some instances a polypeptide linker comprises (GGGGSGGGS)x, wherein x is 1-10. In some embodiments, the linker is a polypeptide linker of 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12 amino acids in length, and longer in length. The linker can comprise SPSTPPTPSPSTPP. The polypeptide linker may be a repeat of (GGGGS)x, (GGGS)x. The linker can comprise glycine, serine, threonine, or proline. In some embodiments, the N-terminus of one targeting moiety is fused to the C-C -terminus of the linker polypeptide and the N-terminus of the linker polypeptide is fused to the N-terminus of the therapeutic moiety.

[0230] In some embodiments, the linker comprises a maleimide group. In some instances, the maleimide group is also referred to as a maleimide spacer. In some instances, the maleimide group further comprises a caproic acid, forming maleimidocaproyl (me). In some cases, the linker comprises maleimidocaproyl (me). In some cases, linker is maleimidocaproyl (me). In other instances, the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-l -carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-l -carboxylate (sulfo-sMCC).

[0231] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some instances, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalysis of thiosuccinimide ring hydrolysis, thereby eliminating maleimide from undergoing an elimination reaction through a retro-Michael reaction. In some instances, the self-stabilizing maleimide is a maleimide group described in Lyon, et al., “Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates,” Nat. Biotechnol. 32(10): 1059-1062 (2014). In some instances, the linker comprises a self-stabilizing maleimide. In some instances, the linker is a self-stabilizing maleimide.

[0232] In some instances, a linker comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more of a carbonyl or dicarbonyl group, oxime group, hydroxylamine group, or protected forms thereof. The TLR-agonist linker derivative or the targeting domain can be the same or different, for example, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different sites in the derivative that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different reactive groups.

[0233] Linkers may comprise a polymer, such as those comprising a water soluble backbone. In some embodiments, polymer backbones that are water-soluble comprise from 2 to about 300 termini. Examples of suitable polymers include, but are not limited to, other poly(alkylene glycols), such as polypropylene glycol) (“PPG”), copolymers thereof (including but not limited to copolymers of ethylene glycol and propylene glycol), terpolymers thereof, mixtures thereof, and the like. Although the molecular weight of each chain of the polymer backbone can vary, it is typically in the range of from about 800 Da to about 100,000 Da, often from about 6,000 Da to about 80,000 Da. The molecular weight of each chain of the polymer backbone may be between about 100 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 100 Da and about 50,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 100 Da and about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 10,000 Da and about 40,000 Da.

[0234] In some embodiments, a polymer used herein terminates on one end with hydroxy or methoxy, i.e., X is H or CH3 ("methoxy PEG"). Alternatively, the polymer can terminate with a reactive group, thereby forming a bifunctional polymer. Typical reactive groups can include those reactive groups that are commonly used to react with the functional groups found in the 20 common amino acids (including but not limited to, maleimide groups, activated carbonates (including but not limited to, p-nitrophenyl ester), activated esters (including but not limited to, N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well as functional groups that are inert to the 20 common amino acids but that react specifically with complementary functional groups (including but not limited to, azide groups, alkyne groups). It is noted that the other end of the polymer, will attach either directly or indirectly to a targeting domain via an amino acid. For instance, this portion is an amide, carbamate, or urea linkage to an amine group.Alternatively, this portion is a maleimide linkage to a thiol group (including but not limited to, the thiol group of cysteine). Alternatively, an alkyne group on the polymer can be reacted with an azide group present in a targeting domain to form a similar product. In some embodiments, a strong nucleophile (including but not limited to, hydrazine, hydrazide, hydroxylamine, semi carb azide) can be reacted with an aldehyde or ketone group present in a targeting domain to form a hydrazone, oxime or semicarbazone, as applicable, which in some cases can be further reduced by treatment with an appropriate reducing agent. Alternatively, the strong nucleophile can be incorporated into the targeting domain via an amino acid and used to react preferentially with a ketone or aldehyde group present in the water-soluble polymer.

[0235] Any molecular mass for a polymer can be used as practically desired, including but not limited to, from about 0.1 Daltons (Da) to 2,500 Da or more. The molecular weight of polymer may be of a wide range, including but not limited to, between about 100 Da and about 5,000 Da or more. In some instances the polymer is 50-5000 Da, 50-3000 Da, 50-2500 Da, 100-2500 Da, 250-2500 Da, 250-5000 Da, or 500-5000 Da. Branched chain polymers, including but not limited to, polymer molecules with each chain having a molecular weight ranging from 0.1-5 kDa, 0.1-4 kDa, 0.1-3 kDa, 0.1-2.5 kDa, 0.1-1.5 kDa.

[0236] Polymers may comprise azide- and acetylene-containing polymer derivatives comprising a water-soluble polymer backbone having an average molecular weight from about 800 Da to about 100,000 Da. The polymer backbone of the water-soluble polymer can be poly(ethylene glycol). However, it should be understood that a wide variety of water-soluble polymersincluding but not limited to poly(ethylene)glycol and other related polymers, including poly(dextran) and polypropylene glycol), are also and that the use of the term PEG or poly(ethylene glycol) is intended to encompass and include all such molecules. The term PEG includes, but is not limited to, poly(ethylene glycol) in any of its forms, including bifunctional PEG, multiarmed PEG, derivatized PEG, forked PEG, branched PEG, pendent PEG (i.e. PEG or related polymers having one or more functional groups pendent to the polymer backbone), or PEG with degradable linkages therein.

[0237] In addition to these forms of polymer, the polymer can also be prepared with weak or degradable linkages in the backbone. For example, polymer can be prepared with ester linkages in the polymer backbone that are subject to hydrolysis. As shown below, this hydrolysis results in cleavage of the polymer into fragments of lower molecular weight: -polymer-CCh-polymer-+H2O apolymer-CCEH+HO-polymer.

[0238] In other embodiments, the polymer is a water-soluble polymer. In other embodiments, the water-soluble polymer is polyethylene glycol (PEG). In some embodiments, the PEG has a molecular weight between 0.1 kDa and 10 kDa. In other embodiments, the PEG has a molecular weight between 0.1 kDa and 5 kDa. In other embodiments, the PEG has a molecular weight between 0.1 kDa and 4 kDa. In other embodiments, the PEG has a molecular weight between 0.1 kDa and 3 kDa. In other embodiments, the PEG has a molecular weight between 0.1 kDa and 2 kDa. In other embodiments, the PEG has a molecular weight between 0.1 kDa and 2.5 kDa. In some embodiments, the poly(ethylene glycol) molecule has a molecular weight of about 0.1 kDa to about 10 kDa. In some embodiments, the polyethylene glycol) molecule has a molecular weight of 0.1 kDa to 50 kDa. In some embodiments, the poly(ethylene glycol) has a molecular weight of 0.1 kDa to 2.5 kDa, or 0.2 to 2.2 kDa, or between 0.5 kDa and 2 kDa. For example, the molecular weight of the polyethylene glycol) polymer in some instances is about 0.5 kDa, or about 1 kDa, or about 2 kDa, or about 2.5 kDa. For example, the molecular weight of the poly(ethylene glycol) polymer in some instances is 0.1 kDa or 0.5 kDa or 1 kDa, or 2.5 kDa. In some embodiments the polyethylene glycol) molecule is a branched PEG. In some embodiments the poly(ethylene glycol) molecule is a branched IK PEG. In some embodiments the poly(ethylene glycol) molecule is a branched 2.5K PEG. In some embodiments the poly(ethylene glycol) molecule is a branched 5K PEG. In some embodiments the poly(ethylene glycol) molecule is a linear PEG. In some embodiments the poly(ethylene glycol) molecule is a linear2.5K PEG. In some embodiments the poly(ethylene glycol) molecule is a linear 10K PEG. In some embodiments the polyethylene glycol) molecule is a linear 2K PEG. In some embodiments the polyethylene glycol) molecule is a linear 0.5K PEG. In some embodiments, the molecular weight of the poly(ethylene glycol) polymer is an average molecular weight. In certain embodiments, the average molecular weight is the number average molecular weight (Mn). The average molecular weight may be determined or measured using GPC or SEC, SDS / PAGE analysis, RP-HPLC, mass spectrometry, or capillary electrophoresis.

[0239] The present disclosure also includes phosphate-based linkers with tunable stability for intracellular delivery of drug conjugates disclosed in US 2017 / 0182181, incorporated by reference herein. The phosphate-based linkers comprise a monophosphate, diphosphate, triphosphate, or tetraphosphate group (phosphate group) covalently linked to the distal end of a linker arm comprising from the distal to the proximal direction a tuning element, optionally a spacer element, and a reactive functional group. The phosphate group of the phosphate-based linker is capable of being conjugated to a payload and the reactive functional group is capable of being conjugated to a cell-specific targeting ligand such as an antibody. The general structure of the phosphate-based linkers is: Phosphate group-Tuning element-Optional spacer element-Functional reactive group A phosphate-based linker conjugated to a payload has the general structure: Payload-Phosphate group-Tuning element-Optional spacer element-Functional reactive group and when conjugated to a targeting ligand has the general structure Payload-Phosphate group-Tuning element-Optional spacer element-Targeting ligand. These phosphate-based linkers have a differentiated and tunable stability in blood vs. an intracellular environment (e.g. lysosomal compartment). The rate at which the phosphate group is cleaved in the intracellular environment to release the payload in its native or active form may be affected by the structure of the tuning element with further effects mediated by substitutions of the phosphate group as well as whether the phosphate group is a monophosphate, diphosphate, triphosphate, or tetraphosphate. Further, these phosphate-based linkers provide the ability to construct conjugates such as antibody-drug conjugates in which the propensity of the conjugate to form aggregates is reduced compared to conjugates in which the same payload is conjugated to the antibody or targeting ligand using a linker that is not a phosphate-based linker as disclosed herein.

[0240] In some embodiments, the linker is 0.1 kDa to 5 kDa. In other embodiments, the linker is 0.1 kDa to 2.5 kDa. In other embodiments, the linker or polymer is linear, branched, multimeric,or dendrimeric. In another embodiment, the linker or polymer is a bifunctional or multifunctional linker or a bifunctional or multifunctional polymer.

[0241] In some instances a targeting moiety is linked to a payload via a water-soluble polymer via methods described herein. In some embodiments, the method comprises contacting an isolated targeting moiety comprising a reactive amino acid side chain with a linker. In some instances, a conjugate is synthesized by reacting a functional group present on the targeting moiety with a reactive group present on the linker. In some instances a conjugate is synthesized by reacting a functional group present on the linker with a reactive group present on the payload. In some instances, a therapeutic-linker moiety is conjugated to a targeting moiety. In some instances, a targeting-linker moiety is conjugated to a therapeutic moiety. In some embodiments, the targeting moiety is linked to a linker comprising a water-soluble polymer.

[0242] In other embodiments, the targeting moiety is conjugated to a therapeutic moiety via a linker. In some instances, the linker comprises a polymer. In some embodiments, the targeting moiety is directly or indirectly conjugated to a linker, polymer, or biologically active molecule. In some embodiments, the linker is a cleavable or non-cleavable linker.Linker Design

[0243] Linkers can be categorized by the cleavage conditions, except in the case of traceless linkers and cyclization-cleavage strategies, which are considered separately. The categories of linkers include acid-labile linkers, nucleophile-labile liners, photolabile linkers, traceless linkers, and cyclization linkers.

[0244] Most acid-labile linkers that have been developed for solid-phase synthesis (SPS) of peptides are derived from mono-alkoxy- or multi-alkoxybenzylic protecting groups and from trityl systems. They were designed to enable mild acidic conditions for carboxylic acid cleavage. The strength of the acid required to induce cleavage is related to the electron donor substituents on the aromatic system that stabilizes the transient resin-bound cation. The greater the resonance stabilization conveyed by additional alkoxy groups or aryl rings, the milder the acidic conditions required for cleavage.

[0245] In the Wang support, cleavage of esters involves concentrated trifluoroacetic acid (TFA) or TFA-dichloromethane (DCM) to provide the corresponding carboxylic acids. The polymericbenzylic alcohol is converted into the reactive Wang trichloroacetimidate [X=OC(NH)CCls], which couples with the chosen alcohol in the presence of catalytic amounts of mild Lewis acid. Alcohols are cleaved from the Wang resin by treatment with 10% TFA-DCM for 30 min at ambient temperature. Conditions under which such Wang ethers are stable include Grignard reactions, ester saponification with lithium hydroxide, Weinreb amide formation, Mitsunobu reactions and enolate alkylations, as well as Heck reactions (S. Hanessian, et al., (1998).Tetrahedron Lett., 39:733-736).

[0246] For the Rink linker, libraries of primary amines have been synthesized by the treatment of Rink amine 3 (X=NH2) resin with aldehydes to form al dimines, which are subsequently reacted with Grignard reagents or lithium reagents to yield amines with two sites of diversity (Katrizky AR, et al., (1997). Tetrahedron Lett., 38:7011-7014). These amines are released from resin by treatment with TFA-water-DCM (5:5:90) for 5 h at room temperature. This strategy allows the production of libraries of amines that are not commercially available and which can themselves be used as building blocks. A-Substituted amides are obtained by reducing the above-mentioned aldimines with Na(CN)BHa to the corresponding amines, followed by acylation with acid chlorides or symmetrical anhydrides (Brown EG, at al., (1997). Tetrahedron Lett., 38:8457). The products are cleaved with TFA-water-DCM (5:1:94) for 20 min at room temperature. The immobilization of alcohols and thiols was recently described in two independent articles. One describes the conversion of Rink acid resin 3 (X=OH) to the corresponding Rinkchloride 3 (X=C1). The chloride can then easily be substituted by various nucleophiles, such as primary and secondary amines, anilines and phenols as well as thiols and alcohols.

[0247] Nucleophile-labile linkers are deemed most simple linkers from which carboxylic acids or alcohols can be cleaved are hydroxyalkyl and carboxylic acid resins, respectively. Postcleavage work-up procedures are necessary to obtain pure products. However, the advantage of this kind of anchorage is the high stability under many reaction conditions.

[0248] Oxy carbonyl resin can be synthesized by the conversion of hydroxymethylated polystyrene beads into the chloroformate solid support to which a premixed solution of 4-m ethoxy pyridine and the chosen Grignard reagent in anhydrous THF is added to yield polymer-bound dihydropyridone. The reaction is quenched after a few minutes with 3 M aqueous HC1-THF (1:1), thus removing unreacted pyridinium. Cleavage of the final products is carried out with NaOH in MeOH-THF (Chen C, et al., (1998). Tetrahedron Lett., 3401-3404).

[0249] Solid-supported dimethylformamidine can be treated with several secondary amines, to yield immobilized amidines through an amine exchange reaction. In a model reaction, N-alkylated piperazines were coupled to yield, which in a second step were alkylated with aromatic aldehydes, resulting in. The resulting secondary alcohol was alkylated in a Williamson reaction with a benzylic halide to generate the corresponding ether. Cleavage of the amino ethers could be accomplished using either aqueous hydrazine-acetic acid or lithium aluminum hydride. Since the building blocks employed are commercially available in great variety, libraries synthesized according to this scheme can incorporate a high degree of diversity (Furth PS, et al., (1997). Tetrahedron Lett., 38:6643-6646).

[0250] Binding of amines to polymeric carriers and release upon nucleophilic treatment has been seen in the art. Secondary amines are coupled onto chloro-Wang resin 1 (X=C1) by stirring it with 10 equiv. of amine in A-methylpyrrolidone at 50°C for 17 h. After synthetic transformations, the polymer-bound amine is quaternized with a-chloroethyl chloroformate to yield an intermediate, followed by nucleophilic attack on the chloride anion at the benzylic carbon atom, which releases the secondary amines in refluxing MeOH as their HC1 salts. The same method works with Merrifield resin as well as with Sheppard resin. When other chloroformates are used for the cleavage step, the corresponding carbamates are obtained in excellent yield and purity. This protocol is remarkably selective because primary amines are not cleaved and A-dibenzyl groups elsewhere in the molecule remain unaffected (Conti P, et al., (1997). Tetrahedron Lett., 38:2915-2918).

[0251] Photolysis is a mild cleavage method that is orthogonal to acidic and basic reaction conditions and is attractive for combinatorial drug discovery because the product can be released directly into neutral aqueous solution suitable for immediate screening. An improved photolabile linker for carboxylic acids, amides and reducing carbohydrates was presented by the group of Fraser-Reid, which is hereby incorporated by reference. Cleavage from this linker occurs by irradiation of the resin in THF for 16 h at 365 nm (Rodebaugh R, et al., (1997). Tetrahedron Lett., 38:7653-7656).

[0252] In terms of traceless linkers, Jin et al. demonstrated how resin-boundaryl sulphonates (R=OAr) can lead to the corresponding aryl products upon reductive cleavage using a mixture of NEts-formic acid-Pd(OAc)2-PPh2(CH2)3PPh2in DMF at 110-140°C for 12 h. The yields range from 5% to 85%, depending on the nature of the substrate. The drawback of low yields and difficult work-up again could be compensated for by the fact that it is one of the rare examples of a traceless linker that can be functionalized on the resin by readily available phenols (Jin S, et al., (1998). Tetrahedron Lett., 39:3651-3654).

[0253] Cyclization-cleavage reactions include linkers based on allylic protecting groups, which are cleaved by palladium(O)-catalysed allyl transfer to nucleophiles, have been used for the immobilization of carboxylic acids, amines and alcohols (Kunz H, et al., (1988)). Agnew chem int ed engl, 27:711-713). The advantages of these linkers are the mild cleavage conditions and the orthogonality to many other protecting groups. However, the drawback of this type of linker is the necessity to purify the products by subsequent chromatographic work-up to remove the catalyst and traces of triphenylphosphine oxide, the oxidized ligand of the metal complex. A strategy that combines Ru-catalysed ring-closing metathesis with concomitant cleavage was used for the synthesis of six- and seven-membered ring systems, such as dihydropyrans, pipecolinic acid derivatives or Freidinger lactams (Piscopio AD, et al., (1997). Tetrahedron Lett, 38:7143-7146). The latter is obtained after the Mitsunobu reaction between A-Boc-2,4-dinitrobenzosulphonamide and cinnamic alcohol resin, synthesized according to the procedure developed by Frechet's group in 1971. The resin-bound A-Boc-A-sulphonamide is deprotected from the Boc group and alkylated with secondary alcohols under Fukuyama-Mitsunobu conditions. After removal of the sulphonyl group by treatment with benzylamine, the amine is acylated with racemic A“-Boc-allylglycine, which provides the penultimate resin-bound diene 37, R3=NHBoc. Cyclization-cleavage with Grubbs' ruthenium catalyst yields the corresponding cycloalkene, in this case, a Freidinger lactam (Piscopio AD, et al., (1997).Tetrahedron Lett, 38:2667-2670). As with the Pd-catalysed cleavage of allylic linkages, the products contain, besides the catalyst itself, impurities of tricyclohexane phosphine oxide and have to be purified by chromatographic work-up (Eggenweiler HM, (1998). Drug Discovery Today, 3(12):552-560).Bioconjugation

[0254] Chemical conjugation can occur by reacting a nucleophilic reactive group of one compound to an electrophilic reactive group of another compound. In some embodiment, the targeting moiety is conjugated to the therapeutic moiety either by reacting a nucleophilic reactive group on the targeting moiety with an electrophilic reactive group on a linker, or by reacting an electrophilic reactive group on the targeting moiety with a nucleophilic reactive moiety on a therapeutic moiety. In some embodiments, a group links the targeting moiety and therapeutic moiety together, the targeting moiety and / or therapeutic moiety can be conjugated to the linker either by reacting a nucleophilic reactive moiety on targeting moiety and / or therapeutic moiety with an electrophilic reactive group on the linker, or by reacting an electrophilic reactive group on targeting moiety and / or therapeutic moiety with a nucleophilic reactive group on the linker. Nonlimiting examples of nucleophilic reactive groups include amino, thiol, and hydroxyl.Nonlimiting examples of electrophilic reactive groups include carboxyl, acyl chloride, anhydride, ester, succinimide ester, alkyl halide, sulfonate ester, maleimido, haloacetyl, and isocyanate. In embodiments where the targeting moiety and therapeutic moiety are conjugated together by reacting a carboxylic acid with an amine, an activating agent can be used to form an activated ester of the carboxylic acid.

[0255] Targeting moi eties, reactive moi eties, and therapeutic moi eties may be linked to one another, directly or through linkers, via suitable click chemistry reactions. Exemplary click chemistry reactions include, but are not limited to formation of esters, thioesters, amides, imines, or oximes; nucleophilic displacement reactions (e.g., nucleophilic displacement of a halide or ring opening of strained ring systems); azide-alkyne Huisgon cycloaddition; thiol-yne addition; and Michael additions (e.g., maleimide addition). In various embodiments, a provided targeting moiety, reactive moiety, therapeutic moiety, or linker may comprise one or more reactive groups as “click chemistry handles” that can join two molecular entities. For example, reactive groups provided herein are capable of joining a reactive moiety directly to a targeting moiety; reactive groups provided herein are capable of joining a targeting moiety to a linker and / or a therapeutic moiety to a linker, and thereby j oining the targeting moiety and the therapeutic moiety.Exemplary coupling reactions between reactive groups include, but are not limited to, formation of esters, thioesters, amides, imines, or oximes; nucleophilic displacement reactions; azidealkyne Huisgon cycloaddition; thiol-yne addition; and Michael additions. Exemplary reactivegroups include: acetals, ketals, hemiacetals, hemiketals, carboxylic acids, strong non-oxidizing acids, strong oxidizing acids, weak acids, acrylates and acrylic acids, acyl halides, sulfonyl halides, chloroformates, alcohols and polyols, aldehydes, alkynes with or without acetylenic hydrogen, amides, imides, amines, phosphines, pyridines, anhydrides, aryl halides, azo, diazo, azido, hydrazines, strong bases, weak bases, carbamates, carbonate salts, chlorosilanes, conjugated dienes, cyanides, diazonium salts, epoxides, esters, sulfate esters, phosphate esters, thiophosphate esters borate esters, ethers, soluble fluoride salts, fluorinated organic compounds, halogenated organic compounds, halogenating agents, aliphatic saturated hydrocarbons, aliphatic unsaturated hydrocarbons, isocyanates and isothiocyanates, ketones, metal hydrides, metal alkyls, metal aryls, silanes, alkali metals, nitrate and nitrite compounds, inorganic, nitrides, phosphides, carbides, silicides, nitriles, nitro, nitroso, nitrate, nitrite compounds, organic, nonredox-active inorganic compounds, organometallics, oximes, peroxides, organic, phenolic salts, phenols and cresols, polymerizable compounds, quaternary ammonium and phosphonium salts, strong reducing agents, weak reducing agents, acidic salts, basic salts, siloxanes, inorganic sulfides, organic sulfides, sulfite and thiosulfate salts, sulfonates, phosphonates, organic thiophosphonates, thiocarbamate esters and salts, and dithiocarbamate esters and salts.

[0256] Intra- and intermolecular cross-linking of peptides and proteins have been recognized as an important strategy to control protein conformations, enhance protein stability, study proteinprotein interactions, and improve pharmacological properties. Disulfide bonds formed between two cysteine or tyrosine side chains serve as a part of natural protein’s secondary or tertiary structures, providing the essential framework for protein function and / or stability (Kubota K, et al., (2018). J Am Chem Soc., 140(8):3128-3133). It has been reported that aryldialkylphosphinebased palladium oxidative addition complexes are able to mediate cysteine or tyrosine- or lysineselective arylations, but bis-palladium complexes have been applied to prepare stapled peptides through the construction of intramolecular cysteine -cysteine and lysine-lysine linkages (Rojas AJ, et al., (2017). S. L. Chem. Sci., 8:4257-4263; Lee HG, et al., (2017). Chem Int. Ed, 56:3177-3181). With the use of this complex bearing an appropriate electrophilic functional group, this bifunctional reagent could react with unprotected peptides or proteins through palladium-mediated cysteine arylation, after which the bound electrophilic group could react with a proximal nucleophilic residue to furnish the desired cross-link (Vinogradova EV, et al., (2015). Nature, 526:687-691). To ensure the avoidance of hydrolysis of the bioconjugation handle priorto the cross-linking step, the electrophilic functional group is required to achieve the high selectivity level transformation. The SH group on the cysteine displaces a leaving group on palladium, ensuring a reductive elimination to provide an S-arylated intermediate. Subsequent nucleophilic attack by a proximal lysine residue is able to provide the cross-linked peptide, as seen in Figure X (Vinogradova EV, et al., (2015). Nature, 526:687-691).

[0257] A study by Karampel et al. utilized an ester bond to attach gemcitabine with a glutaryl linker, which is further attached to the peptide ([D-Lys6]-GnRH) via an amide bond. The gemcitabine structure includes a primary 5 ’-OH and secondary 3 ’-OH, which enabled the conjugation of the targeting peptide at either functional group. Overall, the GSG conjugate allowed higher and sustained levels of gemcitabine in blood for a longer time compared to the direct administration of gemcitabine in mice (Karampelas T, et al., (2014). Bioconjug Chem, 25(4):813-823).

[0258] Another study utilized ester / amide bonds to conjugate paclitaxel to a peptide (Angiopep-2) via a succinyl linker. Here, three molecules of paclitaxel are covalently linked to a single peptide via the side chain of two lysine residues and an N-terminus amine of the peptide to give a peptide-drug conjugate ANG1005. The ester bond in the conjugate is cleaved by the esterases present in lysosomes releasing paclitaxel in the brain. ANG1005 was designed to overcome the main challenge of paclitaxel, which is low BBB permeability due to the multidrug resistance efflux pump P-glycoprotein (P-gp) expression in brain tumor cells. Brain uptake of the conjugate in mouse brains using direct perfusion with [125I]ANG1005 or [3H]paclitaxel showed that total brain uptake of the conjugate was 4.5-fold higher compared to free paclitaxel, and showed that the conjugate could bypass the P-gp system at the BBB when assessed with mdr la-deficient mice (Regina A, et al., (2008). Br J Pharmacol, 155(2): 185-197).

[0259] Another study employed ester and amide bonds to synthesize PDCs of Dox that showed promising in vitro results. In conjugate 3, the primary alcohol at position 14 of Dox was reacted with a glutaryl linker to form an ester, and the other end of the linker was attached to the lysine side chain of the peptide (18-4) via an amide bond. For conjugate 4, the 3’ amino group of Dox was used to form an amide bond with a glutaryl linker, and the other linkage (amide) was the same as conjugate 3. Conjugate 3 had a short half-life in human serum (2 h), most likely due to fast hydrolysis of the ester bond. In contrast, conjugate 4 was relatively stable with a half-life of48 h when incubated with human serum (Soudy R, et al., (2013). J Med Chem, 56(19):7564-7573).

[0260] In general, carbon electrophiles are susceptible to attack by complementary nucleophiles, including carbon nucleophiles, wherein an attacking nucleophile brings an electron pair to the carbon electrophile in order to form a new bond between the nucleophile and the carbon electrophile.

[0261] Non-limiting examples of carbon nucleophiles include, but are not limited to alkyl, alkenyl, aryl and alkynyl Grignard, organolithium, organozinc, alkyl-, alkenyl, aryl- and alkynyl- tin reagents (organostannanes), alkyl-, alkenyl-, aryl- and alkynyl-borane reagents (organoboranes and organoboronates); these carbon nucleophiles have the advantage of being kinetically stable in water or polar organic solvents. Other non-limiting examples of carbon nucleophiles include phosphorus ylids, enol and enolate reagents; these carbon nucleophiles have the advantage of being relatively easy to generate from precursors well known to those skilled in the art of synthetic organic chemistry. Carbon nucleophiles, when used in conjunction with carbon electrophiles, engender new carbon-carbon bonds between the carbon nucleophile and carbon electrophile.

[0262] Non-limiting examples of non-carbon nucleophiles suitable for coupling to carbon electrophiles include but are not limited to primary and secondary amines, thiols, thiolates, and thioethers, alcohols, alkoxides, azides, semi-carb azides, and the like. When used in conjunction with carbon electrophiles, these non-carbon nucleophiles typically generate heteroatom linkages (C-X-C), wherein X is a heteroatom, including, but not limited to, oxygen, sulphur, or nitrogen.Peptide Synthesis

[0263] Once the sequence of amino acids is selected, synthesis of the polypeptide can be achieved using standard peptide synthesis methodology. Formation of peptide bonds and polypeptide synthesis are techniques well-known to one skilled in the art, and encompass both solid phase and solution phase methods; see generally, Bodanszky and Bodanszky, The Practice of Peptide Synthesis, Springer-Verlag, Berlin, 1984; Atherton and Sheppard, SolidPhase Peptide Synthesis: A Practical Approach, IRL Press at Oxford University Press Oxford, England, 1989, and Stewart and Young, Solid phase Peptide Synthesis, 2nd edition, PierceChemical Company, Rockford, 1984, the entire contents of each of which are incorporated herein by reference. In both solution phase and solid phase techniques, the choice of the protecting groups must be considered, as well as the specific coupling techniques to be utilized. For a detailed discussion of peptide synthesis techniques for solution phase and solid phase reactions, see, Bioorganic chemistry: Peptides and Proteins, Hecht, Oxford University Press, New York: 1998, the entire contents of which are incorporated herein by reference.

[0264] The peptides for use in the methods of the present disclosure can be made using conventional solid-phase synthesis from amino acid starting materials, which may include appropriate protecting groups as described herein. These methods for making peptides are well-known in the art.Peptide Storage and Use pH

[0265] While some peptides and proteins are stable at 4 °C, we recommend -20 °C for shortterm storage (1-2 weeks) and -80 °C for longer storage. Peptide sequences containing C, M, or W are prone to air oxidation. All cysteine-containing peptides will slowly oxidize with time, the rate of which is largely dependent on the sequence and storage conditions. It is recommended to purge the air out of the vial and replace it with a blanket of nitrogen or argon. The shelflife of peptide solutions is limited. Proteins and peptides containing N, Q, C, M and W are unstable when stored in solution. Using sterile buffers (pH 5-6) and freezing the aliquots will prolong the storage life of the peptide. Storage at -20 °C or colder is optimal. In general, peptide solutions are stable for up to a week at 4 °C. However, if the peptide sequence has inherent instability, it might be better to freeze the solution when not in use. Peptide solutions at pH>8 should also be frozen when not in use. The most effective way to prevent or minimize peptide degradation is to store the peptide in lyophilized / powder form at -20 °C or preferably at -80 °C. If the peptide is in solution, freeze thaw cycles should be avoided by freezing individual aliquots. Exposure to pH>8 should be avoided. However, if it is necessary to dissolve peptides at pH>8, the solutions should be chilled. Finally, prolonged exposure of lyophilized peptides and solutions (especially at high pH) to atmospheric oxygen should be minimized.Methods of Manufacturing and Reagents Used Therein

[0266] Synthetic schemes for preparing intermediates and compounds of the disclosure are described below. The illustrative schemes described are not intended to limit the methodsavailable to prepare the compounds or imply a specific ordering of steps is required, nor are any of the variable groups intended to limit any of the claims described herein.

[0267] Provided herein are methods of manufacturing compounds of the present disclosure, including reagents and intermediates.

[0268] In some embodiments of manufacturing a compound of the present disclosure, the method of manufacturing comprises reacting a compound of Formula (RXG-II) with a targeting moiety (T) according to present disclosure,wherein:R’ is as defined throughout this disclosure;M is a metal atom selected from palladium, nickel gold, zinc, and copper; andLgis a metal ligand.

[0269] In some embodiments, M is palladium, copper, nickel, gold, or zinc.

[0270] In some embodiments, M is copper.

[0271] In some embodiments, M is nickel.

[0272] In some embodiments, M is zinc.

[0273] In some embodiments, M is palladium.

[0274] In some embodiments, M is palladium(II).

[0275] In some embodiments, M is palladium(O).

[0276] In some embodiments, M is gold (i.e., Au).

[0277] In some embodiments, M is Au(0).

[0278] In some embodiments, M is Au(I).

[0279] In some embodiments, M is Au(III).

[0280] In some embodiments, Lgis monodentate.

[0281] In some embodiments, Lgis bidentate.

[0282] In some embodiments, Lgis tridentate.

[0283] In some embodiments, Lgis neutral.

[0284] In some embodiments, Lgis anionic.

[0285] In some embodiments, each Lgis independently either neutral and / or anionic.

[0286] In some embodiments, Lgis a phosphine.

[0287] In some embodiments, Lgis an oxazole-phosphine.

[0288] In some embodiments, Lgis an amine.

[0289] In some embodiments, Lgis a bidentate phosphine-amine.

[0290] In some embodiments, qq is an integer selected from 0, 1, 2, 3, 4, 5.

[0291] In some embodiments, qq is 0.

[0292] In some embodiments, qq is 1.

[0293] In some embodiments, qq is 2.

[0294] In some embodiments, qq is 3.

[0295] In some embodiments of manufacturing a compound according the present disclosure comprises reacting a compound of Formula (RXG-III) with a targeting moiety (T),wherein:** represents the point of attachment to L or Tx;R” is as defined throughout this disclosure;M is a metal atom selected from palladium, nickel, zinc, gold, and copper; andLgis a metal ligand (e.g., a phosphine).

[0296] In some embodiments, M is palladium, copper, nickel, gold or zinc. In some embodiments, M is palladium. In some embodiments, M is palladium(II). In some embodiments, M is palladium(O).

[0297] In some embodiments, M is palladium, copper, nickel, gold, or zinc.

[0298] In some embodiments, M is copper.

[0299] In some embodiments, M is nickel.

[0300] In some embodiments, M is zinc.

[0301] In some embodiments, M is palladium.

[0302] In some embodiments, M is palladium(II).

[0303] In some embodiments, M is palladium(O).

[0304] In some embodiments, M is gold (i.e., Au).

[0305] In some embodiments, M is Au(0).

[0306] In some embodiments, M is Au(I).

[0307] In some embodiments, M is Au(III).

[0308] In some embodiments, Lgis monodentate.

[0309] In some embodiments, Lgis bidentate.

[0310] In some embodiments, Lgis tridentate.

[0311] In some embodiments, Lgis neutral.

[0312] In some embodiments, Lgis anionic.

[0313] In some embodiments, each Lgis independently either neutral and / or anionic.

[0314] In some embodiments, Lgis a phosphine.

[0315] In some embodiments, Lgis an oxazole-phosphine.

[0316] In some embodiments, Lgis an amine.

[0317] In some embodiments, Lgis a bidentate phosphine-amine.

[0318] In some embodiments, Lgis an oxazole-phosphine.

[0319] In some embodiments, Lgis a phosphine. In some embodiments, Lg is an oxazole-phosphine.

[0320] In some embodiments, qq is an integer selected from 0, 1, 2, 3, 4, 5.

[0321] In some embodiments, qq is 0.

[0322] In some embodiments, qq is 1.

[0323] In some embodiments, qq is 2.

[0324] In some embodiments, qq is 3.

[0325] In some embodiments, qq is an integer selected from 0, 1, 2, 3, 4, 5.

[0326] In some embodiments of Formulae RXG-II, Ila, III, or Illa, Lg is selected from:Table 1. Example electrophilic donation compounds (reagents) of the present disclosure.

[0327] It is understood that any of the compounds of this disclosure may be present in a salt form. In some cases, the salt form of the compound is a pharmaceutically acceptable salt.

[0328] “Pharmaceutically acceptable salt” includes both acid and base addition salts.Pharmaceutically acceptable salts include the acid addition salts (e.g., formed with the free amino groups of the compound) and which are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins.In Situ Covalency

[0329] An established approach to modulating pharmacokinetic and pharmacodynamic profiles, to date only applied to specific small-molecule drugs, is the exploitation of covalent binding of the small molecule to the target of interest.

[0330] In further embodiments, there are ways known in the art to analytically test, assessing directly or indirectly, the pharmacodynamics of the drug in the transgenic mammal to which it has been administered. In still another embodiment, there are ways known in the art to analytically test, assessing directly or indirectly, the pharmacokinetics of the drug in the transgenic mammal to which it has been administered. These techniquesinclude pharmacokinetic assays, pharmacodynamic assays (including measurement of efficacy),toxicological assays, as well as studies of absorption, distribution, excretion and metabolism. When used in place of standard mammals, transgenic mammals provide data that are more predictive of a drug's behavior in a selected species of primate, particularly humans. When used in addition to standard mammals, the differences between transgenic and standard mammals, with respect to drug behavior, indicate the potential role of the transgene in the metabolism of the specific drug under study.

[0331] Pharmacokinetic assays determine the elimination and metabolism of compounds within the body of a mammal over a time course. For example, transgenic mammals according to the present disclosure are administered compound and then blood, or other body fluids or tissues, or excrement (urine or faeces) are collected at various time points following administration.Concentrations of compound(s), or metabolites thereof, are determined by an appropriate analytical method (for example, HPLC using spectrophotometric determination of analyte). Kinetic data are then typically analyzed by graphical and computational means.

[0332] Pharmacodynamic assays determine the activity of compounds within the body of a mammal, normally over a time course. Following administration, the activity of the compound(s) against the target can either use “whole-body” assays (e.g. blood pressure, respiratory rate, electrocardiogram, electromyogram, neurological activity by measuring electromagnetic pulses, etc.) or by imaging techniques (e g. positron emission spectroscopy, nuclear magnetic resonance imaging, echography, etc). Activity of the compound can also be determined by biochemical means. This can include either direct measurement of interaction of the compound with the target, or by measurement of an upstream or downstream marker indicativeof pharmacodynamic activity. For example, according to the present disclosure, transgenic mammals are administered compound and then blood, or other body fluids or tissues, or excrement (urine or feces) are collected at various time points following the administration. Direct measurement of target or marker activity in biological samples can be made by various means (e.g. enzyme assays to determine target or marker activity, Western blot or ELISA techniques to determine either target abundance and / or activity, Northern blot analysis to determine target or marker mRNA levels etc.). Indirect measurement of the target or marker can include the determination of substrate or product levels by various analytical methods (e.g.HPLC using spectrophotometric determination of analyte). Testing of pharmacodynamic activity can also involve challenges to the mammal (e.g. artificially raising blood pressure by chemical ormechanical means, change in diet to promote physiological changes, surgical intervention to produce a disease state, injection of infectious agents etc.). Pharmacodynamic data are then typically analyzed by graphical and computational means, often correlated to tissue compound levels. Included in pharmacodynamic studies is the measurement of drug efficacy in the treatment of disease. For example, the dose required to inhibit tumor growth or eliminate infectious agents.

[0333] As disclosed in W02005055950, one solution to the problem of providing cost-effective glycopeptide therapeutics has been to provide peptides with longer in vivo half-lives. For example, glycopeptide therapeutics with improved pharmacokinetic and pharmacodynamic properties have been produced by attaching synthetic polymers to the peptide backbone. An exemplary polymer that has been conjugated to peptides is poly(ethylene glycol) ("PEG"). The use of PEG to derivatize peptide therapeutics has been demonstrated to reduce the immunogenicity of the peptides. For example, U. S. Pat. No. 4,179,337 (Davis et al.) discloses non-immunogenic polypeptides such as enzymes and peptide hormones coupled to polyethylene glycol (PEG) or polypropylene glycol. In addition to reduced immunogenicity, the clearance time in circulation is prolonged due to the increased size of the PEG conjugate of the polypeptides in question.

[0334] In one embodiment, a PEG-conjugated peptide is a cleavable linker. In another embodiment, a PEG-conjugated peptide is a non-cleavable linker.

[0335] In some aspects, the desired pharmacodynamic effects of therapeutic macromolecules include stimulating or inhibiting a specific response. In some embodiments, the medicinal effect is, but is not limited to, the production or degradation of cytokines, chemokines, signaling molecules or other molecules; inducing proliferation or death of a particular cell type; a particular cell type. Maturation or localization; interaction with enzymes, structural proteins, carrier proteins or receptor proteins; modulation of activity of enzymes, structural proteins or receptor proteins, etc. In some embodiments, the pharmacodynamic effect is to reduce the production of cytokines, such as inflammation-related cytokines such as TNF, IL-1. In some embodiments, the pharmacodynamic effect is to reduce the activity of cytokines. In some embodiments, the pharmacodynamic effect is to reduce the production of unwanted molecules. In some embodiments, the pharmacodynamic effect is to increase the degradation of unwantedmolecules, such as uric acid crystals. In some embodiments, the pharmacodynamic effect is the activity of the enzyme.

[0336] The pharmacodynamic effects of therapeutic macromolecules can be assessed by standard methods. In some aspects, the increased pharmacodynamic effect is achieved through the reduction of inflammation. The level of inflammation is not limited, but the following exemplary methods: scoring inflammatory signs such as redness and swelling; scoring joint signs such as motility, pain or joint destruction; swelling, blood pressure, shortness of breath, etc. Scoring of anaphylactic signs; detecting and / or quantifying cell infiltration by histology, immunohistochemistry, flow cytometry; measuring the concentration of proteins or inflammation-related cytokines such as TNF, IL-1 by ELISA, the expression of a gene or inflammation-related gene assessed by transcriptional analysis; measuring the activity of inflammation-related cytokines, and the like.

[0337] In some aspects, the pharmacodynamic effect is increased through the reduction or degradation of unwanted molecules. In some embodiments, pharmacodynamic effects can be assessed by quantifying, but not limited to, unwanted molecules in tissues or blood samples by methods such as ELISA. In some embodiments, pharmacodynamic effects can be assessed by quantifying molecules produced by the degradation of unwanted molecules, through the use of ELISA. In some aspects, the pharmacodynamic effect is shown through the activity of an enzyme that was not previously present or was not properly present. In some embodiments, the activity of the enzyme can be assessed by detecting the presence or concentration of a product of the enzyme activity.

[0338] In some aspects, reduced doses of therapeutic macromolecules are administered to produce increased pharmacodynamic effects. A reduced dose of therapeutic polymer for this purpose is one of the therapeutic polymers that achieves a medicinal effect in the presence of an anti -therapeutic polymer antibody response by concomitant administration of an immunosuppressive agent dose. The dose required to achieve a similar pharmacological effect with a therapeutic polymer when not co-administered with an immunosuppressant dose in the presence of an anti -therapeutic polymer antibody response is reduced. Reduced doses are determined by administering therapeutic macromolecules at specific doses in the presence of anti -therapeutic macromolecular antibody responses, along with immunosuppressive doses, andassessing the pharmacodynamic effects. The pharmacodynamic effects may then be compared to the pharmacodynamic effects achieved through the administration of the therapeutic macromolecules in the presence of an anti-therapeutic macromolecular antibody response without the combined administration of immunosuppressive agent doses. A smaller dose that achieves a similar pharmacodynamic effect, as determined by such comparison, is the reduced dose.

[0339] The targeting polypeptide moiety and the therapeutic moiety may be directly joined via a peptide bond. In some embodiments, they may be joined via a half-life modulator. In preferred embodiments, the half-life modulator is a polypeptide. Accordingly, the half-life modulator can have two termini, an N-terminus and a C-terminus. In some embodiments, the half-life modulator is joined at one terminus via a peptide bond to the targeting polypeptide domain and is joined at the other terminus via a peptide bond to the activator domain. In certain embodiments, the linker is joined at the N-terminus to the C-terminus of the targeting polypeptide domain and at the C-terminus to the N-terminus of the activator domain. In other embodiments, the linker is joined at the C-terminus to the targeting polypeptide domain and at the N-terminus to the activator domain. Yet, in other embodiments, the half-life modulator is joined at one of the termini of the bi-specific protein. For example, in some embodiments, the half-life modulator is joined at the C-terminus to the N-terminus of the activator domain. In other embodiments, the half-life modulator is joined at the C-terminus of the targeting moiety. In other embodiments, the half-life modulator can be joined at the N-terminus to the C-terminus of the activator domain. Yet in other embodiments, the half-life modulator can be joined at the N-terminus to the C-terminus of the targeting moiety.

[0340] In some embodiments, the half-life modulator can be modified by glycosylation of one or more glyscosylation site present in the half-life modulator. For example, the amino acids asparagine, serine, threonine can be added or removed to alter the glycosylation of the halflife modulator. In some embodiments, glycosylation of the half-life modulator in the bi-specific protein or antibody fragment thereof can modulate the half-life of the bi-specific protein or antibody fragment thereof. In some embodiments, the half-life modulator sequence is modified to reduce glycosylation. Such modification can comprise the substitution of Asn (N) by Gin (Q) or Ala (A), and / or the substitution of Ser (S) or Thr (T) by Ala (A).

[0341] Human serum albumin (HSA) has a naturally long serum half-life, in part due to its binding to FcRN and recycling. HSA is the most abundant protein in the blood and has a demonstrated safety in humans. In some embodiments, the asparagine at position 503 of HSA, which may be deamidated and decrease half-life, can be removed by the N503Q substitution. In some embodiments, the cysteine C34 of HSA may be substituted to serine or alanine (S or A), or non-canonical amino acids to remove the free cysteine and minimize alternate disulfide-bond formation. In some embodiments, the half-life modulator is a modified version of the domain III (mHSA dill) of a modified HSA with the N503Q substitution and an additional terminal glycine. Such a modified version retains the HSA property of binding to FcRn and increased serum halflife.

[0342] In general, the efficacy of recombinant polypeptide pharmaceuticals is often limited by the rapid intrinsic pharmacokinetics of the polypeptide itself, leading to rapid clearance of the polypeptide. An additional advantage of an exemplary antigen-binding polypeptide is prolonged pharmacokinetic elimination half-life due to having a half-life extending domain, e.g., a binding domain that specifically binds HSA. In this regard, exemplary antigen-binding polypeptides have extended serum residence to half-lives. Exemplary polypeptide constructs of this motif, in some embodiments, have half-lives of about 2, 3, about 5, about 7, about 10, about 12, or about 14 days. This contrasts favorably with other binding proteins such as BiTE or DART molecules, which have relatively short elimination half-lives. For example, the BiTE CD19xCD3 bispecific scFv-scFv fusion molecule requires continuous intravenous infusion (iv) drug delivery due to its short elimination half-life. The longer endogenous half-life of exemplary antigen-binding polypeptides of the present disclosure ameliorate this shortcoming, thereby leading to lower dose pharmaceutical formulations, reduced periodic dosing, and / or novel approaches incorporating compounds, which allows for increased therapeutic potential.Table 2. PSMA targeted covalent peptidesTable 3. PSMA-targeted peptide intermediatesor a pharmaceutically acceptable salt thereof.Table 4. Synthetic PTHrP intermediatesTable 5. Synthetically modified residues of amino acid peptide targeting moieties. Example covalent modifications made to a targeting moiety (T) of Formulae I, II, I’, II’, III’, IV’, V’, VI’, or VII’.Table 6. Exemplary amino acid sequences of the present disclosure.Table 7. Example sequences of the disclosure.wherein & represents the point of attachment to R’, R”, and / or L, wherein R’, R”, and L are as defined above.Table 8. Example sequences of the disclosure.[Aib] = 2-aminoisobutyric acid, [hArg] = L-homoarginine

[0343] It will be understood that in some embodiments of the present disclosure, a suitable residue or residues of any one of the sequences of Table 8 can be covalently modified and is attached to R’, R”, and / or L, wherein R’, R”, and / or L are as defined above.

[0344] In some embodiments, the targeting moiety and / or therapeutic moiety of compounds and / or conjugates described herein comprise an amino acid sequence have 95%, 96%, 97%, 98%, or 99% sequence identity to any of the particular sequences or disclosed herein. In some embodiments, the targeting moiety and / or therapeutic moiety of compounds and / or conjugates described herein comprise an amino acid sequence having 1, 2, 3, 4, or 5 substitutions, insertions, or deletions relative thereto.Methods of Treatment

[0345] Conjugates described herein may be used to treat conditions and / or diseases. In some instances, the disease comprises a proliferative disease. In some instances, the proliferative disease comprises cancer. In some instances, the cancer comprises one or more tumors. In someinstances, the cancer comprises solid or liquid tumors. In some instances, conjugates are administered to kill or inhibit growth of a rapidly dividing cell, such as a tumor cell. In some instances, a method of treating a proliferative disease or condition in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a conjugate described herein. In some embodiments, the proliferative disease or condition is a cancer. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the solid tumor cancer is bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer. In some instances, the disease comprises PCa (prostate cancer), CRPCa (castration resistant prostate cancer), solid tumors (neovasculature), NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), ESCC (esophageal cancer) GC (gastric cancer), CRC (colorectal cancer), SCLC (small cell lung cancer), MPM (mesothelioma), PDAC (Pancreatic ductal adenocarcinoma), ALL (Acute Lymphoblastic Leukemia), AML (Acute Myeloid Leukemia), MDS (Myelodysplastic syndromes), MS high tumors, melanoma, DLBCL (diffuse large B cell lymphoma), endometrial cancer, cervical cancer, bladder cancer, BrCa (breast cancer), TNBC (triple negative breast cancer), NE-PCa (Neuroendocrine prostate cancer), GBM (glioblastoma), and RCC (Renal cell carcinoma).

[0346] Conjugates described herein can be used in the treatment of infectious diseases. These infectious diseases include, but are not limited to, Acinetobacter infections, Actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (Acquired immune deficiency syndrome), Amebiasis, Anaplasmosis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacillus cereus infection, Bacterial pneumonia, Bacterial vaginosis, Bacteroides infection, Balantidiasis, Baylisascaris infection, BIC virus infection, Black piedra, Blastocystis hominis infection, Blastomycosis, Bolivian hemorrhagic fever, Borrelia infection, Botulism (and Infant botulism), Brazilian hemorrhagic fever, Brucellosis, Burkholderia infection, Buruli ulcer, Calicivirus infection (Norovirus and Sapovirus), Campylobacteriosis, Candidiasis (Moniliasis; Thrush), Cat-scratch disease, Cellulitis, Chagas Disease (American trypanosomiasis), Chancroid, Chickenpox, Chlamydia, Chlamydophila pneumoniae infection, Cholera, Chromoblastomycosis, Clonorchiasis, Clostridium difficile infection, Coccidioidomycosis, Colorado tick fever,Common cold (Acute viral rhinopharyngitis; Acute coryza), Creutzfeldt- Jakob disease, Crimean-Congo hemorrhagic fever, Cryptococcosis, Cryptosporidiosis, Cutaneous larva migrans, Cyclosporiasis, Cysticercosis, Cytomegalovirus infection, Dengue fever, Dientamoebiasis, Diphtheria, Diphyllobothriasis, Dracunculiasis, Ebola hemorrhagic fever, Echinococcosis, Ehrlichiosis, Enterobiasis (Pinworm infection), Enterococcus infection, Enterovirus infection, Epidemic typhus, Erythema infectiosum (Fifth disease), Exanthem subitum, Fasciolopsiasis, Fasciolosis, Fatal familial insomnia, Filariasis, Food poisoning by Clostridium perfringens, Free-living amebic infection, Fusobacterium infection, Gas gangrene (Clostridial myonecrosis), Geotrichosis, Gerstmann-Straussler-Scheinker syndrome, Giardiasis, Glanders, Gnathostomiasis, Gonorrhea, Granuloma inguinale (Donovanosis), Group A streptococcal infection, Group B streptococcal infection, Haemophilus influenzae infection, Hand, foot and mouth disease (HFMD), Hantavirus Pulmonary Syndrome, Helicobacter pylori infection, Hemolytic-uremic syndrome, Hemorrhagic fever with renal syndrome, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex, Histoplasmosis, Hookworm infection, Human bocavirus infection, Human ewingii ehrlichiosis, Human granulocytic anaplasmosis, Human metapneumovirus infection, Human monocytic ehrlichiosis, Human papillomavirus infection, Human parainfluenza virus infection, Hymenolepiasis, Epstein-Barr Virus Infectious Mononucleosis (Mono), Influenza, Isosporiasis, Kawasaki disease, Keratitis, Kingella kingae infection, Kuru, Lassa fever, Legionellosis (Legionnaires’ disease), Legionellosis (Pontiac fever), Leishmaniasis, Leprosy, Leptospirosis, Listeriosis, Lyme disease (Lyme borreliosis), Lymphatic filariasis (Elephantiasis), Lymphocytic choriomeningitis, Malaria, Marburg hemorrhagic fever, Measles, Melioidosis (Whitmore’s disease), Meningitis, Meningococcal disease, Metagonimiasis, Microsporidiosis, Molluscum contagiosum, Mumps, Murine typhus (Endemic typhus), Mycoplasma pneumonia, Mycetoma, Myiasis, Neonatal conjunctivitis (Ophthalmia neonatorum), (New) Variant Creutzfeldt-Jakob disease (vCJD, nvCJD), Nocardiosis, Onchocerciasis (River blindness), Paracoccidioidomycosis (South American blastomycosis), Paragonimiasis, Pasteurellosis, Pediculosis capitis (Head lice), Pediculosis corporis (Body lice), Pediculosis pubis (Pubic lice, Crab lice), Pelvic inflammatory disease, Pertussis (Whooping cough), Plague, Pneumococcal infection, Pneumocystis pneumonia, Pneumonia, Poliomyelitis, Prevotella infection, Primary amoebic meningoencephalitis, Progressive multifocal leukoencephalopathy, Psittacosis, Q fever, Rabies, Rat-bite fever,Respiratory syncytial virus infection, Rhinosporidiosis, Rhinovirus infection, Rickettsial infection, Rickettsialpox, Rift Valley fever, Rocky mountain spotted fever, Rotavirus infection, Rubella, Salmonellosis, SARS (Severe Acute Respiratory Syndrome), Scabies, Schistosomiasis, Sepsis, Shigellosis (Bacillary dysentery), Shingles (Herpes zoster), Smallpox (Variola), Sporotrichosis, Staphylococcal food poisoning, Staphylococcal infection, Strongyloidiasis, Syphilis, Taeniasis, Tetanus (Lockjaw), Tinea barbae (Barber’s itch), Tinea capitis (Ringworm of the Scalp), Tinea corporis (Ringworm of the Body), Tinea cruris (Jock itch), Tinea manuum (Ringworm of the Hand), Tinea nigra, Tinea pedis (Athlete’s foot), Tinea unguium (Onychomycosis), Tinea versicolor (Pityriasis versicolor), Toxocariasis (Ocular Larva Migrans), Toxocariasis (Visceral Larva Migrans), Toxoplasmosis, Trichinellosis, Trichomoniasis, Trichuriasis (Whipworm infection), Tuberculosis, Tularemia, Ureaplasma urealyticum infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Viral pneumonia, West Nile Fever, White piedra (Tinea blanca), Yersinia pseudotuberculosis infection, Yersiniosis, Yellow fever, Zygomycosis.

[0347] The binding molecules, more therapeutic moiety described in this patent that are against pathogenic strains include, but are not limit, Acinetobacter baumannii, Actinomyces israelii, Actinomyces gerencseriae and Propionib acterium propionicus, Trypanosoma brucei, HIV (Human immunodeficiency virus), Entamoeba histolytica, Anaplasma genus, Bacillus anthracis, Arcanob acterium haemolyticum, Junin virus, Ascaris lumbricoides, Aspergillus genus, Astroviridae family, Babesia genus, Bacillus cereus, multiple bacteria, Bacteroides genus, Balantidium coli, Baylisascaris genus, BK virus, Piedraia hortae, Blastocystis hominis, Blastomyces dermatitides, Machupo virus, Borrelia genus, Clostridium botulinum, Sabia, Brucella genus, usually Burkholderia cepacia and other Burkholderia species, Mycobacterium ulcerans, Caliciviridae family, Campylobacter genus, usually Candida albicans and other Candida species, Bartonella henselae, Group A Streptococcus and Staphylococcus, Trypanosoma cruzi, Haemophilus ducreyi, Varicella zoster virus (VZV), Chlamydia trachomatis, Chlamydophila pneumoniae, Vibrio cholerae, Fonsecaea pedrosoi, Clonorchis sinensis, Clostridium difficile, Coccidioides immitis and Coccidioides posadasii, Colorado tick fever virus, rhinoviruses, coronaviruses, CJD prion, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Ancylostoma braziliense; multiple parasites, Cyclospora cayetanensis, Taenia solium, Cytomegalovirus, Dengue viruses (DEN-1, DEN-2,DEN-3 and DEN-4) - Flaviviruses, Dientamoeba fragilis, Corynebacterium diphtheriae, Diphyllobothrium, Dracunculus medinensis, Ebolavirus, Echinococcus genus, Ehrlichia genus, Enterobius vemiicularis, Enterococcus genus, Enterovirus genus, Rickettsia prowazekii, Parvovirus B19, Human herpesvirus 6 and Human herpesvirus 7, Fasciolopsis buski, Fasciola hepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily, Clostridium perfringens, Fusobacterium genus, Clostridium perfringens; other Clostridium species, Geotrichum candidum, GSS prion, Giardia intestinalis, Burkholderia mallei, Gnathostoma spinigerum and Gnathostoma hispidum, Neisseria gonorrhoeae, Klebsiella granulomatis, Streptococcus pyogenes, Streptococcus agalactiae, Haemophilus influenzae, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71, Sin Nombre virus, Helicobacter pylori, Escherichia coli O157: H7, Bunyaviridae family, Hepatitis A Virus, Hepatitis B Virus, Hepatitis C Virus, Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1, Herpes simplex virus 2, Histoplasma capsulatum, Ancylostoma duodenale and Necator americanus, Hemophilus influenzae, Human bocavirus, Ehrlichia ewingii, Anaplasma phagocytophilum, Human metapneumovirus, Ehrlichia chaffeensis, Human papillomavirus, Human parainfluenza viruses, Hymenolepis nana and Hymenolepis diminuta, Epstein-Barr Virus, Orthomyxoviridae family, Isospora belli, Kingella kingae, Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Kuru prion, Lassa virus, Legionella pneumophila, Legionella pneumophila, Leishmania genus, Mycobacterium leprae and Mycobacterium lepromatosis, Leptospira genus, Listeria monocytogenes, Borrelia burgdorferi and other Borrelia species, Wuchereria bancrofti and Brugia malayi, Lymphocytic choriomeningitis virus (LCMVj, Plasmodium genus, Marburg virus, Measles virus, Burkholderia pseudomallei, Neisseria meningitides, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Rickettsia typhi, Mycoplasma pneumoniae, numerous species of bacteria (Actinomycetoma) and fungi (Eumycetoma), parasitic dipterous fly larvae, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prion, Nocardia asteroides and other Nocardia species, Onchocerca volvulus, Paracoccidioides brasiliensis, Paragonimus westermani and other Paragonimus species, Pasteurella genus, Pediculus humanus capitis, Pediculus humanus corporis, Phthirus pubis, Bordetella pertussis, Yersinia pestis, Streptococcus pneumoniae, Pneumocystis jirovecii, Poliovirus, Prevotella genus, Naegleria fowleri, JC virus, Chlamydophila psittaci, Coxiella burnetii, Rabies virus, Streptobacillus moniliformis and Spirillum minus, Respiratory syncytialvirus, Rhinosporidium seeberi, Rhinovirus, Rickettsia genus, Rickettsia akari, Rift Valley fever virus, Rickettsia rickettsii, Rotavirus, Rubella virus, Salmonella genus, SARS coronavirus, Sarcoptes scabiei, Schistosoma genus, Shigella genus, Varicella zoster virus, Variola major or Variola minor, Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus, Staphylococcus aureus, Streptococcus pyogenes, Strongyloides stercoralis, Treponema pallidum, Taenia genus, Clostridium tetani, Trichophyton genus, Trichophyton tonsurans, Trichophyton genus, Epidermophyton floccosum, Trichophyton rubrum, and Trichophyton mentagrophytes, Trichophyton rubrum, Hortaea werneckii, Trichophyton genus, Malassezia genus, Toxocara canis or Toxocara cati, Toxoplasma gondii, Trichinella spiralis, Trichomonas vaginalis, Trichuris trichiura, Mycobacterium tuberculosis, Francisella tularensis, Ureaplasma urealyticum, Venezuelan equine encephalitis virus, Vibrio colerae, Guanarito virus, West Nile virus, Trichosporon beigelii, Yersinia pseudotuberculosis, Yersinia enterocolitica, Yellow fever virus, Mucorales order (Mucormycosis) and Entomophthorales order (Entomophthoramy cosis), Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Edwardsiella tarda, Yersinia pestis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Pneumocystis carinii, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Clamydia spp.; pathogenic fungi (Aspergillus fumigatus, Candida albicans, Histoplasma capsulatum); protozoa (Entomoeba histolytica, Trichomonas tenas, Trichomonas hominis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or Elelminiths (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms).

[0348] The therapeutic component as a therapeutic moiety in this disclosure for treatment of viral disease include, but are not limited to, antibodies against antigens of pathogenic viruses, including as examples and not by limitation: Poxyiridae, Herpesviridae, Adenoviridae, Papovaviridae, Enteroviridae, Picomaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Non-A / Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, Oncovirus [such as, HBV (Hepatocellular carcinoma), HPV (Cervical cancer, Analcancer), Kaposi's sarcoma-associated herpesvirus (Kaposi's sarcoma), Ep stein -Barr virus (Nasopharyngeal carcinoma, Burkitt's lymphoma, Primary central nervous system lymphoma), MCPy V (Merkel cell cancer), SV40 (Simian virus 40), HCV (Hepatocellular carcinoma), HTLV-I (Adult T-cell leukemia / lymphoma)], Immune disorders caused virus: [such as Human Immunodeficiency Virus (AIDS)]; Central nervous system virus: [such as, JCV (Progressive multifocal leukoencephalopathy), MeV (Subacute sclerosing panencephalitis), LCV (Lymphocytic choriomeningitis), Arbovirus encephalitis, Orthomyxoviridae (probable) (Encephalitis lethargica), RV (Rabies), Chandipura virus, Herpesviral meningitis, Ramsay Hunt syndrome type II; Poliovirus (Poliomyelitis, Post-polio syndrome), HTLV-I (Tropical spastic paraparesis)]; Cytomegalovirus (Cytomegalovirus retinitis, HSV (Herpetic keratitis));Cardiovascular virus [such as CBV (Pericarditis, Myocarditis)]; Respiratory system / acute viral nasophaiyngitis / viral pneumonia: [Epstein-Barr virus (EBV infection / Infectious mononucleosis), Cytomegalovirus; SARS coronavirus (Severe acute respiratory syndrome) Orthomyxoviridae: Influenzavirus A / B / C (Influenza / Avian influenza), Paramyxovirus: Human parainfluenza viruses (Parainfluenza), RSV (Human respiratory syncytial virus), hMPV]; Digestive system virus [MuV (Mumps), Cytomegalovirus (Cytomegalovirus esophagitis); Adenovirus (Adenovirus infection); Rotavirus, Norovirus, Astrovirus, Coronavirus; HBV (Hepatitis B virus), CBV, HAV (Hepatitis A virus), HCV (Hepatitis C virus), HDV (Hepatitis D virus), IIEV (Hepatitis E virus), HGV (Hepatitis G virus)]; Urogenital virus [such as, BK virus, MuV (Mumps)].Pharmaceutically Acceptable Salts

[0349] It is understood that any of the compounds of this disclosure may be present in a salt form. In some cases, the salt form of the compound is a pharmaceutically acceptable salt.

[0350] “Pharmaceutically acceptable salt” includes both acid and base addition salts.Pharmaceutically acceptable salts include the acid addition salts (e.g., formed with the free amino groups of the compound) and which are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Salts derived from organic bases include, but are notlimited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins.

[0351] Salts, solvates, hydrates, and prodrug forms of a compound are of interest. All such forms are embraced by the present disclosure. Thus, the compounds described herein include salts, solvates, hydrates, prodrug and isomer forms thereof, including the pharmaceutically acceptable salts, solvates, hydrates, prodrugs and isomers thereof. In certain embodiments, a compound may be metabolized into a pharmaceutically active derivative.

[0352] A compound of this disclosure may form a solvate with a solvent (including water). Therefore, in one non-limiting embodiment, the present disclosure includes a solvated form of the compound. The term “solvate” refers to a molecular complex of a compound (including a salt thereof) with one or more solvent molecules. Non-limiting examples of solvents are water, ethanol, isopropanol, dimethyl sulfoxide, acetone, and other common organic solvents. The term “hydrate” refers to a molecular complex comprising a compound and water. Pharmaceutically acceptable solvates in accordance with the invention include those wherein the solvent may be isotopically substituted, e.g. D2O, rfc-acetone, t / d-DMSO. A solvate can be in a liquid or solid form.

[0353] The term “prodrug” refers to an agent which is converted into the drug in vivo by some physiological or chemical process (e.g., a prodrug on being brought to the physiological pH is converted to the desired drug form). It is understood that any of the compounds described herein may be present in a prodrug form. A prodrug derivative of a compound generally includes a labile promoiety substituent at a suitable site of the parent compound. The promoiety refers to the group that is removed by enzymatic or chemical reactions, when a prodrug is converted to the drug in vivo.Pharmaceutical Compositions

[0354] A “pharmaceutical composition” is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human. In general, a “pharmaceutical composition” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients via a number of different routes of administration includingoral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, and the like.

[0355] In general, the compositions of the disclosure will include and be administered in a therapeutically effective amount by the desired mode of administration. Suitable dosage ranges depend upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, the indication towards which the administration is directed, and the preferences and experience of the medical practitioner involved. One of ordinary skill in the art of treating such diseases will be able, without undue experimentation and in reliance upon personal knowledge and the disclosure of this application, to ascertain a therapeutically effective amount of the compositions of the disclosure for a given disease.

[0356] In some embodiments, pharmaceutical compositions comprising compounds of the present disclosure can further comprise at least one additional active agent.

[0357] The terms “effective amount,” “pharmaceutically effective amount,” or “therapeutically effective amount” as used herein mean a sufficient amount of the composition to provide the desired utility when administered to a subject having a particular condition. The term “therapeutically effective amount” therefore refers to an amount of therapeutic cells or a composition having therapeutic cells that is sufficient to promote a particular effect when administered to a subject in need of treatment. An effective amount would also include an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example, but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using routine experimentation.

[0358] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. “Pharmaceutically acceptable carrier orexcipient” can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers.

[0359] A pharmaceutically acceptable carrier, diluent, or excipient for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990), and include without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. Carriers and excipients must be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the patient being treated. The carrier can be inert or it can possess pharmaceutical benefits of its own. The amount of carrier employed in conjunction with the disclosed compound is sufficient to provide a practical quantity of material for administration per unit dose of the compound, as described in more detail herein.

[0360] Pharmaceutical compositions are administered in a manner appropriate to the disease to be treated (or prevented). An appropriate dose and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration. Tn general, an appropriate dose and treatment regimen provides the composition(s) in an amount sufficient to provide therapeutic and / or prophylactic benefit (e.g., an improved clinical outcome), or a lessening of symptom severity. Optimal doses are generally determined using experimental models and / or clinical trials. The optimal dose depends upon the body mass, weight, or blood volume of the patient.

[0361] In one embodiment, an injectable pharmaceutical composition described herein, is used in the preparation of medicaments for the treatment of diseases or conditions in a mammal that would benefit from the administration of any one of the injectable pharmaceutical compositions of the conjugates disclosed. Methods for treating any of the diseases or conditions described herein in a mammal in need of such treatment, involves the administration of pharmaceutical compositions that include at least one compound described herein or a pharmaceuticallyacceptable salt, active metabolite, prodrug, or pharmaceutically acceptable solvate thereof, in therapeutically effective amounts to said mammal.ENUMERATED EMBODIMENTSEmbodiment 1. A pharmaceutical composition comprising an effective amount of a targeted therapy comprising:a. [T]- a targeting moiety comprising a first polypeptide comprising a first polypeptide sequence capable of selectively interacting directly or indirectly with a second polypeptide comprising a second polypeptide sequence;b. [R]- a reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, cysteine, histidine, tyrosine, present in the second polypeptide sequence; and c. [Tx]- a therapeutic moiety.Embodiment 2. A pharmaceutical composition comprising an effective amount of a targeted therapy comprising:a. [T]- a targeting moiety comprising a first polypeptide comprising a first polypeptide sequence capable of selectively interacting with a cell surface-associated entity; b. [R]~ a reactive moiety comprising an entity capable of covalent linkage to a reactive atom present in the cell surface-associated entity; andc. [Tx]- a therapeutic moiety.Embodiment 3. A pharmaceutical composition comprising an effective amount of a targeted therapy comprising:a. [T]- a targeting moiety comprising a first polypeptide comprising a first polypeptide sequence capable of selectively interacting with a second polypeptide comprising a second polypeptide sequence;b. [R]- a plurality of reactive moieties Ri to Rnindependently comprising an entity capable of covalent linkage to a lysine, serine, histidine, cysteine or tyrosine present in the second polypeptide sequence; andc. [Tx]- a therapeutic moiety.Embodiment 4. A pharmaceutical composition comprising an effective amount of a therapy comprising:a. [Ri]- a first reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, cysteine or tyrosine present in a targeting moiety comprising a first polypeptide comprising a first polypeptide sequence, wherein the targeting moiety is capable of selectively interacting with a second polypeptide comprising a second polypeptide sequence;b. [R2]- a second reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, cysteine or tyrosine present in the second polypeptide sequence; andc. [Tx]- a therapeutic moiety.Embodiment 5. A pharmaceutical composition comprising an effective amount of a targeted therapy comprising:a. [Rl]- a first reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, cysteine or tyrosine present in a first polypeptide comprising a first polypeptide sequence comprising a target cell surface molecule (e g., a cell surface receptor);b. [Tx]- a therapeutic moiety; andc. [R2]- a second reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, cysteine or tyrosine present in a targeting moiety comprising a second polypeptide sequence, wherein the targeting moiety is capable of selectively- interacting with the first polypeptide.Embodiment 6. The composition of embodiment 1, wherein the first polypeptide comprises an antibody or fragment thereof, or a ligand for a cell surface receptor or fragment thereof.Embodiment 7. The composition of embodiment 1, wherein the first polypeptide comprises a miniprotein or a stapled peptide.Embodiment 8. The composition of embodiment 1, wherein the first polypeptide comprises a macrocyclic peptide.Embodiment 9. The composition of embodiment 1, wherein the second polypeptide comprises a cell surface receptor.Embodiment 10. The composition of embodiment 1, wherein the second polypeptide is synthetic.Embodiment 11. The composition of embodiment 1, wherein the second polypeptide is produced recombinantly.Embodiment 12. The composition of embodiment of 1, formulated for oral administration, subcutaneous administration, or intravenous administration.Embodiment 13. The composition of embodiment 1, wherein the second polypeptide comprises a cell surface receptor selected from a 5-hydroxytryptamine receptor, an acetylcholine receptor, an adenosine receptor, an adenosine A2A receptor, an adenosine A2B receptor, an angiotensin receptor, an apelin receptor, a bile acid receptor, a bombsin receptor, a bradykinin receptor, a cannabinoid receptor, a chemerin receptor, a chemokine receptor, a cholecystokinin receptor, a Class A Orphan receptor, a dopamine receptor, an endothelin receptor, an epidermal growth factor receptor (EGFR). a formyl peptide receptor, a free fatty acid receptor, a galanin receptor, a ghrelin receptor, a glycoprotein hormone receptor, a gonadotropin-releasing hormone receptor, a G protein-coupled receptor, a G protein-coupled estrogen receptor, a histamine receptor, ahydroxy carboxylic acid receptor, a kisspeptin receptor, a leukotriene receptor, a lysophospholipid receptor, a lysophospholipid SIP receptor, a melanin-concentrating hormone receptor, a melanocortin receptor, a melatonin receptor, a motilin receptor, a neuromedin U receptor, a neuropeptide FF / neuropeptide AF receptor, a neuropeptide S receptor, a neuropeptide W / neuropeptide B receptor, a neuropeptide Y receptor, a neurotensin receptor, an opioid receptor, an opsin receptor, an orexin receptor, anoxoglutarate receptor, a P2Y receptor, a platelet-activating factor receptor, a prokineticin receptor, a prolactin-releasing peptide receptor, a prostanoid receptor, a proteinase- activated receptor, a QRFP receptor, a relaxin family peptide receptor, a somatostatin receptor, a succinate receptor, a tachykinin receptor, a thyrotropin-releasing hormone receptor, a trace amine receptor, a urotensin receptor, a vasopressin receptor.Embodiment 14. The composition of embodiment 1, wherein the second polypeptide comprises a cell surface polypeptide.Embodiment 15. The composition of embodiment 1, wherein the second polypeptide comprises a tumor-associated polypeptide, an inflammation associated peptide, lineage specific peptide, or a tissue specific peptide.Embodiment 16. The composition of embodiment 1, wherein upon interaction of the first polypeptide with the second polypeptide, [R] is within 5-20 angstroms of at least one amino acid present in the second polypeptide.Embodiment 17. The composition of embodiment 1, wherein upon interaction of the first polypeptide with the second polypeptide, [R] is within 5-20 angstroms of no more than one lysine, serine, histidine, cysteine or tyrosine.Embodiment The composition of embodiment 1, wherein the targeted therapy comprises a non- canonical amino acid [NCAA],Embodiment 19. The composition of embodiment 1, wherein [T] and [R] are operably linked via a cleavable linker.Embodiment 20. The composition of embodiment 1, wherein [R] and [Tx] are operably linked via a non-cleavable linker.Embodiment The composition of embodiment 1, wherein [R] and [Tx] are operably linked via a cleavable linker.Embodiment 22. The composition of embodiment 1, comprising a plurality of reactive moieties.Embodiment 23. The composition of embodiment 1, further comprising a half-life extension moiety.Embodiment 24. The composition of embodiment 1, comprising a first reactive moiety [Rl] comprising a first entity capable of covalent linkage to a first reaction target comprising a lysine, serine, histidine, cysteine or tyrosine present in the second polypeptide sequence and further comprising a second reactive moiety [R2] comprising a second entity capable of covalent linkage to second reaction target.Embodiment 25. The composition of embodiment 1, comprising a first reactive moiety [Rl] comprising one or more electrophiles.Embodiment 26. The composition of embodiment 1, wherein the targeted therapy is greater than IkDa in molecular mass, or greater than 2kDa, 3kDa, 4kDa or 5kDa in molecular mass.Embodiment 27. The composition of embodiment 1, wherein the targeted therapy comprises a net charge of at least 1.Embodiment 28. The composition of embodiment 1, wherein the targeted therapy is substantially non-cell penetrating to a human cell, cell population, tissue, or organ.Embodiment 29. The composition of embodiment 1, wherein [R] comprises a non- canonical amino acid [NCAA], and wherein the NCAA is capable of forming a covalent bond with a histidine, lysine, or tyrosine residue of the second polypeptide, and wherein the NCAA comprises a fluoro-sulfate moiety.Embodiment 30. The composition of embodiment 1, wherein [Tx] comprises a polypeptide therapeutic comprising an enzyme.Embodiment 31. The composition of embodiment 1, wherein [Tx] comprises a polypeptide therapeutic comprising an antibody or fragment thereof.Embodiment 32. The composition of embodiment 1, wherein [Tx] comprises a polypeptide therapeutic comprising a polypeptide conjugated to a radioligand.Embodiment 33. The composition of embodiment 1, wherein [Tx] comprises a radioligand.Embodiment 34. The composition of embodiment 1, wherein [Tx] comprises a small molecule therapeutic.Embodiment 35. The composition of embodiment 1, wherein the targeted therapy has a lower KON rate than the therapeutic moiety.Embodiment 36. The composition of embodiment 1, wherein the targeted therapy has a lower KOFF rate than the therapeutic moiety when the reactive moiety is covalently linked to the second polypeptide.Embodiment 37. The composition of embodiment 2, wherein the cell surface-associated entity is selected from a carbohydrate, glycoprotein, lipid, nucleic acid, peptide, or polypeptide.Embodiment 38. The composition of embodiment 2, wherein the cell surface-associated entity comprises a post-translationally modified polypeptide residue.Embodiment 39. A composition comprising the pharmaceutical composition of embodiment 1 present in a deactivating material that reduces the reactivity of the reactive moiety prior to administration of the composition to a human subject in need thereof.Embodiment 40. A composition comprising the pharmaceutical composition of embodiment 1 present in a purity level reduce the reactivity of the reactive moiety prior to administration of the composition to a human subject in need thereof.Embodiment 41. A method of treating a human subject suffering from a disease, disorder or condition, comprising administering to the human subject a pharmaceutical composition comprising an effective amount of a targeted therapy comprising:a. [T]- a targeting moiety comprising a first polypeptide comprising a first polypeptide sequence capable of selectively interacting with a second polypeptide comprising a second polypeptide sequence;b. [R]- a reactive moiety comprising an entity capable of covalent linkage to a cysteine or tyrosine present in the second polypeptide sequence; andc. [Tx]- a therapeutic moiety.Embodiment 42. A method of decorating the extracellular surface of a target cell population, comprising administering to the human subject a pharmaceutical composition comprising an effective amount of a targeted therapy comprising:a. [T]- a targeting moiety comprising a first polypeptide comprising a first polypeptide sequence capable of selectively interacting with a second polypeptide comprising a second polypeptide sequence present on the surface of a target cell population; b. [R]- a reactive moiety comprising an entity capable of covalent linkage to a cysteine or tyrosine present in the second polypeptide sequence; andc. [CPM]- a cell painting moiety.wherein the CPM decorates the extracellular surface of the target cell population.Embodiment 43. The composition of embodiment 1, further comprising human material incubated with the therapeutic prior to administration.Embodiment 44. The composition of embodiment 28, wherein the human material is blood, bone marrow, erythrocytes, human cell products, or a human organ for transplant.Embodiment 45. A complex comprising a human material conjugated to a composition comprising:a. [T]- a targeting moiety comprising a first polypeptide comprising a first polypeptide sequence capable of selectively interacting with a second polypeptide comprising a second polypeptide sequence;b. [R]- a reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, cysteine or tyrosine present in the second polypeptide sequence; and c. [Tx]- a therapeutic moiety.Embodiment 46. A complex comprising a human material conjugated to a composition comprising:a. [R]- a reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, cysteine or tyrosine present in a second polypeptide comprising a second polypeptide sequence; andb. [Tx]- a therapeutic moiety.Embodiment 47. A composition comprising:a. [M]- a modulating moiety comprising a first polypeptide comprising a first polypeptide sequence capable of selectively modulating a second polypeptide comprising a second polypeptide sequence, wherein the selectively modulating comprises increasing or decreasing an activity, half-life or signaling of the second polypeptide sequence;b. [R]- a reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, cysteine or tyrosine present in the second polypeptide sequence.Embodiment 48. A composition comprising:a. [M]- a modulating moiety comprising a first polypeptide comprising a first polypeptide sequence capable of selectively modulating a second polypeptide comprising a second polypeptide sequence; andb. [R]- a plurality' of reactive moieties Ri to Rnindependently comprising an entity capable of covalent linkage to a lysine, serine, histidine, cysteine or tyrosine present in the second polypeptide sequence.Embodiment 49. A composition comprising:a. [M]- a modulatory moiety comprising a first polypeptide comprising a first polypeptide sequence, wherein the modulating moiety is capable of selectively modulating a second polypeptide comprising a second polypeptide sequence; b. [Ri]- a first reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, cysteine or tyrosine present in the modulating moiety; and c. [R2]- a second reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, cysteine or tyrosine present in the second polypeptide sequenceEmbodiment 50. A pharmaceutical composition comprising an effective amount of a targeted therapy comprising:a. [BFD]- a bifunctional domain comprising a targeting and therapeutic moiety [TTx] comprising a first polypeptide comprising a first polypeptide sequence capable of selectively interacting with a second polypeptide comprising a second polypeptide sequence, wherein the first polypeptide has a therapeutic activity;b. [R]- a reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, cysteine or tyrosine present in the second polypeptide sequence; andEmbodiment 51. The pharmaceutical composition of embodiment 46, wherein the first polypeptide comprises a cytokine inhibitor, a receptor agonist, or a receptor antagonist.Embodiment 52. A composition comprising:a. [M]- a modulating moiety comprising a first polypeptide comprising a first polypeptide sequence, wherein the modulating moiety is capable of selectively modulating a second polypeptide comprising a second polypeptide sequence b. [Ri]- a first reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, cysteine or tyrosine present in a modulating moiety; and c. [R2]- a second reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, cysteine or tyrosine present in the second polypeptide sequence.Embodiment 53. The composition or pharmaceutical composition of any one of the proceeding embodiments, comprising a therapeutic moiety that is pharmacologically inactive.Embodiment 54. The composition or pharmaceutical composition of any one of the proceeding embodiments, comprising a therapeutic moiety that is substantially cell inpenetrant.Embodiment The composition or pharmaceutical composition of any one of the proceeding embodiments, comprising a therapeutic moiety that is sterically blocked.Embodiment 56. The composition or pharmaceutical composition of any one of the proceeding embodiments, comprising a pharmacodynamic effect on a cell population present in a human subject greater than 4, 8, 12, 16, 20, 24, 48, 72 or greater than 72 hours in duration.Embodiment 57. A pharmaceutical composition comprising an effective amount of a targeted therapy comprising:a. [T]- a targeting moiety comprising a first polypeptide comprising a first polypeptide sequence capable of selectively interacting directly or indirectly with a second polypeptide comprising a second polypeptide sequence;b. [R]- a reactive moiety comprising an entity capable of covalent linkage to a lysine, serine, histidine, cysteine, tyrosine, present in the second polypeptide sequence; and c. [Im]- an inhibitory moiety, wherein the inhibitory moiety irreversibly inhibits the activity of the second polypeptide.Embodiment 58. A conjugate represented by Formula (I) or (II):[T]_[R]_[L2]-[TX] (I)([R]-[Li])m-[T]-([L2]-[Tx])n(II)wherein:each [R] is independently a reactive moiety;[T] is a targeting moiety, wherein the targeting moiety is capable of interacting with a target;each [Tx] is independently a therapeutic moiety;each [Li] is independently a bond or a linker;each [L2] is independently a bond or a linker; andand m and n are each independently an integer from 0 to 5.Embodiment 59. The conjugate of embodiment 58, wherein [T] comprises an antibody, an antibody fragment, a miniprotein, or a peptide, wherein the antibody, antibody fragment, miniprotein, or peptide ligand is capable of interacting with a cell surface receptor.Embodiment 60. The conjugate of embodiment 59, wherein the peptide is a stapled peptide or a macrocyclic peptide.Embodiment 61. The conjugate of any one of embodiments 58-60, wherein [R] is capable of forming a covalent bond with a lysine, serine, histidine, cysteine, or tyrosine residue present in the target.Embodiment 62. The conjugate of any one of embodiments 58-61, wherein [R] comprises an electrophile.Embodiment 63. The conjugate of embodiment 62, wherein electrophile has an electrophilicity index of about 0.8 eV or higher, such as about 1.5 eV or higher.Embodiment 64. The conjugate of embodiment 62 or 63, wherein the electrophile comprises one of the following structures:Embodiment 65. The conjugate of any one of embodiments 58-64, wherein [R] comprises a non-canonical amino acid (NCAA), wherein the NCAA comprises a fluoro-sulfate moiety, and the NCAA is capable of forming a covalent bond with a histidine, lysine, or tyrosine residue present in the target.Embodiment 66. The conjugate of any one of embodiments 58-65, wherein [R] is linked, via [Li], to a cysteine or lysine residue present in [T],Embodiment 67. The conjugate of any one of embodiments 58-66, wherein [Tx] comprises an enzyme, an antibody, an antibody fragment, a peptide, a radioligand, or a small molecule therapeutic.Embodiment 68. The conjugate of any one of embodiments 58-67, wherein [Li], [L2], or [Li] and [L2] are each a cleavable linker.Embodiment 69. The conjugate of any one of embodiments 58-67, wherein [Li], [L2], or [Li] and [L2] are each a non-cleavable linker.Embodiment 70. The conjugate of any one of embodiments 58-69, wherein n is 0 or 1.Embodiment 71. The conjugate of any one of embodiments 58-70, wherein m is 0 or 1.Embodiment 72. The conjugate of any one of embodiments 58-71, wherein the target is a cell surface protein.Embodiment 73. The conjugate of any one of embodiments 58-72, wherein the target is a tumor-associated protein, an inflammation-associated protein, lineage specific protein, or a tissue specific protein.Embodiment 74. The conjugate of any one of embodiments 58-73, wherein the conjugate has a lower KON rate to the target than [T],Embodiment 75. The conjugate of any one of embodiments 58-74, wherein the conjugate has a lower KOFF rate to the target than [Tx],Embodiment 76. The conjugate of any one of embodiments 58-75, further comprising a half-life extension moiety.Embodiment 77. A pharmaceutical composition comprising a conjugate of any one of embodiments 58-76 and a pharmaceutically acceptable carrier.Embodiment 78. A method of treating a human subject suffering from a disease, disorder or condition, comprising administering to the human subject a pharmaceutical composition of embodiment 77.Embodiment 79. A method of delivering a therapeutic agent to a target cell, comprising administering a conjugate of any one of embodiments 58-78.EXAMPLES

[0362] The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice.

[0363] However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0364] Techniques for performing the methods of the present disclosure are well known in the art and described in standard laboratory textbooks, including, for example, Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997); Molecular Cloning: A Laboratory Manual, Third Edition, J. Sambrook and D. W. Russell, eds., Cold Spring Harbor, N. Y., USA, Cold Spring Harbor Laboratory Press, 2001; O'Brian et al., Antibody Phage Display, Methods and Protocols, Humana Press, 2001; Phage Display: A Laboratory Manual, C. F.Barbas III et al. eds., Cold Spring Harbor, N. Y., USA, Cold Spring Harbor Laboratory Press, 2001; and Antibodies, G. Subramanian, ed., Kluwer Academic, 2004. Mutagenesis can, for example, be performed using site-directed mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA 82:488-492 (1985)); DNA Cloning, Vols. 1 and 2, (D. N. Glover, Ed. 1985);Oligonucleotide Synthesis (M. J. Gait, Ed. 1984); PCR Handbook Current Protocols in Nucleic Acid Chemistry, Beaucage, Ed. John Wiley & Sons (1999) (Editor); Oxford Handbook of Nucleic Acid Structure, Neidle, Ed., Oxford Univ Press (1999); PCR Protocols: A Guide to Methods and Applications, Innis et al., Academic Press (1990); PCR Essential Techniques: Essential Techniques, Burke, Ed., John Wiley & Son Ltd (1996); The PCR Technique: RT-PCR, Siebert, Ed., Eaton Pub. Co. (1998); Antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow et al., C. S. H. L. Press, Pub. (1999); Large-Scale Mammalian Cell Culture Technology, Lubiniecki, A., Ed., Marcel Dekker, Pub., (1990). Border et al., Yeast surface display for screening combinatorial polypeptide libraries, Nature Biotechnology, 15(6):553-7 (1997); Border et al., Yeast surface display for directed evolution of protein expression, affinity, and stability, Methods Enzymol., 328:430-44 (2000); ribosome display as described by Pluckthun et al. in U. S. Pat. No. 6,348,315, and Profusion™ as described by Szostak et al. inU. S. Pat. Nos. 6,258,558; 6,261,804; and 6,214,553; and bacterial periplasmic expression as described in US20040058403A1. PCR amplification methods are described in U. S. Pat. Nos. 4,683,192, 4,683,202, 4,800,159, and 4,965,188, and in several textbooks including “PCR Technology: Principles and Applications for DNA Amplification”, H. Erlich, ed., Stockton Press, New York (1989); and “PCR Protocols: A Guide to Methods and Applications”, Innis et al., eds., Academic Press, San Diego, Calif. (1990).

[0365] Although in the foregoing description the disclosure is illustrated with reference to certain embodiments, it is not so limited. Indeed, various modifications of the disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. Thus, while the disclosure is illustrated with reference to biologically active peptide libraries, it extends generally to all peptide and polypeptide libraries.

[0366] All references cited throughout the specification are hereby expressly incorporated by reference.Example 1. Synthesis of a Peptide Conjugate with a Degradable Linker for Delivering Doxorubicin

[0367] The BND3-binding D-peptide [fwhpwammysgggkc], with a cysteine residue added for conjugation, was synthesized via solid-phase peptide synthesis (SPPS) using standard Fmoc chemistry. The peptide was assembled stepwise on an Fmoc-protected resin, with the cysteine residue introduced at the desired site for conjugation. Upon completion of synthesis, the peptide was cleaved from the resin and deprotected using a cleavage cocktail of 95% trifluoroacetic acid (TFA), 2.5% triisopropyl silane (TIS), and 2.5% water. The crude peptide was precipitated with cold diethyl ether, centrifuged, dissolved in acetonitrile / water (0.1% TFA) and lyophilized.

[0368] Doxorubicin-mal eimide (16 mg) was added to a 16.5 mM solution of BND3 -binding D-peptide [fwhpwammysgggkc], in DMF to a final concentration of 16.5 mM (1:1). The reaction mixture was allowed to stir at 36 °C for 6 hours, followed by additional 9 hours at room temperature, at which time LCMS analysis indicated consumption of the starting peptide and formation peptide conjugate. The product was purified by RP-HPLC to give >10 mg of [fwhpwammysgggkc(doxorubicin)].

[0369] The BND3-binding peptide [fwhpwammysgggkc(doxorubicin)] (2 mM) was dissolved in dimethylformamide (DMF) and mixed with N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The reaction was stirred for 1 hour at room temperature, forming a amide bond between the lysine group of the peptide and the SPDP linker. After completion, the linker was reduced to generate a thiol-containing peptide-SPDP conjugate that was purified using high-performance liquid chromatography (HPLC), and lyophilized.

[0370] Next, a lysine-targeting acrylamide warhead (ethyl 2-(bromomethyl)acrylate) was reacted with [fwhpwammysgggk(SPDP)c(doxorubicin)] for attachment to the thiol group of the linker from SPDP. The peptide was dissolved in DMF along with ethyl 2-(bromomethyl)acrylate. The reaction was allowed to proceed for 2 hours at room temperature, resulting in the attachment of the warhead to the peptide-linker conjugate. The product was again purified by HPLC and lyophilized.

[0371] The final peptide conjugate was characterized using reverse-phase HPLC on a C18 column with a water / acetonitrile gradient. The conjugate was further analyzed by electrospray ionization mass spectrometry (ESLMS), which confirmed the expected molecular weight and successful conjugation of the BND3 binder, reactive warhead, degradable linker, and doxorubicin payload.Example 2. Synthesis of a Peptide Conjugate with a Non-Degradable Linker for Delivering Doxorubicin

[0372] The PEG containing BND3-binding peptide [fwhpwammysgggk-PEG4-c]], with an C-terminal cysteine or tyrosine residue for conjugation, was synthesized using standard solid-phase peptide synthesis (SPPS) as described previously. Upon completion of the synthesis, the peptide was cleaved from the resin using TFA, precipitated in diethyl ether, and lyophilized.

[0373] Doxorubicin-mal eimide (16 mg) was added to a 16.5 mM solution of BND3 -binding D-peptide [fwhpwammysgggk-PEG4-c], in DMF to a final concentration of 16.5 mM (1:1). The reaction mixture was allowed to stir at 36 °C for 6 hours, followed by additional 9 hours at room temperature, at which time LCMS analysis indicated consumption of the starting peptide and formation peptide conjugate. The product was purified by RP-HPLC to give >10 mg of [fwhpwammysgggk-PEG4-c(doxorubicin)].

[0374] The BND3-binding peptide [fwhpwammysgggk-PEG4-c(doxorubicin)] (2 mM) was dissolved in dimethylformamide (DMF) and mixed with N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The reaction was stirred for 1 hour at room temperature, forming a amide bond between the lysine group of the peptide and the SPDP linker. After completion, the linker was reduced to generate a thiol -containing peptide-SPDP conjugate that was purified using high-performance liquid chromatography (HPLC), and lyophilized.

[0375] Next, a lysine-targeting acrylamide warhead (ethyl 2-(bromomethyl)acrylate) was reacted with [fwhpwammysgggk(SPDP)-PEG4-c(doxorubicin)] for attachment to the thiol group of the linker from SPDP. The peptide was dissolved in DMF along with ethyl 2-(bromomethyl)acrylate. The reaction was allowed to proceed for 2 hours at room temperature, resulting in the attachment of the warhead to the peptide-linker conjugate. The product was again purified by HPLC and lyophilized.

[0376] The final peptide conjugate was characterized using reverse-phase HPLC with a water / acetonitrile gradient and analyzed by mass spectrometry (ESLMS) to confirm the molecular weight of the peptide-linker-doxorubicin conjugate. The analysis confirmed the successful attachment of the BND3 binding peptide, reactive warhead, non-degradable linker, and doxorubicin.Example 3. Screening for Binding and Activity of a Peptide Conjugate with a Covalent Warhead and Functional Payload in a Biochemical Assay

[0377] The first step is to screen for the binding of the peptide conjugate to the target protein by testing its ability to covalently interact with lysine residues on the BND3 protein. Recombinant BND3 receptor (100 pg / mL) was immobilized on a 96-well plate using standard coating procedures (incubation in PBS overnight at 4°C). After washing with PBS, wells were incubated with increasing concentrations of the peptide conjugate (0, 50, 100, 200 nM) in Tris-HCl buffer (pH 7.5) at 37°C for 1 hour.

[0378] To detect covalent binding, unbound peptide conjugates were removed by washing with PBS. SDS-PAGE followed by silver staining and mass spectrometry confirmed the formation of covalent adducts between BND3 and the peptide conjugate. The presence of the acrylamidewarhead resulted in the formation of covalent bonds with lysine residues on the receptor, confirming successful binding.

[0379] To confirm that the payload (doxorubicin) was delivered and remained functionally active after binding to the target protein, a doxorubicin fluorescence-based detection assay was performed. After binding to BND3, wells were incubated with a fluorescence quenching solution to remove any non-covalently attached doxorubicin. Fluorescence intensity was measured using a plate reader (excitation at 480 nm, emission at 590 nm), and a dose-dependent increase in fluorescence was observed, indicating that the payload had been successfully delivered and retained its fluorescence, a marker of its structural integrity. As controls, wells coated with the target protein but treated with the control peptide (+- warhead and +-payload) showed no significant fluorescence, confirming that the observed signal was specific to the covalent binding and payload delivery by the peptide conjugate.

[0380] To test whether the payload (doxorubicin) retained its cytotoxic function after being delivered by the peptide conjugate. HeLa cells are cultured on a transwell plate and are exposed to the supernatant of well with peptide conjugates. Drug freely diffuses from the lower compartment to the upper compartment through the semi-permeable membrane. Different Hela cells were exposed to various concentrations of the peptide conjugate (0, 50, 100, 200 nM) or control peptides for 24 hours. Cell viability was assessed using a standard MTT assay.Example 4. Synthesis of a Binder-PD-1 Agonist Peptide Conjugate via Solid-Phase Peptide Synthesis, Followed by Covalent Warhead Addition

[0381] The conjugate is designed with a binder sequence that binds a beta cell surface receptor (an antigen-binding moiety derived from mAb43, as described in Kasinathan et al. supra, that binds ZNT8 or CRTH2 / GPR44 (extracellular) blocking peptide (#BLP-PR062), Alomone Labs) at the N-terminus, linked to the PD-1 agonist peptide sequence at the C-terminus, connected by a flexible spacer if necessary. This approach ensures that both functional units are synthesized as a single molecule.

[0382] The synthesis begins by loading an Fmoc-protected resin (compatible with the desired C-terminal functional group) into the peptide synthesizer. The synthesis follows standard Fmoc chemistry protocols. The binder specific to the beta cell surface receptor is synthesized first. Thesequence is elongated from the C-terminal Fmoc-Lysine(alloc) toward the N-terminus, adding one Fmoc-protected amino acid at a time. After completing the binder, the PD-1 agonist peptide sequence is appended directly to the C-terminus of the binder after the removal of the Alloc group on the C-terminal lysine residue. The PD-1 agonist sequence mimics the binding domain of PD-L1 to activate the PD-1 receptor on T cells. After the full peptide is synthesized, the Fmoc group is removed from the final N-terminal amino acid.

[0383] Once the synthesis is complete, the fully assembled Binder-PD-1 agonist conjugate is cleaved from the resin and deprotected using a cleavage cocktail (95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS), 2.5% water). The peptide is precipitated in cold diethyl ether, centrifuged, dissolved in acetonitrile / water (0.1% TFA) and lyophilized to obtain the crude product. The crude peptide is purified using preparative high-performance liquid chromatography (HPLC) with a water / acetonitrile gradient. The purity of the conjugate is confirmed by analytical HPLC, and the molecular weight is verified using electrospray ionization mass spectrometry (ESI-MS).

[0384] Subsequently, an electrophilic warhead is reacted with the Binder-PD-1 agonist conjugate for attachment to the thiol group of a cysteine residue present in the binder.Example 5. Functional Assay to Demonstrate Cell Protection from T Cell Killing

[0385] To confirm that the Binder-PD-1 agonist conjugate binds to the target receptor and irreversibly interacts with extracellular lysine residues, a binding and covalent warhead assay is performed using a primary beta cell culture that expresses the surface target (ZNT8 or GPR44). The cells are incubated with the Binder-PD-1 agonist conjugate at varying concentrations (0, 50, 100, 200 nM) for 1 hour. After washing away unbound conjugates, the cells are lysed, and covalent binding to surface proteins is confirmed by mass spectrometry or Western blot analysis.

[0386] To test whether the PD-1 agonist peptide delivered by the binder protects beta cells from T cell-mediated killing, beta cells are co-cultured with Jurkat T cells (effector cells expressing PD-1) at a 10:1 effector-to-target ratio.

[0387] Beta cells are pre-treated with the Binder-PD-1 agonist conjugate (at various doses 0, 50, 100, 200 nM) for 1 hour, allowing the covalent warhead to irreversibly bind to extracellular lysines and washed 3X with PBS. After incubation with activated Jurkat T cells, beta cells areanalyzed for viability using an MTT assay. The viability is compared to a Binder-PDl agonist conjugate without the covalent warhead and a binder with the covalent warhead but without the PD1 agonism under the same conditions.Example 6. Synthesis of a BND3-Targeting Peptide Conjugate with a Covalent Warhead, Non-Cleavable Linker, and Uricase Payload

[0388] The BND3 peptide is synthesized using standard Fmoc chemistry protocols on a peptide synthesizer. The Fmoc-protected resin is loaded, and amino acids are sequentially added to build the peptide from the C-terminus to the N-terminus.

[0389] After the full sequence is assembled, the Fmoc group is removed from the final N-terminal amino acid. After the synthesis, the peptide is cleaved from the resin using a cleavage cocktail of 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS), and 2.5% water. The cleaved peptide is precipitated with cold diethyl ether, centrifuged, and lyophilized. The crude peptide is purified using preparative high-performance liquid chromatography (HPLC) with a water / acetonitrile gradient. The peptide is characterized by mass spectrometry to confirm the expected molecular weight.

[0390] The purified BND3 peptide is dissolved in PBS (pH 7.4), and maleimide-PEG (MW: 2 kDa) is added. The maleimide group reacts with the cysteine thiol group on the peptide, forming a stable thioether bond. The reaction is allowed to proceed at room temperature for 2 hours.

[0391] The reaction mixture is purified by HPLC to remove unreacted linker and peptide. The purified BND3-PEG conjugate is lyophilized and characterized by HPLC and mass spectrometry. The BND3-PEG conjugate is dissolved in DMSO, and acrylamide is added. The acrylamide warhead reacts with free thiols on the peptide, attaching covalently. The reaction proceeds for 2 hours at room temperature. The resulting BND3(acrylamide)-PEG conjugate is purified by HPLC and lyophilized. The product is characterized by mass spectrometry to confirm the attachment of the warhead.

[0392] Uricase is a tetrameric enzyme that catalyzes the oxidation of uric acid to allantoin, hydrogen peroxide, and carbon dioxide. Recombinant uricase is expressed in E. coli and purified by standard affinity chromatography.

[0393] The BND3(acrylamide)-PEG conjugate is dissolved in PBS (pH 7.4), and purified uricase is added. The PEG linker ensures flexibility and proper presentation of uricase. The mixture is stirred for 3 hours at room temperature, allowing conjugation to occur between the PEG linker and lysine residues on the uricase.

[0394] The final conjugate (BND3(acrylamide)-PEG- uricase) is purified by size-exclusion chromatography to remove unreacted uricase and linker. The product is lyophilized and characterized using SDS-PAGE, HPLC, and mass spectrometry to confirm the successful conjugation of uricase.Example 7. Functional Assay to Assess Uricase Activity

[0395] To confirm that the BND3(acrylamide)-PEG-uricase conjugate binds its target receptor, a surface receptor binding assay is performed using primary red blood cells (RBCs) isolated from patient blood samples. The RBCs are incubated with varying concentrations of the conjugate (0, 50, 100, 200 nM) for 1 hour. After washing to remove unbound conjugate, binding is assessed by flow cytometry using a fluorescent secondary antibody that binds uricase or BND3.

[0396] The ability of the conjugate to irreversibly bind the receptor is tested by incubating cells with the conjugate and then washing thoroughly. Cells are lysed, and the presence of covalently bound conjugate is confirmed by mass spectrometry or Western blot.

[0397] The enzymatic activity of the covalent uricase conjugate is assessed using a uric acid substrate solution. The BND3(acrylamide)-PEG-uricase conjugated red blood cells are incubated with excess uric acid (1 mM) in PBS, and the conversion of uric acid to allantoin is monitored by measuring absorbance using a spectrophotometer. The activity is directly compared to various controls including purified RBCs, isolated uricase enzyme, and non-covalent BND3-uricase all at equal molarity.Example 8. Synthesis of bromo-[dicyclohexyl-[2-(2,6-diisopropoxyphenyl)phenyl]-phosphanyl]-[3-(prop-2-enoylamino)phenyl]palladium(II) (EC301)

[0398] To a solution of 4-bromobenzenesulfonyl fluoride (700 mg, 2.93 mmol, 1 eq) in THF (14 mb) was added bis[(trimethylsilyl)methyl](l,5-cyclooctadiene)palladium(II) (1.25 g, 3.22 mmol, 1.1 eq) and sodium 3 -(2-dicy cl ohexylphosphanylphenyl)-2,4-dimethoxy -benzenesulfonate hydrate (1.71 g, 3.22 mmol, 1.1 eq). The mixture was stirred at 20 °C for 1 h into a glovebox. Then pentane (42 mb) was added dropwise to the mixture. The mixture was stirred at -20 °C for 8 h. LCMS showed the starting reactant was consumed and the desired compound was detected. The mixture was filtered and the filter cake was concentrated under reduced pressure.Compound bromo-[dicyclohexyl-[2-(2,6-diisopropoxyphenyl)phenyl]-phosphanyl]-[3-(prop-2-enoylamino)phenyl]palladium (2.5 g, 2.74 mmol, 94% yield, 94.2% purity) was obtained as a white solid. [M-NaBr+H]+= 755.0. 'HNMR (400 MHz, METHANOL-d48 ppm 7.90 - 8.10 (m, 1 H), 7.10 - 7.60 (m, 8 H), 6.80 - 6.98 (m, 1 H), 3.68 - 3.95 (m, 3 H), 3.50 (s, 3 H), 1.66 -1.94 (m, 8 H), 0.74 - 1.59 (m, 14 H). 38 H's / 38 H's (spectrum / structure).19F NMR (400 MHz, METHANOL-d4) 5 ppm 65.22 (s, 1 F).Example 9. Synthesis of bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-(3-fluorosulfonylphenyl)palladium(II) EC302

[0399] To a solution of 3 -bromobenzenesulfonyl fluoride (700 mg, 2.93 mmol, 1 eq) in THF (14 mb) was added bis[(trimethylsilyl)methyl](l,5-cyclooctadiene)palladium(II) (1.25 g, 3.22 mmol, 1.1 eq) and sSPhos (1.65 g, 3.22 mmol, 1.1 eq). The mixture was stirred at 20 °C for 1 h underglovebox. Then pentane (42 mL) was added dropwise to the mixture. The mixture was stirred at -20 °C for 8 h. LCMS showed the starting reactant was consumed and the desired compound was detected. The mixture was filtered and the filter cake was concentrated under N2.Compound bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-(3-fluorosulfonylphenyl)palladium (EC302, 2.5 g, 2.91 mmol, 99.4% yield, 95.5% purity) was obtained as a white solid. [M-NaBr+H]+= 755.1. 'H NMR (400 MHz, METHANOL-d4) 5 ppm 7.97 (d, J=8.8 Hz, 1 H), 6.70 - 7.58 (m, 9 H), 3.78 (s, 3 H), 3.56 (s, 3 H), 2.21 - 2.34 (m, 2 H), 1.30 - 1.98 (m, 16 H), 0.91 - 1.19 (m, 4 H). 38 H's / 38 H's (spectrum / structure).Example 10. Synthesis of bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-(4-fluorosulfonyloxyphenyl)palladium(II) EC303

[0400] Step 1: 4-Bromophenol (2 g, 11.56 mmol, 1 eq), 2-methyl-l-(2-methylimidazol-l-yl)sulfonyl-imidazole (2.88 g, 12.72 mmol, 1.1 eq) and CS2CO3 (7.53 g, 23.12 mmol, 2 eq) were taken up into a microwave tube in ACN (20 mL). The sealed tube was heated at 120 °C for 15 min under microwave. TLC showed the starting reactant was consumed and two new spots were formed. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by column chromatography (SiCL, Commercial hexanes: Ethyl acetate=10 / l to 0 / 1). Compound (4-bromophenyl) 2-methylimidazole-l-sulfonate (3 g, 9.46 mmol, 81.8% yield) was obtained as a white solid.

[0401] Step 2: To a solution of (4-bromophenyl) 2-methylimidazole-l-sulfonate (3 g, 9.46 mmol, 1 eq) in AcOH (20 mL) was added KHF2 (2.22 g, 28.38 mmol, 935.16 pL, 3 eq). The mixture was stirred at 100 °C for 2 h. TLC showed the starting reactant was consumed and twonew spots were formed. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by column chromatography (SiCh, Commercial hexanes: Ethyl acetate=10 / l to 0 / 1). Compound l-bromo-4-fluorosulfonyloxy-benzene (EC303, 2.6 g, 10.19 mmol, 53.9% yield) was obtained as a colorless oil. 1HNMR (400 MHz, CHLOROFORM-d) 5 ppm 7.54 (d, J=8.8 Hz, 2 H) 7.25 (d, J=9.2 Hz, 2 H). 4 H's / 4 H’s (spectrum / structure).

[0402] Step 3: To a solution of l-bromo-4-fluorosulfonyloxy-benzene (1.5 g, 5.88 mmol, 1 eq) in THF (20 mL) was added sodium 3-(2-dicyclohexylphosphanylphenyl)-2,4-dimethoxy-benzenesulfonate;hydrate (4.68 g, 4.41 mmol, 0.75 equiv.) and bis(trimethylsilylmethyl)palladium;(lZ,5Z)-cycloocta-l,5-diene (3.44 g, 4.41 mmol, 0.75 equiv.). The mixture was stirred at 20 °C for 1 hr. LCMS showed the starting reactant was consumed and the desired compound was detected. The reaction mixture was concentrated under N2. The crude product was purified by reversed-phase HPLC (column: WePure Biotech XP tC18 250x70x10pm; mobile phase: [H20(0.03% TFA)-ACN];gradient:40%-70% B over 20.0 min). Compound bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-(4-fluorosulfonyloxyphenyl)palladium (2.2 g, 2.63 mmol, 59.6% yield, 97.7% purity) was obtained as a white solid. [M-NaBr + H] = 771.1. 1H NMR (400 MHz, METHANOL-d4) 5 ppm 8.11 (d, J=8.8 Hz, 1 H), 7.63 (t, J=7.6 Hz, 1 H), 7.38 - 7.52 (m, 2 H), 6.71 - 7.37 (m, 6 H), 3.79 (s, 3 H), 3.70 - 3.84 (m, 3 H), 1.58 - 2.07 (m, 12 H), 0.67 - 1.55 (m, 10 H). 38 H's / 38 H's (spectrum / structure).Example 11. Synthesis of bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-(3-fluorosulfonyloxyphenyl)palladium(II) EC304

[0403] Step 1: 3 -bromophenol (2 g, 11.56 mmol, 1 eq), 2-methyl-l-(2-methylimidazol-l-yl)sulfonyl-imidazole (2.88 g, 12.72 mmol, 1.1 eq) and CS2CO3 (7.53 g, 23.12 mmol, 2 eq) were taken up into a microwave tube in ACN (20 mL). The sealed tube was heated at 120 °C for 15 min under microwave conditions. TLC showed the starting reactant was consumed and two new spots were formed. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by column chromatography (SiC>2, Commercial hexanes: Ethyl acetate=10 / l to 0 / 1). Compound (3 -bromophenyl) 2-methylimidazole-l -sulfonate (3 g, 8.13 mmol, 70.37% yield, 86% purity) was obtained as a white solid.

[0404] Step 2: To a solution of (3 -bromophenyl) 2-methylimidazole-l -sulfonate (3 g, 8.13 mmol, 70.36% yield, 86% purity) in AcOH (30 mL) was added KHF2 (2.22 g, 28.38 mmol, 935.16 uL, 3 eq). The mixture was stirred at 100 °C for 2 h. TLC showed the starting reactant was consumed and two new spots were formed. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by column chromatography (SiCh, Commercial hexanes: Ethyl acetate=10 / l to 0 / 1). Compound l-bromo-3-fluorosulfonyloxy-benzene (2 g, 7.84 mmol, 82.90% yield, 100% purity) was obtained as a colorless oil.

[0405] Step 3: To a solution of l-bromo-3-fluorosulfonyloxy -benzene (1 g, 3.92 mmol, 1 eq) in THF (10 mL) was added sodium 3-(2-dicyclohexylphosphanylphenyl)-2,4-dimethoxy-benzenesulfonate hydrate (2.29 g, 4.31 mmol, 1.1 eq) and bis[(trimethylsilyl)methyl](l,5-cyclooctadiene)palladium(II) (1.68 g, 4.31 mmol, 1.1 eq). The mixture was stirred at 20 °C for 1 hr. LCMS showed the starting reactant was consumed and the desired compound was detected. The reaction mixture was concentrated under N2. The crude product was purified by reversed-phase HPLC (column: Welch Xtimate C18250*70mm#10pm;mobile phase: [H20(0.03% TFA)-ACN]; gradient: 50%-80% B over 20.0 min). Compound bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-(3-fluorosulfonyloxyphenyl)palladium (EC304, 1.6 g, 1.83 mmol, 46.64% yield, 100% purity) was obtained as a white solid. ’H NMR (400 MHz, METHANOL-d4) 5 ppm 8.11 (d, J=8.8 Hz, 1 H), 7.59 - 7.77 (m, 1 H), 7.39 - 7.58 (m, 2 H), 6.58 - 7.36 (m, 6 H), 3.80 (s, 3 H), 3.71 (s, 3 H), 1.56 - 2.17 (m, 12 H), 1.08 - 1.55 (m, 8 H), 0.81 - 1.06 (m, 2 H). 38 H's / 38 H's (spectrum / structure).Example 12. Synthesis of bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-[4-(phenoxycarbonylamino)phenyl]palladium(II) (EC315)EC315

[0406] Step 1) To a solution of 4-bromoaniline (2.5 g, 14.5 mmol, 1 eq) in THF (25 mL) was added TEA (1.47 g, 14.5 mmol, 2.0 mL, 1 eq) and then phenyl chloroformate (2.96 g, 18.9 mmol, 2.4 mL, 1.3 eq) was added dropwise at 0 °C. The mixture was stirred at 20 °C for 1 h. TLC showed the starting reactant was consumed completely. Another reaction with 2.5 g scale was carried out in parallel. The two reactions (2.5 g x 2) were combined together for work-up. The reaction mixture was poured into H2O 80 mL at 20 °C, and then extracted with EtOAc 80 mL (40 mL x 2). The combined organic layers were dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure to provide a residue that was purified by column chromatography (SiC>2, hexanes: Ethyl acetate = 20 / 1 to 10 / 1). Compound phenyl N-(4-bromophenyl)carbamate (3.8 g, 13.0 mmol, 45% yield) was obtained as a white solid. The material was used without further characterization.

[0407] Step 2) To a solution of phenyl N-(4-bromophenyl)carbamate (2 g, 6.85 mmol, 1 eq) in THF (30 mL) was added bis[(trimethylsilyl)methyl](l,5-cyclooctadiene)palladium(II) (2.93 g, 7.53 mmol, 1.1 eq) and sodium 3-(2-dicyclohexylphosphanylphenyl)-2,4-dimethoxy-benzenesulfonate hydrate (sSPhos) (4.00 g, 7.53 mmol, 1.1 eq). The mixture was stirred at 20 °Cfor 1 h. LCMS showed the starting reactant was consumed completely. To the mixture was added n-pentane (30 mL) and filtered, the filter cake was concentrated under reduced pressure to provide a residue that was purified by prep-HPLC (column: Phenomenex luna Cl 8 (250x70 mm, 15 um); mobile phase: [H2O (0.03% TFA)-ACN]; gradient: 45% - 75% B over 20.0 min).Compound bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-[4-(phenoxycarbonylamino)phenyl]palladium (1.05 g, 1.11 mmol, 16% yield, 96.5% purity) was obtained as a yellow solid. [M-NaBr+H+] = 808.2.!H NMR (400 MHz, METHANOL-^) 5 ppm 9.62 (s, 1 H), 8.14 (d, >8.8 Hz, 1 H), 7.57 - 7.63 (m, 1 H), 7.28 - 7.52 (m, 5 H), 7.14 - 7.27 (m, 5 H), 6.79 - 7.09 (m, 3 H), 3.85 (s, 3 H), 3.66 (s, 3 H), 1.82 - 1.98 (m, 5 H), 1.57 - 1.81 (m, 7 H), 0.96 - 1.50 (m, 10 H). 44 H's / 44 H's (spectrum / structure).Example 13. Synthesis of bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-[3-(phenoxycarbonylamino)phenyl]palladium(II) (EC309)

[0408] Step 1) To a solution of 3 -bromoaniline (5 g, 29.1 mmol, 1 eq) in EtOAc (50 mL) was added pyridine (4.90 g, 62.0 mmol, 2.13 eq) and then added dropwise phenyl chloroformate (4.80 g, 3.85 mL, 30.7 mmol, 1.06 eq) into the mixture at 0 °C. Then the mixture was stirred at 20 °C for 2 h. TLC indicated 3 -bromoaniline was consumed completely and one newspot formed. The reaction mixture was quenched by addition of 1 M HC1 (10 mL) at 20 °C, and then diluted with H2O (30 mL) and extracted with ethyl acetate 120 mL (40 mL x 3), dried over anhydrous Na2SC>4, filtered and concentrated under reduced pressure to provide a residue that was purified by column chromatography (SiC>2, Commercial hexanes: Ethyl acetate=l / O to 15 / 1). Compound phenyl N-(3 -bromophenyl) carbamate (6.1 g, 20.9 mmol, 72% yield) was obtained as a yellow solid. The material was used without further characterization.

[0409] Step 2) To a solution of phenyl N-(3-bromophenyl)carbamate (600 mg, 2.05 mmol, 1 eq) in THF (10 mL) was added bis[(trimethylsilyl)methyl](l,5-cyclooctadiene)palladium(II)(879 mg, 2.26 mmol, 1.1 eq) and sodium3-(2-dicyclohexylphosphanylphenyl)-2,4-dimethoxy-benzenesulfonate hydrate (1.20 g, 2.26 mmol, 1.1 eq). The mixture was stirred at 20 °C for Ih. LCMS showed the starting reactant was consumed and the desired compound was detected, n-Pentane (30 mL) was added, and the subsequent mixture was filtered. The residue was purified by prep-HPLC (column: Waters Xbridge BEH Cl 8250 x 70 mm x 10pm; mobile phase:[H20(0.03% TFA)-ACN];gradient:45%-75% B over 18.0 min). Compound bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-[3-(phenoxycarbonylamino)phenyl]palladium (600 mg, 644.1 pmol, 31% yield, 97.8% purity) was obtained as a white solid. [M-NaBr+H]+=808.2.1H NMR (400 MHz, METHANOL-^) 8 ppm 9.60 (s, 1 H), 8.16 (d, J=8.8 Hz, 1 H), 7.59 (t, J=6.8 Hz, 1 H), 7.45 - 7.52 (m, 1 H), 7.40 (t, J=7.6 Hz, 3 H), 7.19 - 7.31 (m, 3 H), 7.16 (d, J=8.0 Hz, 2 H), 7.09 (d, J=7.2 Hz, 1 H), 7.03 (d, J=8.8 Hz, 1 H), 6.93 (t, J=6.4 Hz, 1 H), 6.72 -6.90 (m, 1 H), 3.84 (s, 3 H), 3.60 (s, 3 H), 1.80 - 2.02 (m, 5 H), 1.55 - 1.78 (m, 7 H), 1.36 - 1.48 (m, 1 H), 0.80 - 1.34 (m, 9 H). 44 H's / 44 H's (spectrum / structure).Example 14. Synthesis of bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-(l-fluorosulfonyIpyrazol-4-yl)palladium(II) (EC313)

[0410] Step 1) To a solution of 4-bromo-lH-pyrazole (2 g, 13.6 mmol, 1 eq) in DCM (10 mL) was added 2,3 -dimethylimidazol-3-ium-l -sulfonyl fluoride trifluoromethanesulfonate (4.47 g, 13.6 mmol, 1 eq) at 0 °C. The mixture was stirred at 20 °C for 4 h. TLC showed the starting reactant was consumed and one new spot was formed. The mixture was concentrated under reduced pressure to provide a residue that was purified by column chromatography(SiCL, hexane: Ethyl acetate=l / O to 3 / 1). Compound 4-bromopyrazole-l -sulfonyl fluoride (1.8 g, 7.86 mmol, 58% yield) was obtained as colorless oil.JH NMR (400 MHz, CHLOROFORM-d) 8 ppm 8.13 (s, 1 H), 7.92 (s, 1 H). 2 H's / 2 H's (spectrum / structure).

[0411] Step 2) To a solution of 4-bromopyrazole-l -sulfonyl fluoride (200 mg, 873 pmol, 1 eq) in THF (10 mL) was added bis[(trimethylsilyl)methyl](l,5-cyclooctadiene)palladium(II) (374 mg, 961 pmol, 1.1 eq) and sodium 3-(2-dicyclohexylphosphanylphenyl)-2,4-dimethoxy-benzenesulfonate hydrate (sSPhos) (510 mg, 961 pmol, 1.1 eq). The mixture was stirred at 20 °C for 1 h. LCMS showed the starting reactant was consumed. To the mixture was added n-pentane (10 mL) and filtered, the filter cake was concentrated under reduced pressure to provide a residue that was purified by prep-HPLC (column: Xbridge OBD C18 100mm x 30mm x 5pm, Kinetex EVO C18 100mm x 30mm x 5pm; mobile phase: [H20(0.03% TFA)-ACN];gradient:40%-80% B over 14.0 min). Compound bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-(l-fluorosulfonylpyrazol-4-yl)palladium (433 mg, 507 pmol, 58% yield, 99.2% purity) was obtained as a white solid. [M-NaBr+H+]=745.1. 'H NMR (400 MHz, METHANOL-^) 8 ppm 8.08 (d, J=8.8 Hz, 1 H), 6.80 -7.77 (m, 7 H), 3.71 (s, 6 H), 1.55 - 2.26 (m, 16 H), 0.90 - 1.40 (m, 6 H). 36 H's / 36 H's (spectrum / structure).19F NMR (400 MHz, METHANOL-d4) 8 ppm 50.71 - 50.84 (m, 1 H), -77.71 (s, 1.4 H, residual TFA).Example 15. Synthesis of bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-[4-(prop-2-enoylamino)phenyl]palladium(II) (EC310)EC310

[0412] Step 1) To a solution of 4-bromoaniline (5.00 g, 29.1 mmol, 1 eq) in DCM (50 mL) was added TEA (5.88 g, 58.1 mmol, 8.09 mL, 2 eq). Then prop-2-enoyl chloride (3.16 g, 34.9 mmol, 1.2 eq) was added dropwise to the mixture at 0 °C. The mixture was stirred at 20 °C for 2 h. TLC showed the starting reactant was consumed and one new spot was formed. The reaction mixture was quenched by addition H2O 500 mL at 0 °C, and then filtered, the filter cake was concentrated under reduced pressure to give a residue. Compound N-(4-bromophenyl)prop-2-enamide (5.8 g, 25.7 mmol, 88% yield) was obtained as a yellow solid and was used in the next step without further characterization.

[0413] Step 2) To a solution of N-(4-bromophenyl)prop-2-enamide (0.75 g, 3.32 mmol,I eq) in THF (10 mL) was added bis[(trimethylsilyl)methyl](l,5-cyclooctadiene)palladium(II) (1.42 g, 3.65 mmol, 1.1 eq) and sodium3-(2-dicyclohexylphosphanylphenyl)-2,4-dimethoxy-benzenesulfonate;hydrate (1.94 g, 3.65 mmol, 1.1 eq). The mixture was stirred at 20 °C for 1 h. LCMS showed most starting reactant was consumed and the desired compound was detected. Another reaction with 0.75 g scale was carried out in parallel. The two reactions (0.75 g x 2) were combined together for work-up. The mixture was filtered and the filter cake was concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18250 x 70 mm x 10 um; mobile phase: [H20(0.03% TFA)-ACN]; gradient:30%-65% B over 20.0 min). Compound bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-[4-(prop-2-enoylamino)phenyl]palladium (1.26 g, 1.35 mmol, 20% yield, 90.3% purity) was obtained as a yellow solid. [M-NaBr+H]+=742.3. 'H NMR (400 MHz, METHANOL-d48 ppm 9.85 (s, 1 H), 8.14 (d, J=9.2 Hz, 1 H), 6.10 - 7.98 (m, II H), 5.52- 5.92 (m, 1 H), 3.84 (s, 3 H), 3.62 (s, 3 H), 1.58 -2.28 (m, 14 H), 1.02 - 1.48 (m, 8 H). 42 H's / 42 H's (spectrum / structure).Example 16. Synthesis of bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-(3-sulfamoylphenyl)palladium(II) (EC306)

[0414] To a solution of 3 -bromobenzenesulfonamide (0.6 g, 2.54 mmol, 1 eq) in THF (20 mL) was added bis[(trimethylsilyl)methyl](l,5-cyclooctadiene)palladium(II) (1.09 g, 2.80 mmol, 1.1 eq) and sodium 3-(2-dicyclohexylphosphanylphenyl)-2,4-dimethoxy-benzenesulfonate»hydrate (sSPhos; 1.48 g, 2.80 mmol, 1.1 eq). The mixture was stirred at 20 °C for 1 h. LCMS showed the starting reactant was consumed and the desired Pd complex was formed. The reaction mixture was concentrated under reduced pressure to remove solvent. The crude product was purified by reversed-phase HPLC (column: WePure Biotech XP tC18 150*40*7pm; mobile phase: [H2O (0.03% TFA)-ACN]; gradient: 10%-50% B over 15.0 min). Compound bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-(3-sulfamoylphenyl)palladium(1.1 g, 1.28 mmol, 50.31% yield, 99.5% purity) was obtained as a yellow solid. [M-NaBr+H+]=752.1. 'H NMR (400 MHz, METHANOL-^) 5 ppm 8.13 (d, J=8.8 Hz, 1 H), 7.49 -7.71 (m, 3 H), 7.35 - 7.46 (m, 2H), 6.89 - 7.32 (m, 4 H), 3.83 (s, 3 H), 3.70 (s, 3 H), 1.55 - 1.98 (m, 12 H), 0.86 - 1.49 (m, 10 H). 38 H's / 40 H's (spectrum / structure).Example 17. Synthesis of bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-[4-[(l-fluorosulfonylazetidine -3-carbonyl)amino]phenyljpalladium(ll) (EC312)

[0415] Step 1) To a solution of l-fluorosulfonylazetidine-3-carboxylic acid (2 g, 10.9 mmol, 1 eq) in DMF (20 mL) was added HATU (4.98 g, 13.1 mmol, 1.2 eq) and DIEA (2.82 g, 21.8 mmol, 3.80 mL, 2 eq) and 4-bromoaniline (1.88 g, 10.9 mmol, 1 eq). The mixture was stirred at 20 °C for 2 h. TLC showed the starting material was consumed. The reaction mixture was quenched by addition of H2O 50 mL at 0 °C, and then extracted with EtOAc 90 mL (30 mL x 3). The combined organic layers were washed with sat. brine 90 mL (30 mL x 3), driedover anhydrous Na2SC>4, filtered and concentrated under reduced pressure to provide a residue that was purified by column chromatography (SiC>2, Commercial hexanes: Ethyl acetate=10 / l to 0 / 1). Compound 3-[(4-bromophenyl)carbamoyl]azetidine-l-sulfonyl fluoride (1 g, 2.97 mmol, 27% yield) was obtained as a yellow solid. The material was used in the next step without additional characterization.

[0416] Step 2) To a solution of 3-[(4-bromophenyl)carbamoyl]azetidine-l-sulfonyl fluoride (500 mg, 1.48 mmol, 1 eq) in THF (10 mL) was added bis[(trimethylsilyl)methyl](l,5-cyclooctadiene)palladium(II) ((l,5-COD)Pd(CH2TMS)2) (635 mg, 1.63 mmol,1.1 eq) and sodium 3 -(2-dicy cl ohexylphosphanylphenyl)-2,4-dimethoxy -benzenesulfonate hydrate (sSPhos) (865 mg, 1.63 mmol, 1.1 eq). The mixture was stirred at 20 °C for 1 h. LCMS showed the starting material was consumed. To the mixture was added n-pentane (10 mL) and filtered, the filter cake was concentrated under reduced pressure to provide a residue that waspurified by prep-HPLC (column: Phenomenex luna Cl 8 (250 x 70mm, 15 pm);mobile phase:[H2O (0.03% TFA)-ACN];gradient:40%-70% B over 20.0 min). Compound bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-[4-[(l-fluorosulfonylazetidine -3-carbonyl)amino]phenyl]palladium (610 mg, 613 pmol, 41% yield, 96.1% purity) was obtained as a yellow solid. [M-NaBr+H]+=853.3. 'H NMR (400 MHz, METHANOL-d4) 5 ppm 8.14 (d, J=8.8 Hz, 1 H), 7.60 (t, J=7.2 Hz, 1 H), 7.16 - 7.52 (m, 5 H), 6.84 - 7.15 (m, 3 H), 4.27 - 4.47 (m, 4 H), 3.84 (s, 3 H), 3.55 - 3.73 (m, 4 H), 1.57 - 2.00 (m, 12 H), 0.92 - 1.47 (m, 10 H). 43 H's / 44 H's (spectrum / structure).19F NMR (400 MHz, METHANOL-^) 5 ppm 26.88 (s, 1 F), -77.79 (s, 2 F, residual TFA).Example 18. Synthesis of bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-[3-[(l-fluorosulfonylazetidine-3-carbonyl)amino]phenyl]palladium(II) (EC314)

[0417] Step 1) To a solution of 3 -bromoaniline (1.69 g, 9.83 mmol, 1.1 mL, 1 eq) and 1-fluorosulfonylazetidine-3-carboxylic acid (1.8 g, 9.83 mmol, 1 eq) in DMF (30 mL) was added TBTU (3.79 g, 11.8 mmol, 1.2 eq) and DIEA (3.81 g, 29.5 mmol, 5.2 mL, 3 eq). The mixture was stirred at 20 °C for 1 h. TLC showed the starting reactant was consumed completely. The reaction mixture was diluted with H2O 50 mL and extracted with EtOAc 50 mL (25 mL x 2). The combined organic layers were washed with brine 50 mL (25 mL x 2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to provide a residue that was purified by column chromatography (SiC>2, Commercial hexanes: Ethyl acetate = 10 / 1 to 2 / 1). Compound 3-[(3-bromophenyl)carbamoyl]azetidine-l -sulfonyl fluoride (2.6 g, 7.71 mmol, 78% yield) was obtained as a yellow solid. The material was used without further characterization

[0418] Step 2) To a solution of 3-[(3-bromophenyl)carbamoyl]azetidine-l-sulfonyl fluoride (0.6 g, 1.78 mmol, 1 eq) in THF (30 mL) was added bis[(trimethylsilyl)methyl](l,5-cyclooctadiene)palladium(II) (762 mg, 1.96 mmol, 1.1 eq) and sodium 3-(2-dicyclohexylphosphanylphenyl)-2,4-dimethoxy -benzenesulfonate hydrate (sSPhos) (1.04 g, 1.96mmol, 1.1 eq). The mixture was stirred at 20 °C for 1 h. LCMS showed the starting reactant was consumed completely. To the mixture was added n-pentane (20 mL) and filtered, the filter cake was concentrated under reduced pressure to provide the compound bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxy sulfonyl -phenyl)phenyl]-phosphanyl]-[3-[(l -fluorosulfonylazetidine-3-carbonyl)amino]phenyl]palladium (0.6 g, 0.58 mmol, 33% yield, 92.6% purity) was obtained as a yellow solid. [M-NaBr+H+] = 853.0. 'HNMR (400 MHz, METHANOL-d4) 5 ppm 8.13 (d, J=8.4 Hz, 1 H), 7.52 - 7.59 (m, 1 H), 7.41 - 7.47 (m, 1 H), 7.15 - 7.37 (m, 4 H), 7.03 (d, J=8.4 Hz, 1 H), 6.88 - 6.93 (m, 1 H), 6.51 - 6.75 (m, 1 H), 4.29 - 4.44 (m, 5 H), 3.80 - 3.91 (m, 3 H), 3.51 - 3.68 (m, 3 H), 1.57 - 1.85 (m, 10 H), 0.94 - 1.40 (m, 12 H). 43 H's / 44 H’s (spectrum I structure).19F NMR (400 MHz, METHANOL-d4) 8 ppm 26.80 - 26.86 (m, 1 F).Example 19. Synthesis of bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-[3-(methylaminomethyl)phenyl]palladium(II) (EC305)

[0419] To a solution of 1 -(3 -brom ophenyl)-N-methyl-m ethanamine (0.8 g, 4.00 mmol, 547.57 pL, 1 eq) in THF (50 mL) was added bis[(trimethylsilyl)methyl](l,5-cyclooctadiene)palladium(II) (1.71 g, 4.40 mmol, 1.1 eq) and sodium 3-(2-dicyclohexylphosphanylphenyl)-2,4-dimethoxy-benzenesulfonate hydrate (2.33 g, 4.40 mmol, 1.1 eq). The mixture was stirred at 20 °C for 1 hr. LCMS showed l-(3-bromophenyl)-N-methyl-methanamine was consumed completely and desired mass was detected. The mixture was added n-pentane and filtered, the filter cake was concentrated under N2. The residue was purified by prep-HPLC (column: WePure Biotech XP tC18250*70*10 pm; mobile phase: [H2O (0.03% TFA) - ACN]; gradient: 15% - 45% B over 18.0 min); to yield the final Pd complex EC305, bromo-[dicyclohexyl-[2-(2,6-dimethoxy-3-sodiooxysulfonyl-phenyl)phenyl]-phosphanyl]-[3-(methylaminomethyl)phenyl]palladium (1 g, 1.22 mmol, 30.45% yield) as a white solid. [M-NaBr+H+]=716.2. ’H NMR (400 MHz, METHANOL-^) 8 ppm 8.14 (d, J=8.8 Hz, 1 H), 7.45 -7.67 (m, 3 H), 7.34 - 7.44 (m, 1 H), 7.14 - 7.26 (m, 1 H), 6.94 - 7.13 (m, 4 H), 4.11 - 3.92 (m, 2H), 3.80 - 3.90 (m, 3 H), 3.43 - 3.63 (m, 3 H), 2.66 - 2.78 (m, 3 H), 1.60 - 2.26 (m, 13 H), 0.98 -1.50 (m, 9 H). 43 H's / 44 H's (spectrum / structure).Example 20. Synthesis of EC201

[0420] Step 1) Resin preparation: To a solution of 2-CTC Resin (5.0 mmol, sub 0.5 mmol / g) and Fmoc-Lys (Dde)-OH (1.0 eq) in DCM (100 mL) was added DIEA (6.0 eq). The mixture was agitated with N2 at 25 °C for 2.5 hrs. The MeOH (10.0 mL) was added in the resin and agitated with N2 at 25 °C for 0.5 h. Then the mixture was filtered to get the resin. The resin was washed with DMF (200 mL x 5).

[0421] Step 2) Deprotection: 20% piperidine in DMF (200 mL) was added and the resin was agitated with N2 at 25 °C for 10 min. The resin was washed with DMF (200 mL x 5) and filtered.

[0422] Step 3)Coupling: A solution of bis(4-nitrophenyl) carbonate (6.0 eq) in DMF (100 mL) was mixed with DIEA (6.0 eq) and added to the resin. The mixture was agitated with N2 at 25 °C for 60 min. The resin was then washed with DMF (200 mL x 5). A solution of NH2-Glu(OtBu)-OtBu HC1 (3.0 eq) and HOBt (3.00 eq) in pyridine (30 mL) was to the resin, the mixture was agitated with N2 at 25 °C for 18 h. The resin was washed with DMF (200 mL x 5).

[0423] Step 4)Dde deprotection: 3% ^ELrELO / DMF (200 mL) was added to the resin and the mixture was agitated with N2 at 25 °C for 15 min. The resin was washed with DMF (200 mL x 5).

[0424] Step 5)Coupling: After Dde removal, a solution of Fmoc-2-Nal-OH (3.0 eq) and HOAt (3.0 eq) in DMF (100 mL), and DIC (3.0 eq) was added to the resin. The mixture was agitated with N2 for 1.5 h at 25°C. The resin was then washed with DMF (200 mL x 5).

[0425] Step 6) Deprotection: 20% piperidine in DMF (200.0 mL) was added to the resin. The mixture was agitated with N2 at 25 °...

Claims

CLAIMSWhat is claimed is:

1. A compound of Formulae (I’):R'— T-L-Tx(r).or a pharmaceutically acceptable salt thereof,wherein:R’ comprises a monovalent reactive group;T is a targeting moiety capable of specifically interacting with a target; Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

2. A compound of Formula (IF)or a pharmaceutically acceptable salt thereof,wherein:R” comprises a divalent reactive group;T is a targeting moiety capable of specifically interacting with a target; Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

3. A compound of Formula (III’)or a pharmaceutically acceptable salt thereof,wherein:R’ comprises a monovalent reactive group;R” comprises a divalent reactive group;T is a targeting moiety capable of specifically interacting with a target; Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

4. A compound of Formula (IV’)T-L-R"-Tx(IV,Xor a pharmaceutically acceptable salt thereof,wherein:R” comprises a divalent reactive group;T is a targeting moiety capable of specifically interacting with a target; Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

5. A compound of Formula (V’)or a pharmaceutically acceptable salt thereof,wherein:R’ comprises a monovalent reactive group;R” comprises a divalent reactive group;T is a targeting moiety capable of specifically interacting with a target; Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

6. A compound of Formula (VI’)or a pharmaceutically acceptable salt thereof,wherein:R’ comprises a monovalent reactive group;T is a targeting moiety capable of specifically interacting with a target;Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

7. A compound of Formula (VIF)(vir),or a pharmaceutically acceptable salt thereof,wherein:R’ comprises a monovalent reactive group;R” comprises a divalent reactive group;T is a targeting moiety capable of specifically interacting with a target;Tx is absent, a therapeutic moiety, or a diagnostic moiety; andL is absent or a linker.

8. The compound according to any one of claims 1, 3, or 5-7, wherein R’ is represented by Formula (II- A):wherein:LAis a bond or chain of 1-60 carbon atoms in length,wherein one or more carbon atoms are optionally substituted with halogen, hydroxyl, cyano, CFH2, CF2H, CF3, alkoxy, C1-C5 alkyl, or -C(=O)-NH2; andwherein one or more carbon atoms of the backbone are optionally replaced by a divalentgroup independently selected from oxygen, alkylamino, carbonyl, 3-14 membered cycloalkylene, 3-8 membered monocyclic heterocyclic group, 5-14 membered bridged heterocyclic group, 5-14 membered spiro heterocyclic group, 5-14 membered heteroarylene, and 7-14 membered heteroaryLfused-heterocyclylene, and wherein each of the 3-14 membered cycloalkylene, 3-8 membered monocyclic heterocyclic group, 5-14 membered bridged heterocyclic group, 5-14 membered spiro heterocyclic group, 5-14 membered heteroarylene, and 7-14 membered heteroaryLfused-heterocyclylene is independently substituted with 0, 1, 2, 3, 4, 5, or 6 Ra;each Rais independently selected from alkylamino, halogen, hydroxyl, C1-C5 alkoxy, C1-C5 alkyl, C1-C5 alkylcarboxyl, C1-4 alkenyl, C3-5 cycloalkyl, C1-5haloalkyl, and an oxo group; andRwis an electrophilic functional group capable of forming a covalent bond with the target.

9. The compound according to claim 8, wherein the compound of Formula (ILA) is represented by a compound of Formula (ILA-1):(ILA-1),whereinLA1and LA2are each independently a bond or chain of 1-30 carbon atoms in length, wherein one or more carbon atoms are optionally substituted with halogen, hydroxyl, cyano, oxo, CFH2, CF2H, CF3, alkoxy, C1-C5 alkyl, or -C(=O)-NH2; andwherein one or more carbon atoms of the backbone are optionally replaced by a divalent group independently selected from oxygen, alkylamino, carbonyl, 3-14 membered cycloalkylene, 3-8 membered monocyclic heterocyclic group, 5-14 membered bridged heterocyclic group, 5-14 membered spiro heterocyclic group, 5-14 membered heteroarylene, and 7-14 membered heteroaryLfused-heterocyclylene, and wherein each of the 3-14 membered cycloalkylene, 3-8 membered monocyclic heterocyclic group, 5-14 membered bridged heterocyclic group, 5-14 membered spiro heterocyclic group, 5-14membered heteroarylene, and 7-14 membered heteroaryl-fused-heterocyclylene is independently substituted with 0, 1, 2, 3, 4, 5, or 6 Ral;each Ralis independently selected from alkylamino, halogen, hydroxyl, C1-C5 alkoxy, C1-C5 alkyl, C1-C5 alkylcarboxyl, C1-4 alkenyl, C3-5 cycloalkyl, C1-5haloalkyl, glycosyl, amino acid, and an oxo group;Ring A is a bond or optionally substituted bivalent 3- to 14-membered ring system; optionally substituted bivalent 3- to 10-membered monocyclic, bicyclic, bridged, or spirocyclic carbocyclic ring system; optionally substituted bivalent 3- to 10-membered monocyclic, bicyclic, bridged, or spirocyclic heterocyclic ring system; optionally substituted bivalent 5- to 10-membered heteroarylene; and optionally substituted bivalent Ce-14 arylene, wherein each of the 3- to 14-membered ring system; 3- to 10-membered monocyclic, bicyclic, bridged, or spirocyclic carbocyclic ring system; 3- to 10-membered monocyclic, bicyclic, bridged, or spirocyclic heterocyclic ring system; 5- to 10-membered heteroarylene; and Ce-14 arylene is optionally independently substituted with 0, 1, 2, 3, 4, 5, or 6 RA; andeach RAis independently selected from alkylamino, halogen, hydroxyl, C1-C5 alkoxy, C1-C5 alkyl, C1-4 alkenyl, C3-5 cycloalkyl, C1-5 haloalkyl, glycosyl, amino acid, and an oxo group.

10. The compound according to claim 8 or 9, wherein Rwis represented by:wherein:X10is a bond or selected from -N(R10)-, -O-, -CH2-, -(CH?)?-, -(CH?)3- and optionally substituted 5- to 10-membered heteroaryl;X20is selected from -C(O)-, -S(O)2-, -S(O)(NR20)-, and optionally substituted 5- to 10- membered heteroaryl;X30is selected from -F, -Cl, and optionally substituted 5- to 10-membered heteroaryl; X40is selected from C, -CH=, -CF=, -CCH3=, -CCF3=, -C(CN)=, and -C(N(R40)2)=; X50is selected from =CH2, =CHCH3, =CHF, =CF2, =CHCF3;X60is selected -N(R10)2-, or -OCi-ealkyl;each of R10, R20, and R40are independently selected from hydrogen, C1-3 alkyl, optionally substituted C1-3 haloalkyl, optionally substituted C1-3 alkoxyl.

11. The compound according to claim 8 or 9, wherein Rwis represented by:wherein:X20is selected from C(O)-, -S(O)2-, -S(O)(NR20)-;X30is selected from -CH=, -CCH3=, -CF=, -CCF3=, -C(CN)=, and -C(N(R30)2)=; X40is selected from -CH=, -CCH3=, -CF=, -CCF3=, -C(CN)=, and -C(N(R30)2)=; X50is selected from -CH2-, -CHCH3-, -CHF-, -CF2-, and -CHCF3-;each R20is independently selected from hydrogen, optionally substituted Ci.3alkyl, optionally substituted Ci-3haloalkyl, optionally substituted Ci-3alkoxyl;each R30is independently selected from hydrogen, optionally substituted Ci-3alkyl, optionally substituted Ci.3haloalkyl, optionally substituted Ci-3alkoxyl; andp is 1 or 2.

12. The compound according to any one of claims 8-11, wherein LA1and LA2are each independently:wherein each nm is independently an integer selected from 0 to 15.

13. The compound according to claim 3, wherein Formula (II-A-1) is represented by:wherein nm is an integer selected from 0 to 15.

14. The compound according to any one of claims 8-13, wherein Rwis selected from:

15. The compound according to any one of claims 8-14, wherein Rwis selected from:

16. The compound according to claim 2-5, or 7, wherein R” is represented by Formula (II-B):wherein:LAand LDare each independently a bond or chain of 1-60 carbon atoms in length,wherein one or more carbon atoms are optionally substituted with halogen, hydroxyl, cyano, CFH2, CF2H, CF3, alkoxy, C1-C5 alkyl, or -C(=O)-NH2; andwherein one or more carbon atoms of the backbone are optionally replaced by a divalent group independently selected from oxygen, alkylamino, carbonyl, 3-14 membered cycloalkylene, 3-8 membered monocyclic heterocyclic group, 5-14 membered bridged heterocyclic group, 5-14 membered spiro heterocyclic group, 5-14 membered heteroarylene, and 7-14 membered heteroaryl-fused-heterocyclylene, and wherein each of the 3-14 membered cycloalkylene, 3-8 membered monocyclic heterocyclic group, 5-14 membered bridged heterocyclic group, 5-14 membered spiro heterocyclic group, 5-14membered heteroarylene, and 7-14 membered heteroaryl-fused-heterocyclylene is independently substituted with 0, 1, 2, 3, 4, 5, or 6 Ra;each Rais independently selected from alkylamino, halogen, hydroxyl, C1-C5 alkoxy, C1-C5 alkyl, C1-C5 alkylcarboxyl, C1-4 alkenyl, C3-5 cycloalkyl, C1-5 haloalkyl, and an oxo group; andRwis an electrophilic functional group capable of forming a covalent bond with the target.

17. The compound according to claim 16, wherein the compound of Formula (IT-B) comprises a Michael acceptor capable of Elcb elimination (retro-Michael) of -Tx or -L- Tx.

18. The compound according to claim 16 or 17, wherein Formula (II-B) is or comprises:wherein* represents the point of attachment to T; andnq is an integer from 0 to 15.

19. The compound according to any one of the preceding claims, wherein L is a chain of 1- 60 carbon atoms in length,wherein one or more carbon atoms are optionally substituted with halogen, hydroxyl, cyano, CFH2, CF2H, CF3, alkoxy, C1-C5 alkyl, or -C(=O)-NH2; andwherein one or more carbon atoms of the backbone are optionally replaced by a divalent group independently selected from oxygen, alkylamino, carbonyl, 3-14 membered cycloalkylene, 3-8 membered monocyclic heterocyclic group, 5-14 membered bridged heterocyclic group, 5-14 membered spiro heterocyclic group, 5-14 membered heteroarylene, and 7-14 membered heteroaryl-fused-heterocyclylene, and wherein each of the 3-14 membered cycloalkylene, 3-8 membered monocyclic heterocyclic group, 5-14 membered bridged heterocyclic group, 5-14 membered spiro heterocyclic group, 5-14 membered heteroarylene, and 7-14 membered heteroaryl-fused-heterocyclylene is independently substituted with 0, 1, 2, 3, 4, 5, or 6 RL; andeach RLis independently selected from alkylamino, halogen, hydroxyl, C1-C5 alkoxy, C1-C5 alkyl, C1-C5 alkyl carboxyl, C1-4 alkenyl, C3-5 cycloalkyl, C1-5haloalkyl, and an oxo group.

20. The compound according any one of the preceding claims, wherein L comprises a mercapto-succinimidyl adduct group, hydrophilic PEG spacer, valine-citrulline (Val-Cit) dipeptide, valine-alanine (Vai-Ala) dipeptide, alanine-alanine (Ala-Ala) dipeptide, acarbohydrate-substituted aryl (e g., phenolic glucuronide), a peptide further substituted with a carbohydrate, a para-aminobenzyl (PAB) spacer, or a combination thereof.

21. The compound according to claim 19 or 20, wherein L is represented by:wherein:# represents the point of attachment to T;* represents the point of attachment to R’; and$ represents the point of attachment to Tx.

22. The compound according to any one of claims 19-21, wherein L is represented by:

23. The compound according to any one of claims 1-3 or 6, wherein -L-Tx is represented by:

24. The compound according to claim 15, wherein -L-Tx is represented by:

25. The compound according to any one of claims 16-21, wherein -L-R”- is represented by:

26. The compound according to any one of claims 16-20, wherein -L-R”-T is represented by:

27. The compound according to any one of claims 1-26, wherein T is a targeting moiety capable of interacting with PSMA, PTHR, PTH1R, PTH2R, PTH3R, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-1, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R,GRPR, STRAP 1, CD70, CD46, CD 166, CLL-1, ADAM9, cKIT, CD36, CD73, ITGaVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, IL-6R, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, ROR1, TROP-2, CX3CR1, CDH17, NECTIN-4, cMET, CD 19, CD20, CD22, CD30, CD33, CD 123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, tau, TREM2, TSLP, mesothelin, PCSK9, ANGPTL3, Lp(a), FGFRlc / p-Klotho, C5, Factor D, MASP-2, properdin, VEGF-A, Ang-2, ROCK, IL-2R, CTLA-4, CD28, CD40L, IL-4Ra, IL-5Ra, IL-10R, IgE, CRTH2, GLP-1, LAG-3, TIGIT, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, pCadherin, CEACAM6, CD47, orEpCAM.

28. The compound according to any one of claims 1-27, wherein T is or comprises29. The compound according to any one of claims 1-27, wherein T is or comprises a sequence selected from Table 6, Table 7, and / or Table 8.

30. The compound according to any one of claims 1-29, wherein Tx is selected from:

31. The compound according to any one of claims 1 -7, wherein the compound is selected from Table 2 or a pharmaceutically acceptable salt thereof.

32. The compound according to any one of claims 1-7, wherein the compound is selected from Table 6 or a pharmaceutically acceptable salt thereof.

33. A pharmaceutical composition comprising a compound according to any one of the preceding claims and a pharmaceutically acceptable carrier.

34. A method of binding a target, the method comprising contacting the target with a compound according to any one of claims 1-32 or a pharmaceutical composition according to claims 20, wherein the target is selected from PSMA, PTHR, PTH1R, PTH2R, PTH3R, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-1, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD 16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD 166, CLL-1, ADAM9, cKIT, CD36, CD73, ITGaVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, IL-6R, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, ROR1, TROP-2, CX3CR1, CDH17, NECTIN-4, cMET, CD19, CD20, CD22, CD30, CD33, CD123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, tau, TREM2, TSLP, mesothelin, PCSK9, ANGPTL3, Lp(a), FGFRlc / p-Klotho, C5, Factor D, MASP-2, properdin, VEGF-A, Ang-2, ROCK, IL-2R, CTLA-4, CD28, CD40L, IL-4Ra, IL-5Ra, IL-10R, IgE, CRTH2, GLP-1, LAG-3, TIGIT, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, pCadherin, CEACAM6, CD47, DPP6, ZNT8, GPR44, PTPRN, GLP1R, and EpCAM.

35. A method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1-32 or a pharmaceutical composition according to claim 33.

36. The method according to claim 35, wherein the disease or disorder is selected from cancer, an autoimmune disease, an inflammatory disease, a neurological disease, neuroinflammation, respiratory disease, asthma, cardiovascular disease, metabolic disease or disorder, ophthalmologic disease, and an infectious disease.

37. The method according to claim 36, wherein the cancer is selected bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eyecancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, thyroid cancer, and prostate cancer.

38. The method according to claim 35, wherein the disease or disorder is selected from acinetobacter infections, actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (acquired immune deficiency syndrome), amebiasis, anaplasmosis, anthrax, arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, bacillus cereus infection, bacterial pneumonia, bacterial vaginosis, bacteroides infection, balantidiasis, baylisascaris infection, BIC virus infection, black piedra, blastocystis hominis infection, blastomycosis, Bolivian hemorrhagic fever, borrelia infection, botulism (and infant botulism), Brazilian hemorrhagic fever, brucellosis, burkholderia infection, buruli ulcer, calicivirus infection (norovirus and sapovirus), campylobacteriosis, candidiasis (moniliasis; thrush), cat-scratch disease, cellulitis, Chagas disease (American trypanosomiasis), chancroid, chickenpox, chlamydia, chlamydophila pneumoniae infection, cholera, chromoblastomycosis, clonorchiasis, Clostridium difficile infection, coccidioidomycosis, Colorado tick fever, common cold (acute viral rhinopharyngitis; acute coryza), Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever, cryptococcosis, cryptosporidiosis, cutaneous larva migrans, cyclosporiasis, cysticercosis, cytomegalovirus infection, Dengue fever, dientamoebiasis, diphtheria, diphyllobothriasis, dracunculiasis, ebola hemorrhagic fever, echinococcosis, ehrlichiosis, enterobiasis (pinworm infection), enterococcus infection, enterovirus infection, epidemic typhus, erythema infectiosum (fifth disease), exanthem subitum, Fasciolopsiasis, Fasciolosis, Fatal familial insomnia, Filariasis, Food poisoning by Clostridium perfringens, Free-living amebic infection, Fusobacterium infection, Gas gangrene (Clostridial myonecrosis), Geotrichosis, Gerstmann-Straussler-Scheinker syndrome, Giardiasis, Glanders, Gnathostomiasis, Gonorrhea, Granuloma inguinale (Donovanosis), Group A streptococcal infection, Group B streptococcal infection, Haemophilus influenzae infection, Hand, foot and mouth disease (HFMD), Hantavirus Pulmonary Syndrome, Helicobacter pylori infection, Hemolytic-uremic syndrome, Hemorrhagic fever with renal syndrome, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex, Histoplasmosis, Hookworm infection, Human bocavirus infection, Human ewingii ehrlichiosis, Human granulocytic anaplasmosis,Human metapneumovirus infection, Human monocytic ehrlichiosis, Human papillomavirus infection, Human parainfluenza virus infection, Hymenolepiasis, Epstein-Barr Virus Infectious Mononucleosis (Mono), Influenza, Isosporiasis, Kawasaki disease, Keratitis, Kingella kingae infection, Kuru, Lassa fever, Legionellosis (Legionnaires’ disease), Legionellosis (Pontiac fever), Leishmaniasis, Leprosy, Leptospirosis, Listeriosis, Lyme disease (Lyme borreliosis), Lymphatic filariasis (Elephantiasis), Lymphocytic choriomeningitis, Malaria, Marburg hemorrhagic fever, Measles, Melioidosis (Whitmore’s disease), Meningitis, Meningococcal disease, Metagonimiasis, Microsporidiosis, Molluscum contagiosum, Mumps, Murine typhus (Endemic typhus), Mycoplasma pneumonia, Mycetoma, Myiasis, Neonatal conjunctivitis (Ophthalmia neonatorum), (New) Variant Creutzfeldt-Jakob disease (vCJD, nvCJD), Nocardiosis, Onchocerciasis (River blindness), Paracoccidioidomycosis (South American blastomycosis), Paragonimiasis, Pasteurellosis, Pediculosis capitis (Head lice), Pediculosis corporis (Body lice), Pediculosis pubis (Pubic lice, Crab lice), Pelvic inflammatory disease, Pertussis (Whooping cough), Plague, Pneumococcal infection, Pneumocystis pneumonia, Pneumonia, Poliomyelitis, Prevotella infection, Primary amoebic meningoencephalitis, Progressive multifocal leukoencephalopathy, Psittacosis, Q fever, Rabies, Rat-bite fever, Respiratory syncytial virus infection, Rhinosporidiosis, Rhinovirus infection, Rickettsial infection, rickettsialpox, Rift Valley fever, Rocky mountain spotted fever, Rotavirus infection, Rubella, Salmonellosis, SARS (Severe Acute Respiratory Syndrome), Scabies, Schistosomiasis, Sepsis, Shigellosis (Bacillary dysentery), Shingles (Herpes zoster), Smallpox (Variola), Sporotrichosis, Staphylococcal food poisoning, Staphylococcal infection, Strongyloidiasis, Syphilis, Taeniasis, Tetanus (Lockjaw), Tinea barbae (Barber’s itch), Tinea capitis (Ringworm of the Scalp), Tinea corporis (Ringworm of the Body), Tinea cruris (Jock itch), Tinea manuum (Ringworm of the Hand), tinea nigra, tinea pedis (Athlete’s foot), tinea unguium (Onychomycosis), tinea versicolor (pityriasis versicolor), toxocariasis (Ocular Larva Migrans), toxocariasis (Visceral Larva Migrans), toxoplasmosis, trichinellosis, trichomoniasis, Trichuriasis (Whipworm infection), tuberculosis, tularemia, ureaplasma urealyticum infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, white piedra (Tinea blanca), yersinia pseudotuberculosis infection, yersiniosis, yellow fever, and zygomy cosis.

39. The method according to claim 34, wherein the disease or disorder is associated with PSMA, PTHR, PTH1R, PTH2R, PTH3R, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-1, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD 166, CLL-1, ADAM9, cKIT, CD36, CD73, ITGaVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, IL-6R, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, ROR1, TROP-2, CX3CR1, CDH17, NECTIN-4, cMET, CD19, CD20, CD22, CD30, CD33, CD 123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, tau, TREM2, TSLP, mesothelin, PCSK9, ANGPTL3, Lp(a), FGFRlc / p-Klotho, C5, Factor D, MASP-2, properdin, VEGF-A, Ang-2, ROCK, IL-2R, CTLA-4, CD28, CD40L, IL-4Ra, IL-5Ra, IL-10R, IgE, CRTH2, GLP-1, LAG-3, TIGIT, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, pCadherin, CEACAM6, CD47, DPP6, ZNT8, GPR44, PTPRN, GLP1R, and EpCAM.

40. A method of manufacturing a compound according to any one of claims 1-32, wherein the method of manufacturing comprises reacting a compound of Formula (RXG-IIa) with a targeting moiety (T) according to any one of claims 1-32,L9M R' (RXG-IIa),wherein:M is a metal atom selected from palladium, nickel, and copper; andLgis a metal ligand (e g., a phosphine).

41. A method of manufacturing a compound according to any one of claims 1-32, wherein the method of manufacturing comprises reacting a compound of Formula (RXG-IIIa) with a targeting moiety (T) according to any one of claims 1-32,wherein:** represents the point of attachment to L or Tx;M is a metal atom selected from palladium, nickel, and copper; andLgis a metal ligand (e.g., a phosphine).

42. A method of delivering a therapeutic component to a target (protein, tissue, organ), the method comprising administering to a subject a compound according to any one of claims 1-32 or a pharmaceutical composition according to claim 33.

43. A compound according to any one of claims 1-32 or a pharmaceutical composition according to claim 33, wherein the compound is manufactured using a compound according to claim 40, 41, or Table 1.

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