Anti-HER2 antibody-drug conjugates and uses thereof

Abstract

Disclosed herein are antibody drug conjugates (ADCs) in which the antibody comprises at least one unnatural amino acid, as well as methods for making such unnatural amino acids and polypeptides. ADCs can contain a wide range of possible functional groups, but typically have at least one oxime, carbonyl, dicarbonyl, and / or hydroxylamine group. Also disclosed herein are unnatural amino acid ADCs that are further modified post-translationally, methods for making such modifications, and methods for purifying such ADCs. Typically, the modified ADCs include at least one oxime, carbonyl, dicarbonyl, and / or hydroxylamine group. Further disclosed are methods for using such unnatural amino acid ADCs and modified unnatural amino acid ADCs, including therapeutic, diagnostic, and other biotechnological uses.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 170,446, filed April 3, 2021, U.S. Provisional Patent Application No. 63 / 194,929, filed May 28, 2021, U.S. Provisional Patent Application No. 63 / 210,481, filed June 14, 2021, U.S. Provisional Patent Application No. 63 / 257,999, filed October 20, 2021, and U.S. Provisional Patent Application No. 63 / 299,879, filed January 14, 2022, each of which is incorporated by reference in its entirety. [Background technology]

[0002] HER2 is a member of the epidermal growth factor receptor family of transmembrane tyrosine kinases. HER2 amplification or overexpression occurs in approximately 25-30% of primary breast cancers and less frequently in many other solid tumor types (Mitri et al., Chemother Res Pract, 2012:743-193) and is associated with negative prognosis, including shorter recurrence-free and overall survival (OS) in breast cancer (Spears et al., Breast Cancer Res Treat. 2012;134(2):701-8) and gastric cancer (Bang et al., Lancet, 2010;376(9742):687-97; Boku et al., Gastric Cancer. 2014;17(1):1-12), as well as shorter OS times in lung, ovarian, colorectal, and pancreatic cancer (Sithanandam et al., Cancer Gene Ther. 2008;15(7):413-48). Summary of the Invention

[0003] One aspect of the present disclosure is an antibody drug conjugate (ADC) comprising: Cytotoxic tubulin analogue 269 having the following structure: [ka] and an anti-HER2 antibody or antibody fragment comprising SEQ ID NO: 2 and SEQ ID NO: 3; During the ceremony, [ka] indicates a single or double bond, and # indicates conjugation to an anti-HER2 antibody or antibody fragment, for antibody drug conjugates (ADCs).

[0004] In some embodiments, the anti-HER2 antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains.

[0005] Another aspect of the present disclosure is an antibody drug conjugate (ADC) comprising: A cytotoxic tubulin analogue having the structure [ka] and an anti-HER2 antibody or antibody fragment comprising SEQ ID NO: 2; wherein the anti-HER2 antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains, wherein [ka] indicates a single or double bond, where # indicates conjugation to an anti-HER2 antibody or antibody fragment, in reference to antibody drug conjugates (ADCs).

[0006] In some embodiments, the anti-HER2 antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain. [ka] represents a double bond, and the tubulin analog is covalently attached to the anti-HER2 antibody via the double bond. In some embodiments, the double bond is between the tubulin analog and one or more members of the non-naturally encoded amino acid sequence. In some embodiments, the ADC comprises two heavy chains, wherein each heavy chain comprises SEQ ID NO: 2 and each heavy chain is individually conjugated to a different tubulin analog having the structure: [ka] (where # indicates attachment to the anti-HER2 antibody or antibody fragment). In some embodiments, the anti-HER2 antibody or antibody fragment is humanized. In some embodiments, the one or more non-naturally encoded amino acids are para-acetylphenylalanine. In some embodiments, the light chain of the anti-HER2 antibody or antibody fragment is any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0007] Yet another aspect of the present disclosure relates to compositions comprising the antibody drug conjugates (ADCs) disclosed herein.

[0008] In some embodiments, the composition further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an immunotherapeutic agent, a chemotherapeutic agent, a hormonal agent, an anti-tumor agent, an immunostimulatory agent, or an immunomodulatory agent, or a combination thereof. In some embodiments, the additional therapeutic agent is a HER2-targeted therapeutic agent. In some embodiments, the additional therapeutic agent is a checkpoint inhibitor, a HER2 kinase inhibitor, a cyclin-dependent kinase inhibitor, a tyrosine kinase inhibitor, a small molecule kinase inhibitor, or a platinum-based therapeutic agent, or a combination thereof.

[0009] Another aspect of the disclosure relates to a method of killing a cell comprising contacting the cell with an ADC disclosed herein.

[0010] In some embodiments, the cells are tumor or cancer cells.

[0011] Yet another aspect of the present disclosure pertains to pharmaceutical compositions comprising the ADCs disclosed herein.

[0012] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0013] Another aspect of the disclosure relates to a method of treating a tumor or cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of an ADC disclosed herein or a pharmaceutical composition disclosed herein.

[0014] In some embodiments, the effective amount is about 0.22, 0.33, 0.44, 0.55, 0.66, 0.77, 0.88, 0.99, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mg / kg (mg / kg) of body weight of a human subject. In some embodiments, the effective amount is about 0.33, 0.66, 0.88, 1.1, 1.3, 1.5, 1.6, 1.7, or 1.8 mg / kg of body weight of a human subject. In some embodiments, administration occurs once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, administration continues for two or more 1, 2, 3, 4, 5, 6, 7, or 8 week cycles. In some embodiments, administration continues for at least 4 weeks. In some embodiments, administration continues for at least 3 weeks. In some embodiments, administration occurs two or more times within a 1, 2, 3, 4, 5, 6, 7, or 8 week or longer cycle. In some embodiments, administration occurs two or more times within a 4 week cycle. In some embodiments, administration occurs two or more times within a 3 week cycle. In some embodiments, the administered dose is the same on different days of administration. In some embodiments, the administered dose is different on different days of administration. In some embodiments, the ADC is administered once every two weeks for more than 8 weeks. In some embodiments, the ADC is administered once every four weeks for more than 8 weeks. In some embodiments, the tumor or cancer is breast cancer, ovarian cancer, gastric cancer, gastroesophageal junction adenocarcinoma, cervical cancer, uterine cancer, endometrial cancer, testicular cancer, prostate cancer, colon cancer, esophageal cancer, bladder cancer, non-small cell lung cancer (NSCLC), urothelial cancer, cholangiocarcinoma, colorectal cancer, pancreatic cancer, or a solid tumor. In some embodiments, the tumor or cancer is breast cancer, ovarian cancer, or gastric cancer.

[0015] In some embodiments, the administration comprises ADC at a dose of about 1.8 mg / kg on day 1 of a first 4-week cycle. In some embodiments, the administration further comprises ADC at a dose of about 1.7 mg / kg, 1.5 mg / kg, 1.3 mg / kg or less on day 1 of a second 4-week cycle. In some embodiments, the administration comprises ADC at a dose of about 1.7 mg / kg on day 1 of a first 4-week cycle. In some embodiments, the administration further comprises ADC at a dose of about 1.5 mg / kg, 1.3 mg / kg or less on day 1 of a second 4-week cycle. In some embodiments, the administration comprises ADC at a dose of about 1.6 mg / kg on day 1 of a first 4-week cycle. In some embodiments, the administration further comprises ADC at a dose of about 1.5 mg / kg, 1.3 mg / kg, 1.1 mg / kg or less on day 1 of a second 4-week cycle. In some embodiments, administration comprises ADC at a dose of about 1.5 mg / kg on day 1 of a first 4-week cycle. In some embodiments, administration further comprises ADC at a dose of about 1.3 mg / kg, 1.1 mg / kg or less on day 1 of a second 4-week cycle.

[0016] In some embodiments, administration continues for two or more 3-week cycles. In some embodiments, administration comprises an ADC at a dose of about 1.8 mg / kg on day 1 of the first 3-week cycle. In some embodiments, administration comprises an ADC at a dose of about 1.7 mg / kg on day 1 of the first 3-week cycle. In some embodiments, administration comprises an ADC at a dose of about 1.5 mg / kg on day 1 of the first 3-week cycle. In some embodiments, administration comprises an ADC at a dose of about 1.3 mg / kg on day 1 of the first 3-week cycle. In some embodiments, administration comprises an ADC at a dose of about 1.1 mg / kg on day 1 of the first 3-week cycle. In some embodiments, administration comprises an ADC at a dose of about 0.88 mg / kg on day 1 of the first 3-week cycle. In some embodiments, administration comprises an ADC at a dose of about 0.66 mg / kg on day 1 of the first 3-week cycle.

[0017] In some embodiments, the method further comprises administering to the human subject an effective amount of an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent, a hormonal agent, an anti-tumor agent, an immunostimulatory agent, an immunomodulatory agent, or an immunotherapeutic agent, or a combination thereof. In some embodiments, the additional therapeutic agent is a checkpoint inhibitor, a HER2 kinase inhibitor, a cyclin-dependent kinase inhibitor, a tyrosine kinase inhibitor, a small molecule kinase inhibitor, or a platinum-based therapeutic agent, or a combination thereof. In some embodiments, the therapeutic agent is a HER2-targeted therapeutic agent. In some embodiments, the method improves or optimizes cancer cell killing. In some embodiments, the method delays tumor or cancer progression or recurrence. In some embodiments, administration is oral, intradermal, intratumoral, intravenous, or subcutaneous. In some embodiments, the tumor or cancer is a HER-2-expressing cancer. In some embodiments, the cancer is a HER-2 low-expressing cancer, a HER2 intermediate-expressing cancer, or a HER2 high-expressing cancer. In some embodiments, the subject to be treated has a HER-2-expressing cancer and cancer metastasis from the same or a different cancer.

[0018] Yet another aspect of the present disclosure relates to a formulation comprising: (i) about 20 mg / ml of an ADC disclosed herein; (ii) about 5 mM histidine buffer at about pH 6.0; (iii) about 6% (w / v) trehalose; and (iv) about 0.02% (w / v) polysorbate 80.

[0019] In some embodiments, the histidine is L-histidine.

[0020] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0021] [Figure 1]1 shows an exemplary antibody drug conjugate (ADC) comprising the cytotoxic tubulin inhibitor AS269 suitable for use in the present disclosure. [Figure 2] Waterfall plots of best change from baseline in total target lesions in the 1.5 mg / kg and 1.3 mg / kg cohorts are shown. [Figure 3] Spider plot change in total target lesions from baseline over time. [Figure 4] Shows sustained response in the cohort dosed at 1.5 mg / kg. [Figure 5] 1 shows a waterfall plot of Pan tumors in the 1.5 mg / kg Q4W cohort. [Figure 6] Shows sustained response in breast cohort dosed at 1.5 mg / kg. [Figure 7] Waterfall plot change from baseline for 1.5 mg / kg and 1.3 mg / kg is shown. [Figure 8] Shows sustained response in breast cohort dosed at 1.3 mg / kg. [Figure 9] Survival probability at 1.5 mg / kg and 1.3 mg / kg Q3W / Q4W is shown. [Figure 10] Survival probability at 1.5 mg / kg Q3W and 1.3 mg / kg Q3W is shown. [Figure 11] Survival probability at 1.5 mg / kg and 1.3 mg / kg Q3W / Q4W is shown. [Figure 12] Survival probability at 1.5 mg / kg Q3W and 1.3 mg / kg Q3W is shown. [Figure 13] A patient with HR+ / HER2-low metastatic breast cancer who achieved a partial response after two cycles of ADC is shown. DETAILED DESCRIPTION OF THE INVENTION

[0022] Overexpression and / or amplification of human epidermal growth factor receptor 2 (HER2) occurs in approximately 20% of breast cancers (BC) and is a major driver of tumor initiation and progression. This HER2 subtype confers potent tumor behavior, and the HER2 receptor remains a valuable target for antibodies, bispecifics, and antibody-drug conjugates (ADCs). Advances in targeted therapy have led to improved prognosis, including survival rates, for patients with HER2-positive breast cancer (HER2+ BC). Novel HER2-targeted therapies are being investigated to overcome drug resistance and help reduce adverse events (e.g., cardiotoxicity). The present disclosure provides a next-generation ADC using a technology platform that conjugates a HER2-specific monoclonal antibody with ambastatin 269 (AS269), a potent cytotoxic tubulin inhibitor. The site-specificity, high homogeneity, and stable covalent bond of the ADC result in its slow release and prolonged peak of serum pAF-AS269, which may contribute to reduced systemic toxicity and increased targeted delivery of the payload to tumor cells at a lower effective dose compared to other HER2 ADCs. The ADC of the present disclosure was designed to inhibit the proliferation of HER2-overexpressing cells through multiple sequential steps, including binding to HER2 on the cancer cell surface, rapid internalization, transport to lysosomes, and release of pAF-AS269 via metabolism within the lysosomes, where it binds to microtubules and induces cancer cell cycle arrest and cell death.

[0023] Disclosed herein are antibody-drug conjugates (ADCs) comprising a targeting moiety, such as an antibody and one or more drugs. The drugs may further comprise one or more linker(s). The ADCs of the present disclosure may also comprise a drug linked to an unnatural amino acid in the targeting moiety. Also included are methods of making such ADCs comprising an unnatural amino acid incorporated into the targeting moiety polypeptide.

[0024] In certain embodiments, a pharmaceutical composition is provided that includes any of the described compounds and a pharmaceutically acceptable carrier, excipient, or binder.

[0025] In further or alternative embodiments, there is a method for detecting the presence of a polypeptide in a patient, the method comprising administering a polypeptide comprising at least one heterocycle-containing non-natural amino acid, wherein the resulting heterocycle-containing non-natural amino acid polypeptide modulates the immunogenicity of the polypeptide relative to a homologous naturally occurring amino acid polypeptide.

[0026] It is to be understood that the methods and compositions described herein are not limited to the particular methodology, protocols, cell lines, constructs, and reagents described herein, as such may vary. It is also to be understood that the terminology used herein is used to describe particular embodiments only, and is not intended to limit the scope of the methods and compositions described herein, which are limited only by the appended claims.

[0027] definition Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of terms should be clear, but in case of potential ambiguity, the definitions provided herein shall take precedence over any dictionary or extrinsic definitions. Furthermore, unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular. In this application, the use of "or" means "and / or" unless otherwise stated. Furthermore, the use of the term "including," as well as other forms such as "includes" and "included," is not limiting.

[0028] Generally, the nomenclatures used in connection with and techniques of cell and tissue culture, molecular biology, immunology, microbiology, gene and protein, and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present disclosure are generally carried out according to conventional methods well known in the art and as described in various general and more detailed references cited and discussed throughout the specification, unless otherwise specified. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used in chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. In order that the present disclosure may be more readily understood, selected terms are defined below.

[0029] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0030] The term "aldol system coupling" or "mixed aldol system coupling" refers to the acid- or base-catalyzed condensation of one carbonyl compound with the enolate / enol of another carbonyl compound, which may or may not be identical, to produce a β-hydroxycarbonyl compound, i.e., an aldol.

[0031] The term "affinity label" as used herein refers to a label that binds reversibly or irreversibly to another molecule in order to modify, destroy, or form a compound with it. Examples of affinity labels include an enzyme and its substrate, or an antibody and its antigen.

[0032] The terms "alkoxy," "alkylamino," and "alkylthio" (or thioalkoxy) are used in their conventional sense to refer to alkyl groups linked to the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.

[0033] The term "alkyl," by itself or as part of another molecule, means, unless otherwise indicated, a straight-chain or branched-chain, or cyclic hydrocarbon radical, or combinations thereof, which may be fully saturated, mono- or polyunsaturated, and having the specified number of carbon atoms (i.e., C1-C6). 10 (meaning 1 to 10 carbons) may include divalent and polyvalent radicals. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers of, e.g., n-pentyl, n-hexyl, n-heptyl, and n-octyl. Unsaturated alkyl groups are those containing one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers. The term "alkyl," unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail herein, such as "heteroalkyl," "haloalkyl," and "homoalkyl."

[0034] The term "alkylene" by itself or as part of another molecule means (-CH2-) n"" means a divalent radical derived from an alkane, as exemplified by the formula: "--", where n can be from 1 to about 24. By way of example only, such groups include, but are not limited to, groups having 10 or fewer carbon atoms, such as, for example, -CH2CH2- and -CH2CH2CH2CH2- structures. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having 8 or fewer carbon atoms. The term "alkylene" is also meant to include groups described herein as "heteroalkylene," unless otherwise specified.

[0035] The term "amino acid" refers to naturally occurring and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to natural amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as a natural amino acid, by way of example only: an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but still retain the same basic chemical structure as a natural amino acid. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium.

[0036] Amino acids may be referred to herein by either their names, their commonly known three letter symbols, or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Additionally, nucleotides may be referred to by their commonly accepted one-letter abbreviations.

[0037] An "amino terminal modification group" refers to any molecule that can be attached to a terminal amine group. By way of example, such a terminal amine group may be at the end of a polymer molecule, including, but not limited to, polypeptides, polynucleotides, and polysaccharides. Terminal modification groups include, but are not limited to, various water-soluble polymers, peptides, or proteins. By way of example only, terminal modification groups include polyethylene glycol or serum albumin. Terminal modification groups may be used to modify the therapeutic properties of the polymer molecule, including, but not limited to, increasing the serum half-life of a peptide.

[0038] As used herein, "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by all or part of antibody genes. Immunoglobulin genes include, but are not limited to, kappa, lambda, alpha, gamma (IgG1, IgG2, IgG3, and IgG4), delta, epsilon, and mu constant region genes, as well as numerous other immunoglobulin variable region genes. As used herein, antibody is meant to include full-length antibodies and antibody fragments, including naturally occurring antibodies in any organism or engineered antibodies (e.g., variants).

[0039] "Antibody fragment" refers to any form of an antibody other than the full-length form. Antibody fragments, as used herein, include smaller components of a full-length antibody and engineered antibodies. Antibody fragments include, but are not limited to, Fv, Fc, Fab and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, and variable region combinations, as well as alternative scaffold non-antibody molecules, bispecific antibodies, etc. (Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9:395-402). Another functional substructure is the single-chain Fv (scFv), which consists of the variable regions of immunoglobulin heavy and light chains covalently linked by a peptide linker (Sz Hu et al., 1996, Cancer Research, 56, 3055-3061). These small (Mr 25,000) proteins generally retain specificity and affinity for an antigen in a single polypeptide and can provide convenient building blocks for larger antigen-specific molecules. Unless otherwise specified, the specification and claims using the terms "antibody" or "antibody(s)" specifically include "antibody fragment" and "antibody fragment(s)."

[0040] As used herein, "antibody drug conjugate" or "ADC" refers to an antibody molecule or fragment thereof covalently linked to one or more biologically active molecule(s). The biologically active molecule(s) may be conjugated to the antibody via a linker, polymer, or other covalent bond.

[0041] As used herein, the terms "aromatic" or "aryl" refer to closed ring structures having at least one ring having a conjugated pi-electron system, and include both carbocyclic aryl and heterocyclic aryl (or "heteroaryl" or "heteroaromatic") groups. A carbocyclic or heterocyclic aromatic group can contain from 5 to 20 ring atoms. The term includes covalently bonded monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups. An aromatic group can be unsubstituted or substituted. Non-limiting examples of "aromatic" or "aryl" groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, anthracenyl, and phenanthracenyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described herein.

[0042] For brevity, the terms "aromatic" or "aryl" when used in combination with other terms (including, but not limited to, aryloxy, arylthioxy, aralkyl, etc.) include both aryl and heteroaryl rings as defined above. Thus, the term "aralkyl" or "alkaryl" is meant to include radicals in which an aryl group is attached to an alkyl group (including, but not limited to, benzyl, phenethyl, pyridylmethyl, etc.), including alkyl groups in which a carbon atom (including, but not limited to, a methylene group) has been replaced with a heteroatom, such as an oxygen atom. Examples of such aryl groups include, but are not limited to, phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, etc.

[0043] As used herein, the term "arylene" refers to a divalent aryl radical. Non-limiting examples of "arylene" include phenylene, pyridinylene, pyrimidinylene, and thiophenylene. Substituents for arylene groups are selected from the group of acceptable substituents described herein.

[0044] A "bifunctional polymer," also referred to as a "bifunctional linker," refers to a polymer containing two functional groups that can specifically react with other moieties to form covalent or non-covalent bonds. Such moieties can include, but are not limited to, natural or unnatural amino acids, or the side groups of peptides containing such natural or unnatural amino acids. The other moieties that can be linked to a bifunctional linker or bifunctional polymer can be the same or different moieties. By way of example only, a bifunctional linker can have a functional group reactive with a group on a first peptide and another functional group reactive with a group on a second peptide, thereby forming a conjugate comprising the first peptide, the bifunctional linker, and the second peptide. Many procedures and linker molecules are known for attaching various compounds to peptides. See, for example, European Patent Application No. 188,256; U.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784, 4,680,338, and 4,569,789, which are incorporated herein by reference in their entireties. A "multifunctional polymer," also referred to as a "multifunctional linker," refers to a polymer containing two or more functional groups capable of reacting with other moieties. Such moieties may include, but are not limited to, natural or unnatural amino acids, or the side groups of peptides containing such natural or unnatural amino acids, to form covalent or noncovalent bonds (including, but not limited to, amino acid side groups). Bifunctional or multifunctional polymers may be of any desired length or molecular weight and may be selected to provide a particular desired spacing or conformation between one or more molecules linked to a compound and the molecule to which it binds or binds to the compound.

[0045] The term "bioavailability" as used herein refers to the rate and extent to which a substance or its active portion is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. Increasing bioavailability refers to increasing the rate and extent to which a substance or its active portion is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. For example, increasing bioavailability can be shown as an increase in the concentration of a substance or its active portion in the blood compared to other substances or active portions. Methods for assessing increased bioavailability are known in the art and can be used to assess the bioavailability of any polypeptide.

[0046] The terms "biologically active molecule," "biologically active moiety," or "biologically active agent," as used herein, refer to any substance that can affect any physical or biochemical property of a biological system, pathway, molecule, or interaction associated with a living organism, including, but not limited to, viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans. In particular, as used herein, biologically active molecules include, but are not limited to, any substance intended for the diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or for otherwise promoting the physical or mental well-being of humans or animals. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, prodrugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles, and micelles. Classes of bioactive agents suitable for use in the methods and compositions described herein include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, antivirals, anti-inflammatory agents, antitumor agents, cardiovascular agents, anxiolytic agents, hormones, growth factors, steroids, microbial toxins, and the like.

[0047] "Modulating biological activity" refers to increasing or decreasing the reactivity of a polypeptide, altering the selectivity of a polypeptide, or increasing or decreasing the substrate selectivity of a polypeptide. Analysis of modified biological activity can be performed by comparing the biological activity of the non-naturally occurring polypeptide with the biological activity of the naturally occurring polypeptide.

[0048] As used herein, the term "biomaterial" refers to materials derived from living organisms, including, but not limited to, materials obtained from bioreactor and / or recombinant methods and techniques.

[0049] As used herein, the term "biophysical probe" refers to a probe capable of detecting or monitoring conformational changes in a molecule. Such molecules include, but are not limited to, proteins, and "biophysical probes" may be used to detect or monitor interactions between proteins and other macromolecules. Examples of biophysical probes include, but are not limited to, spin labels, fluorophores, and photoactivatable groups.

[0050] As used herein, the term "biosynthetically" refers to any method utilizing a translation system (cellular or non-cellular) that includes the use of at least one of the following components: polynucleotides, codons, tRNAs, and ribosomes. As an example, unnatural amino acids may be "biosynthetically incorporated" into unnatural amino acid polypeptides using the methods and techniques described in WO2002 / 085923, which is incorporated herein by reference in its entirety. Additionally, methods for selecting useful unnatural amino acids that may be "biosynthetically incorporated" into unnatural amino acid polypeptides are described in WO2002 / 085923, the contents of which are incorporated herein by reference in their entirety.

[0051] As used herein, the term "biotin analogue" or "biotin mimetic" is any molecule other than biotin that binds with high affinity to avidin and / or streptavidin.

[0052] As used herein, the term "carbonyl" refers to a group containing a moiety selected from the group consisting of -C(O)-, -S(O)-, -S(O)2-, and -C(S)-, including, but not limited to, at least one ketone group, at least one aldehyde group, at least one ester group, at least one carboxylic acid group, and / or at least one thioester group. Such carbonyl groups include ketones, aldehydes, carboxylic acids, esters, and thioesters. Furthermore, such groups may be part of a linear, branched, or cyclic molecule.

[0053] The term "carboxy terminal modification group" refers to any molecule that can be attached to a terminal carboxy group. By way of example, such a terminal carboxy group may be at the end of a polymer molecule, including, but not limited to, polypeptides, polynucleotides, and polysaccharides. Terminal modification groups include, but are not limited to, various water-soluble polymers, peptides, or proteins. By way of example only, terminal modification groups include polyethylene glycol or serum albumin. Terminal modification groups may be used to modify the therapeutic properties of the polymer molecule, including, but not limited to, increasing the serum half-life of a peptide.

[0054] As used herein, the term "chemically cleavable group" (also called "chemically labile") refers to a group that breaks or cleaves upon exposure to an acid, base, oxidizing agent, reducing agent, chemical initiator, or radical initiator.

[0055] As used herein, "cofolding" refers to a refolding process, reaction, or method that utilizes at least two molecules that interact with each other and convert an unfolded or improperly folded molecule into a properly folded molecule. By way of example only, "cofolding" uses at least two polypeptides that interact with each other and convert an unfolded or improperly folded polypeptide into a native, properly folded polypeptide. Such polypeptides may contain natural amino acids and / or at least one unnatural amino acid.

[0056] As used herein, a "conjugate" refers to a polypeptide that is bound, e.g., covalently attached, directly or via a linker to a compound or compound linker described herein. A "targeting moiety" refers to a structure that has selective affinity for a target molecule compared to other non-target molecules. A targeting moiety of the present disclosure binds to a target molecule. A targeting moiety may include, for example, an antibody, a peptide, a ligand, a receptor, or a binding portion thereof. The target biological molecule may be a cellular biological receptor or other structure, such as a tumor antigen. As used herein, the terms "conjugate of the present invention," "conjugate of the present disclosure," "targeting moiety conjugate," "targeting conjugate," or "targeting moiety-active molecule conjugate" refer to a targeting polypeptide, or a portion, analog, or derivative thereof, that binds to a target present on a cell or its subunit conjugated to a biologically active molecule, portion, or analog thereof, including, but not limited to, a cytotoxic tubulin inhibitor such as AS269. As used herein, the term "tumor targeting moiety conjugate" or "tumor targeting moiety-biologically active molecule conjugate" refers to a tumor-targeting polypeptide or portion, analog, or derivative thereof that binds to a target present on a tumor cell or a subunit thereof conjugated to a biologically active molecule or portion, or analog thereof, including, but not limited to, a cytotoxic tubulin inhibitor such as AS269. Unless otherwise specified, the terms "compound of the invention," "compound of the disclosure," "composition of the disclosure," and "composition of the invention" are used as alternatives to the term "conjugate of the invention."

[0057] The term "conservatively modified variants" applies to both natural and non-natural amino acids and natural and non-natural nucleic acid sequences, as well as combinations thereof. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to natural and non-natural nucleic acids that encode identical or substantially identical natural and non-natural amino acid sequences, or, if the natural and non-natural nucleic acids do not encode natural and non-natural amino acid sequences, to essentially identical sequences. By way of example, due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any particular protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon may be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are a type of conservatively modified variation. Thus, by way of example, every natural or non-natural nucleic acid sequence herein that encodes a natural or non-natural polypeptide also describes every possible silent variation of the natural or non-natural nucleic acid. One of skill in the art will recognize that each codon (except AUG, which is normally the only codon for methionine, and TGG, which is normally the only codon for tryptophan) of a natural or non-natural nucleic acid can be altered to obtain a functionally identical molecule. Accordingly, each silent variation of natural and non-natural nucleic acids that encodes a natural and non-natural polypeptide is implicit in each described sequence.

[0058] With respect to amino acid sequences, individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, or delete a single natural and unnatural amino acid, or a small percentage of natural and unnatural amino acids in the encoded sequence, are "conservatively modified variants," and the alterations result in the deletion of an amino acid, the addition of an amino acid, or the substitution of a natural and unnatural amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar natural amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the methods and compositions described herein.

[0059] Conservative substitution tables providing functionally similar amino acids are known to those skilled in the art. The following eight groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). (See, e.g., Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman & Co.; 2nd edition (December 1993)).

[0060] The terms "cycloalkyl" and "heterocycloalkyl," by themselves or in combination with other terms, represent, unless otherwise stated, cyclic forms of "alkyl" and "heteroalkyl," respectively. Thus, cycloalkyl or heterocycloalkyl include saturated, partially unsaturated, and fully unsaturated ring bonds. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Heteroatoms can include, but are not limited to, oxygen, nitrogen, or sulfur. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. Furthermore, the term encompasses polycyclic structures, including, but not limited to, bicyclic and tricyclic ring structures. Similarly, the term "heterocycloalkylene" by itself or as part of another molecule means a divalent radical derived from a heterocycloalkyl, and the term "cycloalkylene" by itself or as part of another molecule means a divalent radical derived from a cycloalkyl.

[0061] The term "cyclodextrin" as used herein refers to a cyclic carbohydrate consisting of at least six to eight glucose molecules in a ring formation. The outer portion of the ring contains water-soluble groups, and the center of the ring is a relatively nonpolar cavity that can accommodate small molecules.

[0062] As used herein, the term "cytotoxic" refers to a compound that damages cells.

[0063] As used herein, a "denaturing agent" or "denaturant" refers to any compound or material that causes the reversible unfolding of a polymer. By way of example only, a "denaturing agent" or "denaturant" may cause the reversible unfolding of a protein. The strength of a denaturing agent or denaturant is determined by both the properties and the concentration of the particular denaturing agent or denaturant. For example, denaturing agents or denaturants include, but are not limited to, chaotropes, detergents, organics, water-miscible solvents, phospholipids, or combinations thereof. Non-limiting examples of chaotropes include, but are not limited to, urea, guanidine, and sodium thiocyanate. Non-limiting examples of detergents include, but are not limited to, strong detergents such as sodium dodecyl sulfate, or polyoxyethylene ethers (e.g., Tween or Triton detergents), sarkosyl, mild nonionic detergents (e.g., digitonin), mild cationic detergents such as N-2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents (e.g., sodium cholate or sodium deoxycholate), or zwitterionic detergents, including, but not limited to, sulfobetaine (Zwittergent), 3-(3-chloroamidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and 3-(3-chloroamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO). Non-limiting examples of organic water-miscible solvents include, but are not limited to, acetonitrile, lower alkanols (particularly C2-C4 alkanols such as ethanol or isopropanol), or lower alkanediols (C2-C4 alkanediols such as ethylene glycol) that may be used as denaturants.Non-limiting examples of phospholipids include, but are not limited to, naturally occurring phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine and phosphatidylinositol or synthetic phospholipid derivatives or variants such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

[0064] The term "diamine," as used herein, refers to a group / molecule containing at least two amine functional groups, including, but not limited to, hydrazine, amidine, imine, 1,1-diamine, 1,2-diamine, 1,3-diamine, and 1,4-diamine groups. Furthermore, such groups may be part of linear, branched, or cyclic molecules.

[0065] As used herein, the term "detectable label" refers to a label that can be observed using analytical techniques, including, but not limited to, fluorescence, chemiluminescence, electron-spin resonance, ultraviolet / visible absorption spectroscopy, mass spectrometry, nuclear magnetic resonance, magnetic resonance, and electrochemical methods.

[0066] The term "dicarbonyl," as used herein, refers to a group containing at least two moieties selected from the group consisting of -C(O)-, -S(O)-, -S(O)2-, and -C(S)-, including, but not limited to, 1,2-dicarbonyl, 1,3-dicarbonyl, and 1,4-dicarbonyl groups, as well as at least one ketone group, at least one aldehyde group, at least one ester group, at least one carboxylic acid group, and / or at least one thioester group. Such dicarbonyl groups include diketones, ketoaldehydes, ketoacids, ketoesters, and ketothioesters. Furthermore, such groups may be part of linear, branched, or cyclic molecules. The two moieties in a dicarbonyl group may be the same or different and may contain substituents that will form, for example, an ester, ketone, aldehyde, thioester, or amide with either of the two moieties.

[0067] As used herein, the term "drug" refers to any substance used in the prevention, diagnosis, mitigation, treatment, or cure of a disease or condition.

[0068] As used herein, the term "effective amount" refers to a sufficient quantity of an agent or compound being administered to relieve to some extent one or more of the symptoms of the disease or condition being treated. That result can be a reduction and / or alleviation of the signs, symptoms, or pathogenesis of a disease, or any other desired alteration of a biological system. For example, the agent or compound administered can include, but is not limited to, a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-amino acid polypeptide. Compositions containing such natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides can be administered for prophylactic, enhancing, and / or therapeutic treatments. An appropriate "effective" amount in any individual case can be determined using techniques, such as dose escalation studies.

[0069] The terms "enhance" or "enhancing" mean to increase or prolong either the potency or duration of a desired effect. By way of example, "enhancing" the effect of a therapeutic agent refers to the ability to increase or prolong either the potency or duration of the therapeutic agent's effect during treatment of a disease, disorder, or condition. As used herein, an "enhancing-effective amount" refers to an amount sufficient to enhance the effect of the therapeutic agent in treating a disease, disorder, or condition. When used in a patient, the effective amount for this use will depend on the severity and course of the disease, disorder, or condition, previous treatments, the patient's health status and response to the drugs, and the judgment of the treating physician.

[0070] As used herein, the term "eukaryote" refers to organisms belonging to the plant genetic eukaryote domain, such as, but not limited to, animals (including, but not limited to, mammals, insects, reptiles, birds, etc.), ciliates, plants (including, but not limited to, monocots, dicots, and algae), fungi, yeasts, flagellates, microsporidia, and protists.

[0071] As used herein, the term "fatty acid" refers to a carboxylic acid having a hydrocarbon side chain of about C6 or greater.

[0072] As used herein, the term "fluorophore" refers to a molecule that emits a photon upon excitation and is thereby fluorescent.

[0073] As used herein, the terms "functional group," "active moiety," "activating group," "leaving group," "reactive site," "chemically reactive group," and "chemically reactive moiety" refer to the portion or unit of a molecule at which a chemical reaction occurs. These terms are somewhat synonymous in the chemical arts and are used herein to refer to the portion of a molecule that performs a function or activity and is reactive with other molecules.

[0074] The term halogen includes fluorine, chlorine, iodine and bromine.

[0075] The term "haloacyl," as used herein, refers to an acyl group containing a halogen moiety, including, but not limited to, -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like.

[0076] The term "haloalkyl," as used herein, refers to an alkyl group containing a halogen moiety, including, but not limited to, -CF3 and -CH2CF3.

[0077] As used herein, the term "heteroalkyl" refers to a straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of an alkyl group and at least one heteroatom selected from the group consisting of O, N, Si, and S, wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, and S, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. Additionally, up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

[0078] The term "heterocyclic-based bond" or "heterocyclic bond" refers to the moiety formed from the reaction of a dicarbonyl group with a diamine group. The resulting reaction product is a heterocycle containing a heteroaryl or heterocycloalkyl group. The resulting heterocyclic group serves as a chemical linkage between the non-natural amino acid or non-natural amino acid polypeptide and another functional group. In one embodiment, the heterocyclic bond comprises a nitrogen-containing heterocyclic bond, including, by way of example, a pyrazole bond, a pyrrole bond, an indole bond, a benzodiazepine bond, and a pyrazolone bond.

[0079] Similarly, the term "heteroalkylene" refers to a divalent radical derived from heteroalkyl, as exemplified, but not limited to, -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-. For heteroalkylene groups, the same or different heteroatoms can also occupy either or both of the chain termini (including, but not limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, etc.). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. By way of example, the formula -C(O)R'- represents both -C(O)R'- and -R'C(O)-.

[0080] The terms "heteroaryl" or "heteroaromatic," as used herein, refer to an aryl group containing at least one heteroatom selected from N, O, and S, where the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen atom(s) may be optionally quaternized. A heteroaryl group may be substituted or unsubstituted. A heteroaryl group may be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.

[0081] The term "homoalkyl," as used herein, refers to an alkyl group that is a hydrocarbon group.

[0082] As used herein, the term "identical" refers to two or more sequences or subsequences that are the same. Furthermore, as used herein, the term "substantially identical" refers to two or more sequences that have a percentage of contiguous units that are the same when compared and aligned for maximum correspondence over a comparison window or designated region, as determined using a comparison algorithm or by manual alignment and visual inspection. By way of example only, two or more sequences may be "substantially identical" if the contiguous units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. Such percentages describe the "percent identity" of two or more sequences. Sequence identity may exist over a region that is at least about 75-100 contiguous units long, over a region that is about 50 contiguous units long, or, if not specified, over the entire sequence. This definition also refers to the complement of a test sequence. By way of example only, two or more polypeptide sequences are identical if the amino acid residues are the same; whereas, two or more polypeptide sequences are "substantially identical" if the amino acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. Identity can exist over a region that is at least about 75 to about 100 amino acids in length, over a region that is about 50 amino acids in length, or, if not specified, over the entire polypeptide sequence. Further, by way of example only, two or more polynucleotide sequences are identical if the nucleic acid residues are identical; however, two or more polynucleotide sequences are "substantially identical" if the nucleic acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. Identity can exist over a region that is at least about 75 to about 100 nucleic acids in length, over a region that is about 50 nucleic acids in length, or, if not specified, over the entire sequence of the polynucleotide sequence.

[0083] For sequence comparison, typically, one sequence serves as a reference sequence, and test sequences are compared with it.When using sequence comparison algorithm, test sequences and reference sequences are input into computer, subsequence coordinates are designated as needed, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity of test sequences to reference sequences based on program parameters.

[0084] As used herein, the term "immunogenicity" refers to the antibody response to administration of a therapeutic drug. Immunogenicity to a therapeutic non-natural amino acid polypeptide can be obtained using quantitative and qualitative assays to detect anti-non-natural amino acid polypeptide antibodies in biological fluids. Such assays include, but are not limited to, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), luminescence immunoassays (LIA), and fluorescence immunoassays (FIA). Analysis of immunogenicity to a therapeutic non-natural amino acid polypeptide involves comparing the antibody response upon administration of the therapeutic non-natural amino acid polypeptide with the antibody response upon administration of the therapeutic natural amino acid polypeptide.

[0085] As used herein, the term "isolated" refers to the separation and removal of a component of interest from components that are not of interest. An isolated material may be in a dry or semi-dry state or in a solution, including, but not limited to, an aqueous solution. An isolated component may be in a homogeneous state, or it may be part of a pharmaceutical composition that includes additional pharmaceutically acceptable carriers and / or excipients. Purity and homogeneity may be determined using analytical chemistry techniques, including, but not limited to, polyacrylamide gel electrophoresis or high performance liquid chromatography. Additionally, a component of interest is described herein as substantially purified if it is isolated and is the predominant species present in a preparation. As used herein, the term "purified" may refer to a component of interest that is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% pure, or greater. By way of example only, a nucleic acid or protein is "isolated" if it is free from at least some of the cellular components with which it is naturally associated, or if it is concentrated to a level greater than its in vivo or in vitro production. Also, by way of example, a gene is isolated if it is separated from open reading frames which flank it and encode proteins other than the gene of interest.

[0086] As used herein, the term "label" refers to an easily detectable substance that is incorporated into a compound so that its physical distribution can be detected and / or monitored.

[0087] As used herein, the term "bond" or "linker" refers to a bond or chemical moiety formed from a chemical reaction between a payload, drug, and / or linker and a functional group of another molecule. Such bonds can include, but are not limited to, covalent and non-covalent bonds, including, but not limited to, oximes, esters, carbonates, phosphate imine esters, hydrazones, acetals, orthoesters, peptide bonds, and oligonucleotide linkages. A hydrolytically stable bond means that the bond is substantially stable in water and does not react with water at useful pH values, including, but not limited to, stability under physiological conditions for extended periods of time, perhaps even indefinitely. A hydrolytically unstable or degradable bond means that the bond is degradable in water or in aqueous solutions, including, for example, blood. An enzymatically unstable or degradable bond means that the bond can be degraded by one or more enzymes. By way of example only, PEG and related polymers can include degradable bonds in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. Such degradable bonds include, but are not limited to, ester bonds formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on bioactive agents; such ester groups typically hydrolyze under physiological conditions to release the bioactive agent. Other hydrolytically degradable bonds include, but are not limited to, carbonate bonds; imine bonds resulting from the reaction of amines with aldehydes; phosphate ester bonds formed by the reaction of alcohols with phosphate groups; hydrazone bonds, which are the reaction product of hydrazides with aldehydes; acetal bonds, which are the reaction product of aldehydes with alcohols; orthoester bonds, which are the reaction product of formates with alcohols; peptide bonds formed by amine groups, including, but not limited to, at the termini of polymers such as the carboxyl groups of PEG and peptides; and oligonucleotide bonds formed by phosphoramidite groups, including, but not limited to, at the termini of polymers and the 5' hydroxyl groups of oligonucleotides.Linkers include, but are not limited to, short linear, branched, multi-armed molecules, or dendrimeric molecules, e.g., polymers. In some embodiments of the present disclosure, the linker may be branched. In other embodiments, the linker may be a bifunctional linker. In some embodiments, the linker may be a trifunctional linker. Many different cleavable linkers are known to those of skill in the art. See U.S. Pat. Nos. 4,618,492; 4,542,225; and 4,625,014. Mechanisms for releasing drugs from these linker groups include, for example, irradiation of photolabile bonds and acid-catalyzed hydrolysis. U.S. Pat. No. 4,671,958 includes a description of immunoconjugates containing linkers that are cleaved at target sites in vivo by, for example, proteolytic enzymes of a patient's complement system. The length of the linker can be predetermined or selected depending on the desired spatial relationship between the polypeptide and the molecule to which it is linked. Given the numerous methods reported for attaching various radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and other agents to antibodies, one of skill in the art can determine the appropriate method for attaching a given agent or molecule to a polypeptide.

[0088] The term "modified," as used herein, refers to the presence of an alteration to a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. Such an alteration or modification may be obtained by post-synthetic modification of a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide, or by co-translational or post-translational modification of a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. The form "modified or unmodified" means that the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide under consideration is optionally modified; i.e., the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide under consideration can be modified or unmodified.

[0089] As used herein, the term "modulated serum half-life" refers to a positive or negative change in the circulating half-life of a modified biologically active molecule compared to its unmodified form. Exemplary modified biologically active molecules include, but are not limited to, natural amino acids, non-natural amino acids, natural amino acid polypeptides, or non-natural amino acid polypeptides. For example, serum half-life is measured by collecting blood samples at various time points after administration of the biologically active molecule or modified biologically active molecule and determining the concentration of the molecule in each sample. The serum half-life can be calculated by correlating serum concentration with time. For example, a modified serum half-life may be an increased serum half-life, which may allow for improved dosing regimens or avoid toxic effects. Such an increase in serum half-life may be at least about 2-fold, at least about 3-fold, at least about 5-fold, or at least about 10-fold. Methods for assessing the increased serum half-life of any polypeptide are well known to those skilled in the art.

[0090] As used herein, the term "modulated therapeutic half-life" refers to a positive or negative change in the half-life of a therapeutically effective amount of a modified biologically active molecule compared to its unmodified form. Exemplary modified biologically active molecules include, but are not limited to, natural amino acids, non-natural amino acids, natural amino acid polypeptides, or non-natural amino acid polypeptides. For example, therapeutic half-life is measured by measuring the pharmacokinetic and / or pharmacodynamic properties of the molecule at various time points after administration. An increase in therapeutic half-life may enable a particular beneficial dosing regimen, a particular beneficial total dose, or avoid undesirable effects. For example, an increase in therapeutic half-life may result from increased potency, increased or decreased binding of the modified molecule to its target, increased or decreased another parameter or mechanism of action of the unmodified molecule, or increased or decreased destruction of the molecule by enzymes, such as proteases. Methods for assessing the increased therapeutic half-life of any polypeptide are well known to those skilled in the art.

[0091] "Unnatural amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. Other terms that may be used synonymously with the term "non-natural amino acid" are "non-naturally encoded amino acid," "unnatural amino acid," "non-naturally-occurring amino acid," and various hyphenated and non-hyphenated versions thereof. The term "unnatural amino acid" includes, but is not limited to, amino acids that occur naturally by modification of naturally encoded amino acids (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine) but that are not themselves incorporated into a growing polypeptide chain by the translation complex. Examples of such amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine. Furthermore, the term "unnatural amino acid" includes, but is not limited to, amino acids that do not occur in nature and can be obtained synthetically or by modification of unnatural amino acids. In some embodiments, the unnatural amino acid comprises a lysine analog, such as N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, or allyloxycarbonyl lysine. In some embodiments, the unnatural amino acid comprises a sugar moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine. Examples of such amino acids also include those in which the naturally occurring N- or O-bond between the amino acid and the sugar is replaced with a covalent bond not normally found in nature, including, but not limited to, an alkene, oxime, thioether, amide, etc.Examples of such amino acids also include sugars not normally found in naturally occurring proteins, such as 2-deoxyglucose and 2-deoxygalactose. Specific examples of unnatural amino acids include, but are not limited to, p-acetyl-L-phenylalanine, p-propargyloxyphenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methylphenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylaniline, p-azido-L p-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propargyloxy-L-phenylalanine, 4-azido-L-phenylalanine, para-azidoethoxyphenylalanine, and para-azidomethyl-phenylalanine. In some embodiments, the unnatural amino acid is selected from the group consisting of para-acetyl-phenylalanine, 4-azido-L-phenylalanine, para-azidoethoxyphenylalanine, or para-azidomethylphenylalanine.

[0092] As used herein, the term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single- or double-stranded form. By way of example only, such nucleic acids and nucleic acid polymers include, but are not limited to, (i) analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized similarly to naturally occurring nucleotides; (ii) oligonucleotide analogs, including but not limited to PNAs (peptide nucleic acids), analogs of DNA used in antisense technology (phosphorothioates, phosphoramidates, etc.); and (iii) conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions), as well as complementary sequences and explicitly indicated sequences. By way of example, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0093] As used herein, the term "oxidizing agent" refers to a compound or material that can remove electrons from a compound being oxidized. Exemplary oxidizing agents include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythreitol, and oxygen. A wide variety of oxidizing agents are suitable for use in the methods and compositions described herein.

[0094] As used herein, the term "pharmaceutically acceptable" refers to a substance, such as, but not limited to, a salt, carrier, or diluent, that does not abrogate the biological activity or properties of the compound and that is relatively non-toxic, i.e., the substance may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0095] As used herein, the term "polyalkylene glycol" or "poly(alkene glycol)" refers to linear or branched polymeric polyether polyols. Such polyalkylene glycols include, but are not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof. Other exemplary embodiments are listed in commercial supplier catalogs, such as, for example, Shearwater Corporation's catalog "Polyethylen Glycol and Derivatives for Biomedical Applications" (2001). By way of example only, such polymeric polyether polyols have average molecular weights of from about 0.1 kDa to about 100 kDa. By way of example, such polymeric polyether polyols include, but are not limited to, molecular weights of from about 100 Da to about 100,000 Da or more. The molecular weight of the polymer may be from about 100 Da to about 100,000 Da, including, but not limited to, about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30, Examples of molecular weights include about 10,000 Da, about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, about 1,000 Da, about 900 Da, about 800 Da, about 700 Da, about 600 Da, about 500 Da, 400 Da, about 300 Da, about 200 Da, and about 100 Da. In some embodiments, the molecular weight of the polymer is about 100 Da to about 50,000 Da. In some embodiments, the molecular weight of the polymer is about 100 Da to about 40,000 Da. In some embodiments, the molecular weight of the polymer is from about 1,000 Da to about 40,000 Da, hi some embodiments, the molecular weight of the polymer is from about 2,000 to about 50,000 Da.In some embodiments, the molecular weight of the polymer is from about 5,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the polymer is from about 10,000 Da to about 40,000 Da. In some embodiments, the poly(ethylene glycol) molecule is a branched polymer. The molecular weight of the branched PEG can be from about 1,000 Da to about 100,000 Da, including, but not limited to, about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about Examples of branched PEGs include about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da, about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, and about 1,000 Da. In some embodiments, the molecular weight of the branched PEG is about 1,000 Da to about 50,000 Da. In some embodiments, the molecular weight of the branched PEG is about 1,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the branched PEG is about 5,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the branched PEG is about 5,000 Da to about 20,000 Da. In other embodiments, the molecular weight of the branched PEG is about 2,000 to about 50,000 Da.

[0096] As used herein, the term "polymer" refers to a molecule made up of repeating subunits, including, but not limited to, a polypeptide, a polynucleotide, or a polysaccharide, or a polyalkylene glycol.

[0097] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description of a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. These terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are unnatural amino acids. Furthermore, such "polypeptide," "peptide," and "protein" include amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0098] The term "post-translationally modified" refers to any modification of a natural or unnatural amino acid that occurs after such amino acid is translationally incorporated into a polypeptide chain. Such modifications include, but are not limited to, co-translational in vivo modifications, co-translational in vitro modifications (e.g., modifications in cell-free translation systems), post-translational in vivo modifications, and post-translational in vitro modifications.

[0099] As used herein, the term "prodrug" or "pharmaceutically acceptable prodrug" refers to an agent that is converted into the parent drug in vivo or in vitro, does not abrogate the biological activity or properties of the drug, and is relatively non-toxic; i.e., the substance can be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. A prodrug is generally a drug precursor that, after administration to a subject and subsequent absorption, is converted into an active or more active species by some process, such as conversion by a metabolic pathway. Some prodrugs have a chemical group present in the prodrug that weakens the activity of the prodrug and / or confers solubility or some other property to the drug. When the chemical group is cleaved and / or modified from the prodrug, the active drug is generated. Prodrugs are converted into active drugs in the body by enzymatic or non-enzymatic reactions. Prodrugs can provide improved physicochemical properties, such as better solubility, enhanced delivery properties, such as specific targeting of certain cells, tissues, organs, or ligands, and improved therapeutic value of the drug. Advantages of such prodrugs include, but are not limited to, (i) easier administration compared to the parent drug, (ii) the prodrug may be oral bioavailable, whereas the parent drug is not, and (iii) the prodrug has improved solubility in pharmaceutical compositions compared to the parent drug. Prodrugs include pharmacologically inactive or reduced-activity derivatives of active drugs. Prodrugs can be designed to control the amount of a drug or biologically active molecule that reaches the desired site of action by manipulating the drug's properties, e.g., physicochemical, biopharmaceutical, or pharmacokinetic properties. An example of a prodrug is, but is not limited to, a non-natural amino acid polypeptide, which is administered as an ester ("prodrug") to facilitate transport across cell membranes where water solubility is detrimental to mobility, but is subsequently metabolically hydrolyzed to the active carboxylic acid once inside the cell, where water solubility is beneficial.Prodrugs are designed as reversible drug derivatives and are used as modifiers to enhance drug transport to site-specific tissues.

[0100] As used herein, the term "prophylactically effective amount" refers to an amount of a composition comprising at least one non-natural amino acid polypeptide or at least one modified non-natural amino acid polypeptide, administered prophylactically to a patient, that will relieve to some extent one or more of the symptoms of the disease, condition, or disorder being treated. In such prophylactic applications, such amount may depend on the patient's state of health, weight, etc. It will be well understood that it is within the skill of the art to determine such prophylactically effective amounts by routine experimentation, including, but not limited to, a dose escalation clinical trial.

[0101] As used herein, the term "protected" refers to the presence of a "protecting group" or moiety that prevents reaction of a chemically reactive functional group under certain reaction conditions. The protecting group varies depending on the type of chemically reactive group being protected. By way of example only, (i) if the chemically reactive group is an amine or hydrazide, the protecting group may be selected from tert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc), (ii) if the chemically reactive group is a thiol, the protecting group may be orthopyridyl disulfide, and (iii) if the chemically reactive group is a carboxylic acid, such as butanoic or propionic acid, or a hydroxyl group, the protecting group may be benzyl or an alkyl group, such as methyl, ethyl, or tert-butyl.

[0102] By way of example only, blocking / protecting groups may be selected from: [ka]

[0103] Further protecting groups include, but are not limited to, photolabile groups such as Nvoc and MeNvoc, as well as other protecting groups known in the art. Other protecting groups are described in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, which is incorporated herein by reference in its entirety.

[0104] The term "recombinant host cell," also known as a "host cell," refers to a cell that contains an exogenous polynucleotide, where the method used to insert the exogenous polynucleotide into the cell includes, but is not limited to, direct uptake, transduction, f-mating, or other methods known in the art for generating recombinant host cells. By way of example only, such an exogenous polynucleotide may be a non-integrating vector, including, but not limited to, a plasmid, or may be integrated into the host genome.

[0105] The term "redox active agent" as used herein refers to a molecule that oxidizes or reduces another molecule, thereby reducing or oxidizing the redox active agent. Examples of redox active agents include, but are not limited to, ferrocene, quinones, Ru, 2+ / 3+ Complex, Co 2+ / 3+ Complexes and Os 2+ / 3+ Complexes are included.

[0106] As used herein, the term "reducing agent" refers to a compound or substance that can add electrons to a compound being reduced. Examples of reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2-aminoethanethiol), and reduced glutathione. Such reducing agents may be used, by way of example only, to maintain sulfhydryl groups in a reduced state and to reduce intramolecular or intermolecular disulfide bonds.

[0107] As used herein, "refolding" refers to any process, reaction, or method that converts an improperly folded or unfolded state into a native or properly folded conformation. By way of example only, refolding converts a disulfide bond-containing polypeptide from an improperly folded or unfolded state into a native or properly folded conformation with respect to the disulfide bonds. Such disulfide bond-containing polypeptides may be natural amino acid polypeptides or non-natural amino acid polypeptides.

[0108] The term "safety" or "safety profile" used herein refers to the side effects that may be associated with the administration of a drug, relative to the number of times the drug is administered.For example, a drug that is administered multiple times and has only mild or no side effects is said to have a good safety profile.The methods used to evaluate the safety profile of any polypeptide are known in the art.

[0109] As used herein, the phrases "selectively hybridize" or "specifically hybridize" refer to a molecule that binds, replicates, or hybridizes to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture, including, but not limited to, total cellular or library DNA or RNA.

[0110] The phrase "stringent hybridization conditions" refers to the hybridization of sequences of DNA, RNA, PNA, or other nucleic acid mimics, or combinations thereof, under conditions of low ionic strength and high temperature. For example, under stringent conditions, a probe will hybridize to its target subsequence in a complex mixture of nucleic acids (including, but not limited to, total cellular or library DNA or RNA), but will not hybridize to other sequences in the complex mixture. Stringent conditions are sequence-dependent and will vary depending on the environment. For example, longer sequences will hybridize specifically at higher temperatures. Stringent hybridization conditions include, but are not limited to, the following: (i) about 5-10°C lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength and pH; (ii) for short probes (including, but not limited to, about 10 to about 50 nucleotides), a salt concentration of about 0.01M to about 1.0M at about pH 7.0 to about pH 8.3, and a temperature of at least about 30°C, and for long probes (including, but not limited to, more than 50 nucleotides), a temperature of at least about 60°C; (iii) the addition of a destabilizing agent, including, but not limited to, formamide; (iv) incubation in 50% formamide, 5xSSC, and 1% SDS at 42°C, or incubation in 5xSSC, about 1% SDS at 65°C, followed by a wash in 0.2xSSC and about 0.1% SDS at 65°C for about 5 to about 120 minutes. By way of example only, detection of selective or specific hybridization includes, but is not limited to, a positive signal over a background of at least 2. An extensive guide to nucleic acid hybridization is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993).

[0111] The term "subject," as used herein, refers to an animal that has been the object of treatment, observation, or experiment. By way of example only, a subject may be a mammal, including, but not limited to, a human.

[0112] As used herein, the term "substantially purified" refers to a component of interest that may be substantially or essentially free from other components that normally accompany or interact with the component of interest prior to purification. By way of example only, a preparation of the component of interest may be "substantially purified" if it contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating components. Thus, a "substantially purified" component of interest may have a purity level of about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more. By way of example only, a natural or non-natural amino acid polypeptide may be purified from a native or host cell in the case of a recombinantly produced natural or non-natural amino acid polypeptide. By way of example, a preparation of a natural or non-natural amino acid polypeptide may be "substantially purified" if the preparation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminants. By way of example, when a natural or non-natural amino acid polypeptide is recombinantly produced by a host cell, the natural or non-natural amino acid polypeptide may be present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cells. By way of example, when a natural or non-natural amino acid polypeptide is recombinantly produced by a host cell, the natural or non-natural amino acid polypeptide can be present in the culture at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L or less of the dry weight of the cells.By way of example, a "substantially purified" natural or non-natural amino acid polypeptide can have a purity level of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more as determined by suitable methods, including but not limited to, SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0113] The term "substituents," also called "non-interfering substituents," refers to groups that can be used to replace another group on a molecule. Such groups include, but are not limited to, halo, C-C 10 Alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl, C1-C 10 Alkoxy, C5-C 12 Aralkyl, C3-C 12 Cycloalkyl, C4-C 12 Cycloalkenyl, phenyl, substituted phenyl, toluoyl, xylenyl, biphenyl, C2-C 12 Alkoxyalkyl, C5-C 12 Alkoxyaryl, C5-C 12 Aryloxyalkyl, C7-C 12 Oxyaryl, C1-C6 alkylsulfinyl, C1-C 10 Alkylsulfonyl, -(CH2) m -O-(C1-C 10 alkyl), where m is 1 to 8, aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic radical, nitroalkyl, —NO2, —CN, —NRC(O)—(C1-C 10 alkyl), -C(O)-(C1-C 10 alkyl), C2-C 10 Alkylthioalkyl, -C(O)O-(C1-C 10 alkyl), -OH, -SO2, =S, -COOH, -NR2, carbonyl, -C(O)-(C1-C 10alkyl)-CF3, -C(O)-CF3, -C(O)NR2, -(C1-C 10 Aryl)-S-(C6-C 10 aryl), -C(O)-(C6-C 10 aryl), -(CH2) m -O-(CH2) m -O-(C1-C 10 alkyl), where each m is 1-8, -C(O)NR, -C(S)NR, -SONR, -NRC(O)NR, -NRC(S)NR, salts thereof, and the like. Each R group in the foregoing list includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl, or alkaryl. When substituent groups are specified by their conventional chemical formula written from left to right, they are equally inclusive of chemically identical substitutes that would result from writing the structure from right to left, e.g., -CHO- is equivalent to -OCH-.

[0114] By way of example only, substituents for alkyl and heteroalkyl radicals (including groups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) include, but are not limited to, -OR, ═O, ═NR, ═N—OR, -NR, -SR, -halogen, -SiR, -OC(O)R, -C(O)R, -COR, -CONR, -OC(O)NR, -NRC(O)R, -NRC(O)NR, -NR(O)R, -NR—C(NR)═NR, -S(O)R, -S(O)R, -S(O)NR, -NRSOR, -CN, and -NO. Each R group in the above list includes, but is not limited to, hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (including, but not limited to, aryl substituted with 1 to 3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or aralkyl groups). When two R groups are attached to the same nitrogen atom, they can combine with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR2 is meant to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl.

[0115] By way of example, substituents for aryl and heteroaryl groups include, but are not limited to, -OR, =O, =NR, =N-OR, -NR2, -SR, -halogen, -SiR3, -OC(O)R, -C(O)R, -COR, -CONR2, -OC(O)NR2, -NRC(O)R, -NRC(O)NR2, -NR(O)2R, -NR-C(NR2)=NR, -S(O)R, -S(O)2R, -S(O)2NR2, -NRSO2R, -CN, -NO2, -R, -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy, and fluoro(C1-C4)alkyl, in numbers ranging from 0 to the total number of open valences on the aromatic ring system, and each R group in the foregoing list includes, but is not limited to, hydrogen, alkyl, heteroalkyl, aryl, and heteroaryl.

[0116] As used herein, the term "therapeutically effective amount" refers to an amount of a composition comprising at least one unnatural amino acid polypeptide and / or at least one modified unnatural amino acid polypeptide administered to a patient already suffering from a disease, condition, or disorder sufficient to cure or at least partially inhibit or alleviate to some extent one or more symptoms of the disease, disorder, or condition being treated. The effectiveness of such compositions will depend on factors including, but not limited to, the severity and course of the disease, disorder, or condition, previous treatments, the patient's health status and response to drugs, and the judgment of the treating physician. By way of example only, a therapeutically effective amount may be determined by routine experimentation, including, but not limited to, a dose escalation clinical trial.

[0117] The term "thioalkoxy," as used herein, refers to a sulfur-containing alkyl group linked to the molecule via an oxygen atom.

[0118] As used herein, the term "toxic moiety" or "toxic group" refers to a compound capable of causing harm, injury, or death. Toxic moieties include, but are not limited to, auristatins, DNA minor groove binders, DNA minor groove alkylators, enediynes, lexitropsins, duocarmycins, taxanes, puromycins, cytotoxic tubulin inhibitors, maytansinoids, vinca alkaloids, AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, and cyanomorpholino-doxorubicin. , echinomycin, combretastatin, calicheamicin, maytansine, DM-1, netropsin, podophyllotoxin (e.g., etoposide, teniposide, etc.), baccatin and its derivatives, antitubulin agents, cryptophysin, combretastatin, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, epothilone A, epothilone B, nocodazole, colchicine, colcimide, estramustine, cemadotin, discodermolide, maytansine, eleutherobin, meclometin, Retamine, cyclophosphamide, melphalan, carmustine, lomustine, semustine, streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide, itarabin, cytosine arabinoside, fluorouracil, floxuridine, 6-thioguanine, 6-mercaptopurine, pentosulfonylurea, statins, 5-fluorouracil, methotrexate, 10-propargyl-5,8-dideazafolate, 5,8-dideazatetrahydrofolate, leucovorin, fludarabine phosphate, pentostatin, gemcitabine, Ara-C, paclitaxel, docetaxel, deoxycoformycin, mitomycin C, L-asparaginase, azathioprine, brequinar, antibiotics (e.g., anthracyclines, gentamicin, cephalothin, vancomycin, telavancin,Daptomycin, azithromycin, erythromycin, roxithromycin, furazolidone, amoxicillin, ampicillin, carbenicillin, flucloxacillin, methicillin, penicillin, ciprofloxacin, moxifloxacin, ofloxacin, doxycycline, minocycline, oxytetracycline, tetracycline, streptomycin, rifabutin, ethambutol, rifaximin, etc.), antiviral drugs (e.g., abacavir, acyclovir, Ampligen, Sidof fovir, delaviridine, didanosine, efavirenz, entecavir, phosphonet, ganciclovir, ibacitabine, Immunovir, idoxuridine, inosine, lopinavir, methisazone, nexavir, nevirapine, oseltamivir, penciclovir, stavudine, trifluridine, truvada, valacyclovir, zanamivir, etc.), daunorubicin hydrochloride, daunomycin, rubidomycin, cerubicin, idarubicin, doxorubicin, epirubicin and morpholino derivatives, phenoxyzolidinone Cyclopeptides (e.g., dactinomycin), basic glycopeptides (e.g., bleomycin), anthraquinone glycosides (e.g., plicamycin, mithramycin), anthracenediones (e.g., mitoxantrone), azirinopyrroloindoledione (e.g., mitomycin), macrocyclic immunosuppressants (e.g., cyclosporine, FK-506, tacrolimus, prograf, rapamycin, etc.), navelbene, CPT-11, anastrazole, letrazole, capecitabine, riloxaf Taxanes include fluoxetine, ifosfamide, droloxafine, allocolchicine, halichondrin B, colchicine, colchicine derivatives, maytansine, rhizoxin, paclitaxel, paclitaxel derivatives, docetaxel, thiocolchicine, tritylcysterol, vinblastine sulfate, vincristine sulfate, cisplatin, carboplatin, hydroxyurea, N-methylhydrazine, epidophyllotoxin, procarbazine, mitoxantrone, leucovorin, and tegafur. "Taxane" also includes paclitaxel and any active taxane derivative or prodrug.

[0119] The terms "treat," "treating," and "treatment," as used herein, include alleviating, attenuating, or ameliorating the symptoms of a disease or condition, preventing additional symptoms, ameliorating or preventing the underlying metabolic cause of the symptoms, inhibiting a disease or condition, e.g., arresting the onset of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, alleviating the conditions caused by a disease or condition, or arresting the symptoms of a disease or condition. The terms "treat," "treating," or "treatment" include, but are not limited to, prophylactic and / or therapeutic treatments.

[0120] As used herein, the term "water-soluble polymer" refers to any polymer that is soluble in an aqueous solvent. Such water-soluble polymers include, but are not limited to, polyethylene glycol, polyethylene glycol propionaldehyde, mono C1-C6 10-alkoxy or its aryloxy derivatives (as described in U.S. Pat. No. 5,252,714, incorporated herein by reference), monomethoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran derivatives, such as dextran sulfate, polypropylene glycol, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives (including, but not limited to, methylcellulose and carboxymethylcellulose), serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycol and derivatives thereof, polyvinyl ethyl ether, and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide], or mixtures thereof. By way of example only, coupling of such water-soluble polymers to natural amino acid polypeptides or non-natural polypeptides can result in changes, including but not limited to, increased water solubility, increased or modulated serum half-life, increased or modulated therapeutic half-life relative to the unmodified form, increased bioavailability, modulated biological activity, extended circulation time, modulated immunogenicity, modulated physically associated characteristics, including but not limited to, aggregation and multimerization, altered receptor binding, activity modulator or other targeting polypeptide binding, altered binding to one or more binding partners, and dimerization or multimerization of the modified targeting polypeptide receptor. Furthermore, such water-soluble polymers may or may not have biological activity of their own, and may be utilized as linkers to attach targeting polypeptides to other substances, including but not limited to, one or more targeting polypeptides or one or more biologically active molecules.

[0121] General description of synthesis / testing Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology within the skill of the art are employed.

[0122] The compounds provided herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides, and reagents for making the foregoing compounds) include compounds that are identical to those set forth in the various formulas and structures provided herein, except that one or more atoms have been replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that may be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, e.g., 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 35 S, 18 F, 36 Certain isotopically labeled compounds described herein, such as 3 H and 14 Compounds incorporating radioactive isotopes such as C are useful in drug and / or substrate tissue distribution assays. Additionally, deuterium, i.e., 2 Substitution with isotopes such as H may offer certain therapeutic advantages due to increased metabolic stability, such as increased in vivo half-life or reduced dosage requirements.

[0123] Some of the compounds herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, as well as reagents for producing the foregoing compounds) possess an asymmetric carbon atom and can therefore exist as enantiomers or diastereomers. Diastereomeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by known methods, such as, for example, chromatography and / or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture to a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers, and converting the individual diastereomers to the corresponding pure enantiomers (e.g., by hydrolysis). All such isomers, including diastereomers, enantiomers, and mixtures thereof, are considered part of the compositions described herein.

[0124] In further or additional embodiments, the compounds described herein (including but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) are used in the form of prodrugs. In further or additional embodiments, the compounds described herein (including but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) are metabolized when administered to an organism in need of production of a metabolite, which is then used to achieve a desired effect, including a desired therapeutic effect. In further or additional embodiments are active metabolites of non-natural amino acids and "modified or unmodified" non-natural amino acid polypeptides.

[0125] The methods and formulations described herein include the use of N-oxides, crystalline forms (also known as polymorphs), or pharmaceutically acceptable salts of the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides. In certain embodiments, the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides may exist as tautomers. All tautomers are included within the scope of the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides presented herein. Additionally, the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides described herein can exist in unsolvated forms as well as solvated forms with pharmaceutically acceptable solvents, such as water, ethanol, and the like. The solvated forms of the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides presented herein are also considered to be disclosed herein.

[0126] Some of the compounds described herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, as well as reagents for producing the foregoing compounds) can exist in several tautomeric forms. All such tautomeric forms are considered to be part of the compositions described herein. Also, for example, all enol-keto forms of any of the compounds described herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, as well as reagents for producing the foregoing compounds) are considered to be part of the compositions described herein.

[0127] Some of the compounds herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, as well as reagents for producing any of the foregoing compounds) may be acidic and may form salts with pharmaceutically acceptable cations. Some of the compounds herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, as well as reagents for producing the foregoing compounds) may be basic and may therefore form salts with pharmaceutically acceptable anions. All such salts, including disalts, are within the scope of the compositions described herein and may be prepared by conventional methods. For example, salts may be prepared by contacting acidic and basic entities in either aqueous, non-aqueous, or partially aqueous media. Salts are recovered using at least one of the following techniques: filtration, precipitation with a non-solvent followed by filtration, evaporation of the solvent, or, in the case of aqueous solutions, lyophilization.

[0128] Pharmaceutically acceptable salts of the non-natural amino acid polypeptides disclosed herein can be formed when an acidic proton present in the parent non-natural amino acid polypeptide is replaced with a metal ion, such as an alkali metal ion, alkaline earth ion, or aluminum ion, or coordinates with an organic base. In addition, salt forms of the disclosed non-natural amino acid polypeptides can be prepared using salts of the starting materials or intermediates. The non-natural amino acid polypeptides described herein can be prepared as pharmaceutically acceptable acid addition salts (a type of pharmaceutically acceptable salt) by reacting the free base form of the non-natural amino acid polypeptides described herein with a pharmaceutically acceptable inorganic or organic acid. Alternatively, the non-natural amino acid polypeptides described herein can be prepared as pharmaceutically acceptable base addition salts (a type of pharmaceutically acceptable salt) by reacting the free acid form of the non-natural amino acid polypeptides described herein with a pharmaceutically acceptable inorganic or organic acid.

[0129] Types of pharmaceutically acceptable salts include, but are not limited to: (1) acid addition salts, such as those formed with inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with acids, e.g., acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, and 2-naphthalenesulfonic acid. (2) Acid addition salts formed with organic acids such as 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and muconic acid; (3) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or by coordination with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.

[0130] The corresponding counterions of pharmaceutically acceptable salts of non-natural amino acid polypeptides can be analyzed and identified using a variety of methods, including, but not limited to, ion exchange chromatography, ion chromatography, capillary electrophoresis, inductively coupled plasma, atomic absorption spectrometry, mass spectrometry, or any combination thereof. Additionally, the therapeutic activity of such pharmaceutically acceptable salts of non-natural amino acid polypeptides can be tested using the techniques and methods described in the Examples.

[0131] Reference to a salt should be understood to include its solvent addition forms or crystalline forms, particularly solvates or polymorphs. Solvates contain either stoichiometric or non-stoichiometric amounts of solvent and are often formed during the crystallization process using pharmaceutically acceptable solvents such as water or ethanol. When the solvent is water, hydrates are formed, or when the solvent is alcohol, alcoholates are formed. Polymorphs include different crystal packing arrangements of the same elemental composition of a compound. Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardnesses, crystal shapes, optical and electrical properties, stability, and solubility. A single crystalline form may predominate depending on various factors, such as the recrystallization solvent, crystallization rate, and storage temperature.

[0132] Screening and characterization of polymorphs and / or solvates of pharmaceutically acceptable salts of non-natural amino acid polypeptides can be achieved using a variety of techniques, including, but not limited to, thermal analysis, X-ray diffraction, spectroscopy, vapor sorption, and microscopy. Thermal analysis methods address thermochemical decomposition or thermophysical processes (including, but not limited to, polymorphic transitions), and such methods are used to analyze relationships between polymorphic forms, determine mass loss, find glass transition temperatures, or for excipient compatibility testing. Such methods include, but are not limited to, differential scanning calorimetry (DSC), modulated differential scanning calorimetry (MDCS), thermogravimetric analysis (TGA), and thermogravimetric and infrared analysis (TG / IR). X-ray diffraction methods include, but are not limited to, single crystal and powder diffractometers and synchrotron radiation sources. Various spectroscopic techniques used include, but are not limited to, Raman, FTIR, UVIS, and NMR (liquid and solid). Various microscopy techniques include, but are not limited to, polarized light microscopy, scanning electron microscopy (SEM) with energy dispersive X-ray analysis (EDX), environmental scanning electron microscopy with EDX (in a gas or water vapor atmosphere), IR microscopy, and Raman microscopy.

[0133] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0134] Site-specific anti-HER2 antibody-drug conjugates

[0135] Using a clinically validated, unique conjugation technology, drugs can be site-specifically attached to antibodies via a stable oxime bond formed by conjugating para-acetyl-phenylalanine (pAF), a non-natural amino acid that can be incorporated into antibodies, with an aminooxy-containing drug-linker. This allows for exactly two drug-linker conjugation sites specific to a single antibody. To screen for the optimal conjugation site, pAF can be incorporated into the most surface-exposed site of an antibody, which is advantageous compared to other site-specific conjugation technologies.

[0136] In some cases, the present disclosure provides next-generation site-specific anti-HER2 ADCs, including humanized HER2-targeting monoclonal antibodies (mAbs) conjugated to cytotoxic tubulin inhibitors, using a non-natural amino acid incorporation technology platform. The ADCs synthesized in this manner are homogeneous and highly stable, enabling more efficient drug delivery to target tumor cells, maximizing on-target efficacy, and minimizing off-target toxicity, thereby broadening the therapeutic window.

[0137] In some cases, the ADC contains a short, non-cleavable aminooxy-PEG4 linker attached to the N-terminus of monomethyl auristatin F (MMAF) to generate the cytotoxic payload, the cytotoxic tubulin inhibitor linker derivative AS269. MMAF is a highly potent synthetic auristatin derivative that inhibits cell proliferation by interfering with tubulin polymerization. In some cases, the ADC contains two AS269 cytotoxic payloads site-specifically conjugated to a trastuzumab-based antibody containing an unnatural amino acid. In some cases, the ADC can exhibit antitumor activity by optimizing the number and position of the payloads and the chemical bond conjugating the payloads to the antibody. In some cases, AS269 is a generally non-cell-permeable tubulin inhibitor specifically designed to form a highly stable covalent bond with the antibody and kill only tumor cells upon entry into the cell when assisted by a conjugated targeting antibody, thereby limiting off-target effects on normal tissues. In some cases, AS269 exhibits limited permeability across cell membranes without conjugation to a targeting antibody, thereby reducing off-target toxicity. In some cases, AS269 is a weak substrate of the multidrug resistance pump (MDR), thereby retaining and concentrating the drug within cancer cells, which may lead to more potent killing of cancer cells. Combining the unique properties of AS269 with the optimized number and position of payloads and chemical linkages that conjugate the payload to the antibody with a DAR of 2 may result in in vivo stability, improved efficacy, and reduced exposure to the payload in serum, which may contribute to the antitumor activity and tolerability profile observed with the ADC. In some cases, the ADC comprises a structure synthesized according to Figure 1. In some cases, the ADC comprises AS269 as a payload and an anti-HER2 antibody comprising one or more unnatural amino acids disclosed herein.In some cases, the AS269 payload is specifically and stably conjugated to the unnatural amino acid pAF at a unique site in the heavy chain of the mAb (one payload per heavy chain), and the anti-HER2 antibody comprises the heavy chain constant region of an anti-HER2 Fab having the corresponding amino acid sequence, SEQ ID NO: 2 (Kabat numbering), and the light chain sequence having the corresponding amino acid sequence, SEQ ID NO: 3. In some cases, the ADC comprises AS269 as a payload and an anti-HER2 antibody comprising the heavy or light chain constant region of an anti-HER2 Fab having those Kabat numbering and the corresponding amino acid heavy chain sequence, SEQ ID NO: 2, and light chain sequences SEQ ID NOs: 4-11.

[0138] Cytotoxic Tubulin Inhibitor Linker Derivatives

[0139] At one level, described herein are tools (methods, compositions, techniques) for generating and using targeting polypeptides of ADCs or analogs that include at least one unnatural amino acid or modified unnatural amino acid bearing a carbonyl, dicarbonyl, oxime, aminooxy, or hydroxylamine group. Such targeting polypeptides of ADCs that include unnatural amino acids can include additional functionality, including, but not limited to, polymers; water-soluble polymers; derivatives of polyethylene glycol; second proteins or polypeptides or polypeptide analogs; antibodies or antibody fragments; and any combination thereof. It should be noted that the various functions described above do not imply that components of one function cannot be classified as components of another function. Indeed, overlap occurs depending on the specific situation. As just one example, water-soluble polymers overlap to a certain extent with derivatives of polyethylene glycol, but this overlap is not complete, and therefore both functions are listed above.

[0140] One aspect includes methods for selecting and designing cytotoxic tubulin inhibitor linker derivatives and targeting polypeptides to be modified using the methods, compositions, and techniques described herein. Novel cytotoxic tubulin inhibitor linker derivatives and targeting polypeptides can be designed de novo, by way of example only, as part of a high-throughput screening process (in which large numbers of polypeptides can be designed, synthesized, characterized, and / or tested), or de novo based on the researcher's goals. Novel cytotoxic tubulin inhibitor linker derivatives and targeting polypeptides can also be designed based on the structure of known or partially characterized polypeptides. Principles for selecting which amino acid(s) to replace and / or modify are described separately herein. The selection of which modifications to use is also described herein and can be used to meet the needs of the experimenter or end user. Such needs may include, but are not limited to, manipulating the therapeutic effect of the polypeptide, improving the safety profile of the polypeptide, modulating the pharmacokinetics, pharmacology, and / or pharmacodynamics of the polypeptide, for example, by way of example only, increasing water solubility, bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending circulation time. Additionally, such modifications include, by way of example only, providing additional functionality to the polypeptide, incorporating antibodies, and any combination of the foregoing modifications.

[0141] Also described herein are cytotoxic tubulin inhibitor linker derivatives and targeting polypeptides that have been or can be modified to contain oxime, carbonyl, dicarbonyl, aminooxy, or hydroxylamine groups. This aspect includes methods for making, purifying, characterizing, and using such cytotoxic tubulin inhibitor linker derivatives and targeting polypeptides.

[0142] The cytotoxic tubulin inhibitor linker derivative or targeting polypeptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10 or more of the following: carbonyl or dicarbonyl groups, oxime groups, aminooxy groups, hydroxylamine groups, or protected forms thereof. The cytotoxic tubulin inhibitor linker derivative or targeting polypeptide may be the same or different, for example, there may 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 a derivative that comprises 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.

[0143] As described herein, the disclosure provides targeting polypeptides coupled to another molecule having the formula "targeting polypeptide-LM," where L is a linking group or chemical bond, and M is any other molecule, including, but not limited to, another targeting polypeptide. In some embodiments, L is stable in vivo. In some embodiments, L is hydrolyzable in vivo. In some embodiments, L is metastable in vivo.

[0144] The targeting polypeptide and M can be linked to each other through L using standard linking agents and procedures known to those of skill in the art. In some embodiments, the targeting polypeptide and M are directly fused and L is a bond. In other embodiments, the targeting polypeptide and M are fused via a linking group L. For example, in some embodiments, the targeting polypeptide and M are linked to each other via a peptide bond, optionally via a peptide or amino acid spacer. In some embodiments, the targeting polypeptide and M are linked to each other by chemical conjugation, optionally via a linking group (L). In some embodiments, L is directly conjugated to each of the targeting polypeptide and M.

[0145] Chemical conjugation can occur by reacting a nucleophilic reactive group of one compound with an electrophilic reactive group of another compound. In some embodiments, when L is a bond, the targeting polypeptide is conjugated to M by reacting a nucleophilic reactive moiety on the targeting polypeptide with an electrophilic reactive moiety on Y, or by reacting an electrophilic reactive moiety on the targeting polypeptide with a nucleophilic reactive moiety on M. In embodiments where L is a group linking the targeting polypeptide and M together, the targeting polypeptide and / or M can be conjugated to L by reacting a nucleophilic reactive moiety on the targeting polypeptide and / or M with an electrophilic reactive moiety on L, or by reacting an electrophilic reactive moiety on the targeting polypeptide and / or M with a nucleophilic reactive moiety on L. Non-limiting examples of nucleophilic reactive groups include amino, thiol, and hydroxyl. Non-limiting examples of electrophilic reactive groups include carboxyl, acyl chloride, anhydride, ester, succinimide ester, alkyl halide, sulfonate ester, maleimide, haloacetyl, and isocyanate. In embodiments in which the targeting polypeptide and M are conjugated together by reaction of a carboxylic acid with an amine, an activating agent may be used to form an activated ester of the carboxylic acid.

[0146] The activated ester of a carboxylic acid may be, for example, N-hydroxysuccinimide (NHS), tosylate (Tos), mesylate, triflate, carbodiimide, or hexafluorophosphate. In some embodiments, the carbodiimide is 1,3-dicyclohexylcarbodiimide (DCC), 1,1'-carbonyldiimidazole (CDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), or 1,3-diisopropylcarbodiimide (DICD). In some embodiments, the hexafluorophosphate is selected from the group consisting of hexafluorophosphate benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), and O-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluorophosphate (HBTU).

[0147] In some embodiments, the targeting polypeptide comprises a nucleophilic reactive group (e.g., an amino group, a thiol group, or a hydroxyl group on the side chain of lysine, cysteine, or serine) that can be conjugated to an electrophilic reactive group on M or L. In some embodiments, the targeting polypeptide comprises an electrophilic reactive group (e.g., a carboxylate group on the side chain of Asp or Glu) that can be conjugated to a nucleophilic reactive group on M or L. In some embodiments, the targeting polypeptide is chemically modified to include a reactive group that can be directly conjugated to M or L. In some embodiments, the targeting polypeptide is modified at the N- or C-terminus to include a natural or unnatural amino acid having a nucleophilic side chain. In exemplary embodiments, the N- or C-terminal amino acid of the targeting polypeptide is selected from the group consisting of lysine, ornithine, serine, cysteine, and homocysteine. For example, the N- or C-terminal amino acid of the targeting polypeptide may be modified to include a lysine residue. In some embodiments, targeting polypeptides are modified at the N- or C-terminal amino acid to include natural or unnatural amino acids with electrophilic side chains, such as, for example, Asp and Glu. In some embodiments, internal amino acids of targeting polypeptides are substituted with natural or unnatural amino acids with nucleophilic side chains, as described hereinabove. In exemplary embodiments, the substituted internal amino acids of targeting polypeptides are selected from the group consisting of lysine, ornithine, serine, cysteine, and homocysteine. For example, internal amino acids of targeting polypeptides may be substituted with lysine residues. In some embodiments, internal amino acids of targeting polypeptides are substituted with natural or unnatural amino acids with electrophilic side chains, such as, for example, Asp and Glu.

[0148] In some embodiments, M comprises a reactive group that can be directly conjugated to the targeting polypeptide or L. In some embodiments, M comprises a nucleophilic reactive group (e.g., amine, thiol, hydroxyl) that can be conjugated to an electrophilic reactive group on the targeting polypeptide or L. In some embodiments, M comprises an electrophilic reactive group (e.g., carboxyl group, activated form of carboxyl group, compound with a leaving group) that can be conjugated to a nucleophilic reactive group on the targeting polypeptide or L. In some embodiments, M is chemically modified to comprise either a nucleophilic reactive group that can be conjugated to an electrophilic reactive group on the targeting polypeptide or L. In some embodiments, M is chemically modified to comprise an electrophilic reactive group that can be conjugated to a nucleophilic reactive group on the targeting polypeptide or L.

[0149] In some embodiments, conjugation can be achieved using organosilanes, such as aminosilanes treated with glutaraldehyde; carbonyldiimidazole (CDI) activation of silanol groups; or dendrimers. Various dendrimers are known in the art, including poly(amidoamine) (PAMAM) dendrimers synthesized by the divergent method starting from ammonia or ethylenediamine initiator core reagents; a subclass of PAMAM dendrimers based on a trisaminoethyleneimine core; radially layered dendrimers, which are inverted unimolecular micelles consisting of a hydrophilic, nucleophilic polyamidoamine (PAMAM) interior and a hydrophobic organosilicon (OS) exterior phase; These include (layered) poly(amidoamine-organosilicon) dendrimers (PAMAMOS); poly(propyleneimine) (PPI) dendrimers (generally poly-alkylamines with primary amines as end groups, while the dendrimer interior consists of many tertiary trispropyleneamines); poly(propyleneamine) (POPAM) dendrimers; diaminobutane (DAB) dendrimers; amphiphilic dendrimers; micellar dendrimers, which are unimolecular micelles of water-soluble hyperbranched polyphenylenes; polylysine dendrimers; and dendrimers based on polybenzyl ether hyperbranched backbones.

[0150] In some embodiments, conjugation may be carried out by olefin metathesis. In some embodiments, both M and the targeting polypeptide, M and L, or the targeting polypeptide and L contain alkene or alkyne moieties that can undergo metathesis. In some embodiments, a suitable catalyst (e.g., copper, ruthenium) is used to accelerate the metathesis reaction. Suitable methods for carrying out olefin metathesis reactions are described in the art. See, for example, Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000), Walensky et al., Science 305:1466-1470 (2004), and Blackwell et al., Angew, Chem., Int. Ed. 37:3281-3284 (1998).

[0151] In some embodiments, conjugation may be performed using click chemistry. The "click reaction" is broad and easy to perform, uses only readily available reagents, and is insensitive to oxygen and water. In some embodiments, the click reaction is a cycloaddition reaction between an alkynyl group and an azide group to form a triazolyl group. In some embodiments, the click reaction uses a copper or ruthenium catalyst. Suitable methods for performing click reactions are described in the art. For example, Kolb et al.,Drug Discovery Today 8:1128(2003);Kolb et al.,Angew.Chem.Int.Ed.40:2004(2001);Rostovtsev et al.,Angew.Chem.Int.Ed.41:2596(2002);Tornoe et al. al.,J.Org.Chem.67:3057(2002);Manetsch et al.,J.Am.Chem.Soc.126:12809(2004);Lewis et al. al.,Angew.Chem.Int.Ed.41:1053(2002);Speers,J.Am.Chem.Soc.125:4686(2003);Chan et al.Org.Lett.6:2853(2004);Zhang et al. See Waser et al., J. Am. Chem. Soc. 127:15998 (2005); and Waser et al., J. Am. Chem. Soc. 127:8294 (2005).

[0152] Indirect conjugation via high affinity specific binding partners, such as streptavidin / biotin, or avidin / biotin, or lectin / carbohydrate, is also contemplated.

[0153] In some embodiments, the targeting polypeptide and / or M is functionalized to contain a nucleophilic reactive group or an electrophilic reactive group with an organic derivatizing agent. This derivatizing agent can react with selected side chains or N- or C-terminal residues of the targeting amino acid on the targeting polypeptide and functional groups on M. Reactive groups on the targeting polypeptide and / or M include, for example, aldehyde, amino, ester, thiol, α-haloacetyl, maleimide, or hydrazino groups. Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine ​​residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, or other agents known in the art. Alternatively, the targeting polypeptide and / or M can be indirectly linked to each other via an intermediate carrier, such as a polysaccharide or polypeptide carrier. An example of a polysaccharide carrier is aminodextran. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and mixed polymers of these amino acids with others, such as serine, to impart desirable solubility characteristics to the resulting loaded carrier.

[0154] Cysteinyl residues most commonly react with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetylphosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0155] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

[0156] Lysinyl and amino-terminal residues react with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include transaminase-catalyzed reactions with imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and glyoxylate.

[0157] Arginyl residues are modified by reaction with one or several conventional reagents, such as phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed under alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the lysine and arginine epsilon-amino groups.

[0158] The specific modification of tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetyloxyimidizole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively.

[0159] Carboxyl side groups (aspartyl or glutamyl) can be selectively modified by reaction with carbodiimides (RN=C=N-R'), where R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0160] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), deamidation of asparagine or glutamine, acetylation of N-terminal amines, and / or amidation or esterification of C-terminal carboxylic acid groups.

[0161] Another type of covalent modification involves chemically or enzymatically coupling glycosides to peptides. The sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as those of cysteine, (d) free hydroxyl groups, such as those of serine, threonine, or hydroxyproline, (e) aromatic residues, such as those of tyrosine or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 1987 / 05330 and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0162] In some embodiments, L is a bond. In these embodiments, the targeting polypeptide and M are conjugated together by reacting a nucleophilic reactive moiety on the targeting polypeptide with an electrophilic reactive moiety on M. In alternative embodiments, the targeting polypeptide and M are conjugated together by reacting an electrophilic reactive moiety on the targeting polypeptide with a nucleophilic moiety on M. In an exemplary embodiment, L is an amide bond formed upon reaction of an amine on the targeting polypeptide (e.g., the ε-amine of a lysine residue) with a carboxyl group on M. In alternative embodiments, the targeting polypeptide and / or M are derivatized with a derivatizing agent prior to conjugation.

[0163] In some embodiments, L is a linking group. In some embodiments, L is a bifunctional linker and comprises only two reactive groups prior to conjugation to the targeting polypeptide and M. In embodiments in which both the targeting polypeptide and M have electrophilic reactive groups, L comprises two of the same or two different nucleophilic groups (e.g., amine, hydroxyl, thiol) prior to conjugation to the targeting polypeptide and M. In embodiments in which both the targeting polypeptide and M have nucleophilic reactive groups, L comprises two of the same or two different electrophilic groups (e.g., carboxyl group, activated form of carboxyl group, compound with leaving group) prior to conjugation to the targeting polypeptide and M. In embodiments in which one of the targeting polypeptide or M has a nucleophilic reactive group and the other of the targeting polypeptide or M has an electrophilic reactive group, L comprises one nucleophilic reactive group and one electrophilic group prior to conjugation to the targeting polypeptide and M.

[0164] L can be any molecule having at least two reactive groups that can react with each of the targeting polypeptide and M (prior to conjugation to the targeting polypeptide and M). In some embodiments, L has only two reactive groups and is bifunctional. L (prior to conjugation to the peptide) can be represented by Formula VI: [ka] (wherein A and B are independently nucleophilic or electrophilic reactive groups). In some embodiments, A and B are both nucleophilic groups or both electrophilic groups. In some embodiments, one of A or B is a nucleophilic group and the other of A or B is an electrophilic group. Non-limiting combinations of A and B are shown in Table 1 below.

[0165] [Table 1] [Table 2] [Table 3] [Table 4]

[0166] In some embodiments, A and B may comprise alkene and / or alkyne functional groups suitable for olefin metathesis reactions. In some embodiments, A and B comprise moieties suitable for click chemistry (e.g., alkenes, alkynes, nitriles, azides). Other non-limiting examples of reactive groups (A and B) include pyridyldithiols, aryl azides, diazirines, carbodiimides, and hydrazides.

[0167] In some embodiments, L is hydrophobic. Hydrophobic linkers are known in the art. See, for example, Bioconjugate Techniques, G.T. Hermanson (Academic Press, San Diego, CA, 1996), which is incorporated by reference in its entirety. Suitable hydrophobic linking groups known in the art include, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoic acid. Prior to conjugation of the composition to the peptide, the hydrophobic linking group comprises at least two reactive groups (A and B), as described herein and shown below: [ka]

[0168] In some embodiments, the hydrophobic linking group comprises either a maleimide group or an iodoacetyl group and either a carboxylic acid or an activated carboxylic acid (e.g., an NHS ester) as the reactive group. In these embodiments, the maleimide group or iodoacetyl group can be coupled to a thiol moiety on the targeting polypeptide or M, and the carboxylic acid or activated carboxylic acid can be coupled to an amine on the targeting polypeptide or M, with or without the use of a coupling reagent. Any coupling agent known to those of skill in the art may be used to couple the carboxylic acid to a free amine, such as DCC, DIC, HATU, HBTU, TBTU, and other activators described herein. In certain embodiments, the hydrophilic linking group comprises an aliphatic chain of 2 to 100 methylene groups, where A and B are carboxyl groups or derivatives thereof (e.g., succinic acid). In other specific embodiments, L is iodoacetic acid. [ka]

[0169] In some embodiments, the linking group is hydrophilic, such as a polyalkylene glycol. Prior to conjugation of the composition to the peptide, the hydrophilic linking group comprises at least two reactive groups (A and B), as described herein and shown below: [ka]

[0170] In certain embodiments, the linking group is polyethylene glycol (PEG). In certain embodiments, PEG has a molecular weight of about 100 daltons to about 10,000 daltons, e.g., about 500 daltons to about 5000 daltons. In some embodiments, PEG has a molecular weight of about 10,000 daltons to about 40,000 daltons.

[0171] In some embodiments, the hydrophilic linking group comprises either a maleimide group or an iodoacetyl group and either a carboxylic acid or an activated carboxylic acid (e.g., an NHS ester) as the reactive group. In these embodiments, the maleimide group or iodoacetyl group can be coupled to a thiol moiety on the targeting polypeptide or M, and the carboxylic acid or activated carboxylic acid can be coupled to an amine on the targeting polypeptide or M, with or without the use of a coupling reagent. Any suitable coupling agent known to those of skill in the art may be used to couple the carboxylic acid to an amine, such as DCC, DIC, HATU, HBTU, TBTU, and other activators described herein. In some embodiments, the linking group is maleimide-polymer(20-40 kDa)-COOH, iodoacetyl-polymer(20-40 kDa)-COOH, maleimide-polymer(20-40 kDa)-NHS, or iodoacetyl-polymer(20-40 kDa)-NHS.

[0172] In some embodiments, the linking group is comprised of an amino acid, a dipeptide, a tripeptide, or a polypeptide, wherein the amino acid, dipeptide, tripeptide, or polypeptide comprises at least two activating groups as described herein. In some embodiments, the linking group (L) comprises a moiety selected from the group consisting of amino, ether, thioether, maleimide, disulfide, amide, ester, thioester, alkene, cycloalkene, alkyne, trizoyl, carbamate, carbonate, cathepsin B cleavable, and hydrazone.

[0173] In some embodiments, L comprises a chain length of 1 to about 60 atoms, or 1 to 30 or more atoms, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms. In some embodiments, the chain atoms are all carbon atoms. In some embodiments, the chain atoms in the backbone of the linker are selected from the group consisting of C, O, N, and S. The chain atoms and linker may be selected according to their expected solubility (hydrophilicity) to provide a more soluble conjugate. In some embodiments, L provides a functional group that undergoes cleavage by an enzyme or other catalyst or hydrolysis conditions found in the target tissue, organ, or cell. In some embodiments, the length of L is sufficient to reduce the potential for steric hindrance.

[0174] In some embodiments, L is stable in biological fluids, such as blood or blood fractions. In some embodiments, L is stable in serum for at least 5 minutes, e.g., less than 25%, 20%, 15%, 10%, or 5% of the conjugate is cleaved when incubated in serum for 5 minutes. In other embodiments, L is stable in serum for at least 10, or 20, or 25, or 30, or 60, or 90, or 120 minutes, or 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, or 24 hours. In these embodiments, L does not contain a functional group that is susceptible to hydrolysis in vivo. In some exemplary embodiments, L is stable in serum for at least about 72 hours. Non-limiting examples of functional groups that cannot undergo significant hydrolysis in vivo include amides, ethers, and thioethers. For example, the following compound (Q and Y represent two groups connected by the linker group L) does not undergo significant hydrolysis in vivo: [ka]

[0175] In some embodiments, L is hydrolyzable in vivo. In these embodiments, L comprises a functional group that can undergo hydrolysis in vivo. Non-limiting examples of functional groups that can undergo hydrolysis in vivo include esters, anhydrides, and thioesters. For example, the following compound (Q and Y represent two groups connected by the linker group L) contains an ester group and can therefore undergo hydrolysis in vivo: [ka]

[0176] In some exemplary embodiments, L is unstable, undergoing substantial hydrolysis within 3 hours and complete hydrolysis within 6 hours in plasma at 37° C. In some exemplary embodiments, L is not unstable.

[0177] In some embodiments, L is metastable in vivo. In these embodiments, L includes a functional group (e.g., an acid-labile functional group, a reduction-labile functional group, or an enzyme-labile functional group) that can be chemically or enzymatically cleaved in vivo, optionally over a period of time. In these embodiments, L may include, for example, a hydrazone moiety, a disulfide moiety, or a cathepsin-cleavable moiety. Without intending to be bound by any particular theory, when L is metastable, the targeting polypeptide-LM conjugate is stable in the extracellular environment, e.g., stable in serum for the period described above, but is unstable in the intracellular environment or conditions that mimic the intracellular environment, and therefore is cleaved upon entering the cell. In some embodiments, when L is metastable, L is stable in serum for at least about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, or 48 hours, e.g., at least about 48, 54, 60, 66, or 72 hours, or for about 24-48, 48-72, 24-60, 36-48, 36-72, or 48-72 hours.

[0178] In another embodiment, the polymer derivatives of the present disclosure comprise a polymer backbone having the following structure: X-CH2CH2O-(CH2CH2O) n -CH2CH2-O-(CH2) m -WN=N=N, (where: W is an aliphatic or aromatic linker moiety containing 1 to 10 carbon atoms; n is from 1 to about 4000, X is a functional group as described above, and m is from 1 to 10).

[0179] The azide-containing polymer derivatives of the present disclosure can be prepared by various methods known in the art and / or disclosed herein. In one method, illustrated below, a water-soluble polymer backbone having an average molecular weight of about 800 Da to about 100,000 Da, wherein a first end is attached to a first functional group and a second end is attached to a suitable leaving group, is reacted with azide anion (which can be paired with any of several suitable counterions, including sodium, potassium, tert-butylammonium, etc.). The leaving group undergoes nucleophilic displacement and is replaced with an azide moiety, yielding the desired azide-containing polymer;

[0180] X-Polymer-LY+N3 - →X-Polymer-L N3

[0181] As shown, suitable polymer backbones for use in the present disclosure have the formula X-polymer-LY, where the polymer is poly(ethylene glycol), X is a functional group that does not react with azide groups, and Y is a suitable leaving group. Examples of suitable functional groups include, but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl, amine, aminooxy, protected amine, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine, and vinylpyridine, and ketone. Examples of suitable leaving groups include, but are not limited to, chloride, bromide, iodide, mesylate, tresylate, and tosylate.

[0182] Another method for preparing the azide-containing polymer derivatives of the present disclosure involves contacting a linking agent having an azide functionality with a water-soluble polymer backbone having an average molecular weight of from about 800 Da to about 100,000 Da, where the linking agent has chemical functionality that selectively reacts with chemical functionality on the polymer to form an azide-containing polymer derivative product, with the azide separated from the polymer backbone by the linking group.

[0183] An exemplary reaction scheme is shown below: X-polymer-Y+N-linker-N=N=N→PG-X-polymer-linker-N=N=N, where The polymer is poly(ethylene glycol), X is a capping group such as alkoxy or a functional group as described above; and Y is a functional group that does not react with azide functional groups but reacts efficiently and selectively with N functional groups).

[0184] Examples of suitable functional groups include, but are not limited to, Y, which is a carboxylic acid, carbonate, or active ester when N is an amine; Y, which is a ketone when N is a hydrazide or aminooxy moiety; and Y, which is a leaving group when N is a nucleophile. Purification of the crude product can be achieved by known methods, including, but not limited to, precipitation of the product, optionally followed by chromatography.

[0185] A more specific example for the case of a polymeric diamine in which one of the amines is protected with a protecting group moiety such as tert-butyl-Boc, and the resulting monoprotected polymeric diamine is reacted with a linking moiety bearing an azide functionality, is shown below: BocHN-polymerNH2+HO2C-(CH2)3-N=N=N

[0186] In this case, the amine group can be coupled to a carboxylic acid group using thionyl chloride or carbodiimide reagents and various activating agents, such as N-hydroxysuccinimide or N-hydroxybenzotriazole, to create an amide bond between the monoamine polymer derivative and the azide group-containing linker moiety. After successful amide bond formation, the resulting N-tert-butyl-Boc-protected azide-containing derivative can be used directly to modify biologically active molecules or can be further constructed to introduce other useful functional groups. For example, the Nt-Boc group can be hydrolyzed by treatment with strong acid to generate an omega-amino-polymer-azide. The resulting amine can be used as a synthetic handle to introduce other useful functional groups, such as maleimide groups, activated disulfides, and activated esters, to create valuable heterobifunctional reagents.

[0187] Heterobifunctional derivatives are particularly useful when it is desirable to attach different molecules to each end of a polymer, for example, omega-N-amino-N-azide polymers allow for the attachment of a molecule bearing an activated electrophilic group, such as an aldehyde, ketone, activated ester, or activated carbonate, to one end of the polymer and a molecule bearing an acetylene group to the other end of the polymer.

[0188] In another embodiment of the present disclosure, A is an aliphatic linker of 1 to 10 carbon atoms or a substituted aryl ring of 6 to 14 carbon atoms, X is a functional group that does not react with the azide group, and Y is a suitable leaving group.

[0189] Multiple targeting polypeptides may be linked by a linker polypeptide, where the linker polypeptide is optionally 6-14, 7-13, 8-12, 7-11, 9-11, or 9 amino acids in length. Other linkers include, but are not limited to, small polymers such as PEG, which may be multi-armed, allowing multiple targeting polypeptide molecules to be linked together. Multiple targeting polypeptides and modified targeting polypeptides may be linked to each other in a head-to-head configuration via their N-termini using such linkers or by direct chemical bonding between the respective N-termini of each polypeptide. For example, two targeting polypeptides may be linked to form a dimer by a chemical bond between their N-terminal amino groups or modified N-terminal amino groups. Multiple targeting polypeptides may also be linked at their respective N-termini using a linking molecule designed to contain multiple chemical functional groups for binding to the N-terminus of each targeting polypeptide. Furthermore, multiple targeting polypeptides may be linked by bonds between amino acids other than the N-terminal or C-terminal amino acids. Examples of covalent bonds that can be used to form dimers and multimers of the targeting polypeptides described herein include, but are not limited to, disulfide bonds, sulfhydryl bonds, or thiol bonds. In addition, certain enzymes, such as sortase, can be used to form covalent bonds between a targeting polypeptide and a linker that includes the N-terminus of the targeting polypeptide.

[0190] The linker may have a wide range of molecular weights or molecular lengths. Linkers with larger or smaller molecular weights can be used to provide the desired spatial relationship or conformation between the targeting polypeptide and the conjugate, or, if present, between the conjugate and its binding partner. Linkers with longer or shorter molecular lengths can be used to provide the desired space or flexibility between the targeting polypeptide and its conjugate, or between the conjugate and its binding partner.

[0191] In some embodiments, the present disclosure provides a water-soluble bifunctional linker having a dumbbell structure comprising: a) an azide, alkyne, hydrazine, hydrazide, aminooxy, hydroxylamine, or carbonyl-containing moiety at at least a first end of the polymer backbone; and b) at least a second functional group at a second end of the polymer backbone. The second functional group may be the same as or different from the first functional group. In some embodiments, the second functional group does not react with the first functional group. In some embodiments, the present disclosure provides a water-soluble compound comprising at least one arm of a branched molecular structure. For example, the branched molecular structure may be dendritic.

[0192] In exemplary embodiments, the polymer is linked to the targeting polypeptide or modified targeting polypeptide via a linker. For example, the linker may comprise one or two amino acids attached to the polymer at one end—e.g., an albumin-binding moiety—and attached to any available position on the polypeptide backbone at the other end. Additional exemplary linkers include hydrophilic linkers, such as chemical moieties containing at least five non-hydrogen atoms, 30-50% of which are either N or O. Further exemplary linkers that may link the polymer to the targeting polypeptide or modified targeting polypeptide are disclosed in US2012 / 0295847 and WO / 2012 / 168430, each of which is incorporated herein by reference in its entirety.

[0193] Optionally, multiple targeting polypeptides or modified targeting polypeptide molecules may be linked by a linker polypeptide, wherein the linker polypeptide is optionally 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, and optionally the N-terminus of one targeting polypeptide is fused to the C-terminus of the linker polypeptide and the N-terminus of the linker polypeptide is fused to the N-terminus of another targeting polypeptide. Further exemplary linker polypeptides that may be utilized are disclosed in WO / 2013 / 004607, the entire contents of which are incorporated herein by reference.

[0194] As used herein, the terms "electrophilic group," "electrophile," and the like refer to an atom or group of atoms that can accept an electron pair to form a covalent bond. As used herein, "electrophilic group" includes, but is not limited to, halides, carbonyl, and epoxide-containing compounds. Typical electrophiles can be halides, such as thiophosgene, glycerin dichlorohydrin, phthaloyl chloride, succinyl chloride, chloroacetyl chloride, chlorosuccinyl chloride, and the like; ketones, such as chloroacetone, bromoacetone, and the like; aldehydes, such as glyoxal, and the like; isocyanates, such as hexamethylene diisocyanate, toluene diisocyanate, meta-xylylene diisocyanate, cyclohexylmethane-4,4-diisocyanate, and the like, and derivatives of these compounds.

[0195] As used herein, the terms "nucleophilic group," "nucleophile," and the like refer to an atom or group of atoms that have a pair of electrons capable of forming a covalent bond. This type of group can be an ionizable group that reacts as an anionic group. As used herein, "nucleophilic groups" include, but are not limited to, hydroxyl, primary amine, secondary amine, tertiary amine, and thiol.

[0196] Table 2 shows various starting electrophiles and nucleophiles that may be combined to create desired functional groups. The information provided is meant to be illustrative and not limiting of the synthetic techniques described herein.

[0197] [Table 5] [Table 6]

[0198] Generally, carbon electrophiles are susceptible to attack by complementary nucleophiles, including carbon nucleophiles, where the attacking nucleophile donates an electron pair to the carbon electrophile to form a new bond between the nucleophile and the carbon electrophile.

[0199] Non-limiting examples of carbon nucleophiles include alkyl, alkenyl, aryl, and alkynyl Grignards, organolithium, organozinc, alkyl-, alkenyl-, aryl-, and alkynyl-tin reagents (organostannanes), and alkyl-, alkenyl-, aryl-, and alkynyl-borane reagents (organoboranes and organoboronic acids), which have the advantage of being kinetically stable in water or polar organic solvents. Other non-limiting examples of carbon nucleophiles include phosphorus ylide, enol, and enolate reagents, which have the advantage of being relatively easy to generate from precursors well known to those skilled in the art of synthetic organic chemistry. When used in combination with a carbon electrophile, a carbon nucleophile generates a new carbon-carbon bond between the carbon nucleophile and the carbon electrophile.

[0200] 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, thioethers, alcohols, alkoxides, azides, semicarbazides, etc. These non-carbon nucleophiles, when used in combination with carbon electrophiles, typically produce heteroatom bonds (CXC), where X is a heteroatom, including, but not limited to, oxygen, sulfur, or nitrogen.

[0201] In some cases, the polymers used in the present disclosure terminate at one end with hydroxy or methoxy, i.e., X is H or CH3 ("methoxyPEG"). Alternatively, the polymer may terminate with a reactive group, thereby forming a bifunctional polymer. Exemplary reactive groups may include reactive groups commonly used to react with 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 esters), activated esters (including, but not limited to, N-hydroxysuccinimide, p-nitrophenyl esters), and aldehydes), as well as functional groups that are inert toward the 20 common amino acids but specifically react with complementary functional groups (including, but not limited to, azide groups, alkyne groups). Note that the other end of the polymer, represented by Y in the above formula, is directly or indirectly attached to a targeting polypeptide via a naturally occurring or non-naturally encoded amino acid. For example, Y may be an amide, carbamate, or urea linkage to an amine group of the polypeptide (including, but not limited to, the epsilon amine of lysine or the N-terminus). Alternatively, Y may be a maleimide linkage to a thiol group (including, but not limited to, the thiol group of cysteine). Alternatively, Y may be a linkage to a residue that is not generally accessible through the 20 common amino acids. For example, an azide group on a polymer may react with an alkyne group on a targeting polypeptide to form a Huisgen [3+2] cycloaddition product. Alternatively, an alkyne group on a polymer may react with an azide group present in a targeting polypeptide to form a similar product. In some embodiments, strong nucleophiles (including, but not limited to, hydrazine, hydrazide, hydroxylamine, semicarbazide) may react with aldehyde or ketone groups present in the targeting polypeptide to form hydrazones, oximes, or semicarbazones as needed, which may be further reduced in some cases by treatment with an appropriate reducing agent.Alternatively, strong nucleophiles may be incorporated into the targeting polypeptide via a non-naturally encoded amino acid, which may be used to preferentially react with ketone or aldehyde groups present in the water-soluble polymer.

[0202] Any molecular weight for the polymer may be used as practically desired, including, but not limited to, from about 100 Daltons (Da) to 100,000 Da or more, optionally including, but not limited to, 0.1 to 50 kDa or 10 to 40 kDa. The molecular weight of the polymer may range widely, including, but not limited to, from about 100 Da to about 100,000 Da or more. The polymer may be from about 100 Da to 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, In some embodiments, the polymer is from about 100 Da to about 50,000 Da. Branched polymers may also be used, including, but not limited to, polymer molecules having a molecular weight in the range of 1 to 100 kDa (including, but not limited to, 1 to 50 kDa or 5 to 20 kDa) for each chain. The molecular weight of each chain of the branched polymer may be, but is not limited to, from about 1,000 Da to about 100,000 Da or more.The molecular weight of each chain of the branched polymer may be from about 1,000 Da to 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, 5 ... Examples of molecular weights include 0 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, and 1,000 Da. In some embodiments, the molecular weight of each chain of the branched polymer is from about 1,000 Da to about 50,000 Da. In some embodiments, the molecular weight of each chain of the branched polymer is from about 1,000 Da to about 40,000 Da. In some embodiments, the molecular weight of each chain of the branched polymer is from about 5,000 Da to about 40,000 Da. In some embodiments, the molecular weight of each chain of the branched polymer is from about 5,000 Da to about 20,000 Da. A wide range of polymer molecules are described, including but not limited to, in the Shearwater Polymers, Inc. catalog and the Nektar Therapeutics catalog, both of which are incorporated herein by reference.

[0203] The present disclosure provides, in some embodiments, azide- and acetylene-containing polymer derivatives comprising a water-soluble polymer backbone having an average molecular weight of 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 polymers, including but not limited to poly(ethylene glycol) and other related polymers including poly(dextran) and poly(propylene glycol), are also suitable for use in the practice of the present disclosure, and the use of the term PEG or poly(ethylene glycol) is intended to encompass and include all such molecules. The term PEG includes poly(ethylene glycol) in any of its forms, including but not limited to bifunctional PEG, multi-armed PEG, derivatized PEG, forked PEG, branched PEG, pendant PEG (i.e., PEG or related polymers having one or more functional groups dependent from the polymer backbone), or PEG containing degradable linkages therein.

[0204] In addition to these forms of polymers, polymers may be prepared with weak or degradable bonds in the backbone. For example, polymers may be prepared with ester bonds in the polymer backbone that are hydrolyzed, which cleaves the polymer into lower molecular weight fragments, as shown below: -Polymer-CO2-Polymer-+H2O → Polymer-CO2H+HO-Polymer-

[0205] Many polymers are also suitable for use in the present disclosure. In some embodiments, water-soluble polymer backbones having from 2 to about 300 termini are particularly useful in the present disclosure. Examples of suitable polymers include, but are not limited to, other poly(alkylene glycols), such as poly(propylene glycol) ("PPG"), their copolymers (including, but not limited to, copolymers of ethylene glycol and propylene glycol), their terpolymers, mixtures thereof, and the like. The molecular weight of each chain of the polymer backbone can vary, but 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 from about 100 Da to 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. Examples of molecular weights include 1, 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 from about 100 Da to about 50,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is from about 100 Da to about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is from about 1,000 Da to about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is from about 5,000 Da to about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is from about 10,000 Da to about 40,000 Da.

[0206] In one feature of this embodiment of the present disclosure, the intact polymer conjugate is minimally degraded upon administration prior to hydrolysis, such that hydrolysis of the cleavable bond is effective to govern the rate of sustained release of the active targeting polypeptide into the bloodstream, as opposed to enzymatic degradation prior to release of the targeting polypeptide into the systemic circulation.

[0207] Suitable physiologically cleavable bonds include, but are not limited to, esters, carbonates, carbamates, sulfates, phosphates, acyloxyalkyl ethers, acetals, and ketals. Such conjugates should have physiologically cleavable bonds that are stable during storage and administration. For example, a targeting polypeptide or modified targeting polypeptide linked to a polymer should maintain its integrity when prepared as a final pharmaceutical composition, when dissolved in an appropriate delivery vehicle (if used), and when administered regardless of route.

[0208] Structure and synthesis of cytotoxic tubulin inhibitor linker derivatives: electrophilic and nucleophilic groups Cytotoxic tubulin inhibitor derivatives with linkers containing hydroxylamine (also called aminooxy) groups can react with various electrophilic groups to form conjugates (including, but not limited to, conjugates with PEG or other water-soluble polymers). Similar to hydrazines, hydrazides, and semicarbazides, the enhanced nucleophilicity of the aminooxy group allows it to react efficiently and selectively with various molecules containing carbonyl or dicarbonyl groups, including, but not limited to, ketones, aldehydes, or other functional groups with similar chemical reactivity. See, for example, Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34(9):727-736 (2001). However, while reaction with a hydrazine group produces the corresponding hydrazone, oximes are generally produced by reaction of an aminooxy group with a carbonyl- or dicarbonyl-containing group, such as a ketone, aldehyde, or other functional group with similar chemical reactivity. In some embodiments, cytotoxic tubulin inhibitor derivatives having linkers containing azides, alkynes, or cycloalkynes allow for linking of molecules via cycloaddition reactions (e.g., 1,3-dipolar cycloaddition, azide-alkyne, Huisgen cycloaddition, etc.) (as described in U.S. Pat. No. 7,807,619, the contents of which are incorporated herein by reference to the extent relevant to the reaction).

[0209] Thus, in certain embodiments described herein, there are cytotoxic tubulin inhibitor derivatives having linkers containing hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, ketoamine, ketoalkyne, and enedione hydroxylamine groups, hydroxylamine-like groups (which have similar reactivity to and are structurally similar to hydroxylamine groups), masked hydroxylamine groups (which can be readily converted to hydroxylamine groups), or protected hydroxylamine groups (which have similar reactivity to hydroxylamine groups upon deprotection). In some embodiments, the cytotoxic tubulin inhibitor derivatives having linkers include azides, alkynes, or cycloalkynes.

[0210] Such cytotoxic tubulin inhibitor linker derivatives or targeting polypeptides may be in the form of a salt or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide or polynucleotide, and may optionally be post-translationally modified.

[0211] In certain embodiments, the cytotoxic tubulin inhibitor linker derivative compounds are stable in aqueous solution under mildly acidic conditions for at least one month. In certain embodiments, the ADCs disclosed herein are stable under mildly acidic conditions for at least two weeks. In certain embodiments, the ADCs disclosed herein are stable under mildly acidic conditions for at least five days. In certain embodiments, such acidic conditions are pH 2-8. In certain embodiments, the ADCs disclosed herein have a shelf life of one, two, three, or more years as liquid and lyophilized powder formulations for injection when stored at -25°C to -15°C and 2°C to 8°C, respectively.

[0212] The methods and compositions provided and described herein include polypeptides containing unnatural amino acids bearing at least one carbonyl or dicarbonyl group, oxime group, hydroxylamine group, or protected or masked forms thereof. The introduction of at least one reactive group into a cytotoxic tubulin inhibitor linker derivative or targeting polypeptide can enable the application of conjugation chemistries that involve specific chemical reactions with one or more targeting polypeptide(s) (but do not react with normally present amino acids). Once incorporated, the targeting polypeptide of the ADC side chain may be modified using the chemical methodologies described herein or chemical methodologies appropriate for the specific functional groups or substituents present in the cytotoxic tubulin inhibitor linker derivative or targeting polypeptide.

[0213] The cytotoxic tubulin inhibitor linker derivatives and targeting polypeptide methods and compositions described herein provide for conjugates of substances having a wide variety of functional groups, substituents or moieties with other substances, including, but not limited to, polymers, water-soluble polymers; derivatives of polyethylene glycol; second proteins or polypeptides or polypeptide analogs; antibodies or antibody fragments; and any combination thereof.

[0214] In certain embodiments, the cytotoxic tubulin inhibitor linker derivatives, targeting polypeptides, ADCs, linkers, and reagents (described herein) are stable in aqueous solution under mildly acidic conditions (including, but not limited to, pH 2-8). In other embodiments, such compounds are stable under mildly acidic conditions for at least one month. In other embodiments, such compounds are stable under mildly acidic conditions for at least two weeks. In other embodiments, such compounds are stable under mildly acidic conditions for at least five days.

[0215] Another aspect of the compositions, methods, techniques, and strategies described herein is a method for studying or using any of the above-described "modified or unmodified" non-natural amino acid targeting polypeptides. This aspect includes, by way of example only, therapeutic, diagnostic, assay-based, industrial, cosmetic, plant biology, environmental, energy production, consumer product, and / or military uses that would benefit from targeting polypeptides comprising "modified or unmodified" non-natural amino acid polypeptides or proteins.

[0216] The present disclosure provides ADC molecules comprising at least one unnatural amino acid. In certain embodiments of the present disclosure, the ADCs comprising at least one unnatural amino acid comprise at least one post-translational modification. In one embodiment, the at least one post-translational modification is selected from the group consisting of, but not limited to, a label, a dye, a linker, another ADC polypeptide, a polymer, a water-soluble polymer, a derivative of polyethylene glycol, a photocrosslinker, a radionuclide, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound, a resin, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, DNA, RNA, an antisense polynucleotide, a sugar, a cyclodextrin, an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that interacts covalently or non-covalently with other molecules, a photocaged moiety, a moiety excitable by actinic radiation, a photoisomerizable moiety, a hydroxyl group ... The present invention also includes the attachment of a molecule comprising a first reactive group to at least one unnatural amino acid comprising a first reactive group, the molecule comprising a cleavable moiety, biotin, a derivative of biotin, a biotin analog, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, an extended side chain, a carbon-linked sugar, a redox-active agent, an aminothioacid, a toxic moiety, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chemiluminescent group, an electron-dense group, a magnetic group, an intercalating group, a chromophore, an energy transfer agent, a biologically active agent, a detectable label, a small molecule, a quantum dot, a nanotransmitter, a radionucleotide, a radiotransmitter, a neutron capture agent, or any combination of the above, or any other desired compound or substance, using chemical methods known to those of skill in the art to be suitable for the particular reactive group. For example, the first reactive group is an alkynyl moiety (including but not limited to, the unnatural amino acid p-propargyloxyphenylalanine, where the propargyl group is sometimes also referred to as an acetylene moiety) and the second reactive group is an azide moiety, and [3+2] cycloaddition chemistry is utilized.In another example, the first reactive group is an azido moiety (including, but not limited to, the unnatural amino acid p-azido-L-phenylalanine, or pAZ (sometimes referred to herein)) and the second reactive group is an alkynyl moiety. Certain embodiments of the modified ADCs of the present disclosure use at least one unnatural amino acid (including, but not limited to, an unnatural amino acid comprising a keto functional group) comprising at least one post-translational modification, where the at least one post-translational modification comprises a saccharide moiety. In certain embodiments, the post-translational modification occurs in vivo in a eukaryotic cell or a non-eukaryotic cell. A linker, polymer, water-soluble polymer, or other molecule may attach the molecule to the polypeptide. In additional embodiments, the linker attached to the ADC is of sufficient length to allow for dimer formation. The molecule may also be directly linked to the polypeptide.

[0217] In certain embodiments, the ADC protein comprises at least one post-translational modification carried out in vivo by one host cell type that is not normally carried out by another host cell type. In certain embodiments, the protein comprises at least one post-translational modification carried out in vivo by a eukaryotic cell that is not normally carried out by a non-eukaryotic cell. Examples of post-translational modifications include, but are not limited to, glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linkage modification, and the like.

[0218] In some embodiments, an ADC comprises one or more non-naturally encoded amino acids for glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, or glycolipid-linkage modification of a polypeptide. In some embodiments, an ADC comprises one or more non-naturally encoded amino acids for glycosylation of a polypeptide. In some embodiments, an ADC comprises one or more naturally encoded amino acids for glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, or glycolipid-linkage modification of a polypeptide. In some embodiments, an ADC comprises one or more naturally encoded amino acids for glycosylation of a polypeptide.

[0219] In some embodiments, an ADC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation of the polypeptide. In some embodiments, an ADC comprises one or more deletions that enhance glycosylation of the polypeptide. In some embodiments, an ADC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation at different amino acids of the polypeptide. In some embodiments, an ADC comprises one or more deletions that enhance glycosylation at different amino acids of the polypeptide. In some embodiments, an ADC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation at different amino acids of the polypeptide. In some embodiments, an ADC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation at naturally encoded amino acids of the polypeptide. In some embodiments, an ADC comprises one or more naturally encoded amino acid additions and / or substitutions that enhance glycosylation at different amino acids of the polypeptide. In some embodiments, the ADC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation of naturally encoded amino acids of the polypeptide, hi some embodiments, the ADC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation of non-naturally encoded amino acids of the polypeptide.

[0220] In one embodiment, the post-translational modification comprises the attachment of an oligosaccharide to asparagine via a GlcNAc-asparagine linkage (including, but not limited to, the oligosaccharide being (GlcNAc-Man)2-Man-GlcNAc-GlcNAc, etc.). In another embodiment, the post-translational modification comprises the attachment of an oligosaccharide (including, but not limited to, Gal-GalNAc, Gal-GlcNAc, etc.) to serine or threonine via a GalNAc-serine, GalNAc-threonine, GlcNAc-serine, or GlcNAc-threonine linkage. In certain embodiments, proteins or polypeptides of the disclosure may comprise secretion or localization sequences, epitope tags, FLAG tags, polyhistidine tags, GST fusions, and / or the like. Examples of secretory signal sequences include, but are not limited to, prokaryotic secretory signal sequences, eukaryotic secretory signal sequences, eukaryotic secretory signal sequences 5'-optimized for bacterial expression, novel secretory signal sequences, pectate lyase secretory signal sequences, Omp A secretory signal sequences, and phage secretory signal sequences. Examples of secretory signal sequences include, but are not limited to, STII (prokaryotes), Fd GIII and M13 (phage), Bgl2 (yeast), and the transposon-derived signal sequence bla. Any such sequence may be modified to provide a desired result with the polypeptide, including, but not limited to, replacing one signal sequence with a different signal sequence, replacing one leader sequence with a different leader sequence, etc.

[0221] A protein or polypeptide of interest can comprise 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 unnatural amino acids. The unnatural amino acids can be the same or different, for example, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different sites in the protein that contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different unnatural amino acids. In certain embodiments, at least one, but not all, of a particular amino acid present in a naturally occurring version of the protein is substituted with an unnatural amino acid.

[0222] The present disclosure provides methods and compositions based on ADCs containing at least one non-naturally encoded amino acid. The incorporation of at least one non-naturally encoded amino acid into an ADC allows for the application of specific conjugation chemistries, including, but not limited to, those using one or more non-naturally encoded amino acids, without reacting with the 20 commonly occurring amino acids. In some embodiments, ADCs containing non-naturally encoded amino acids are attached to a water-soluble polymer, e.g., polyethylene glycol (PEG), or a linker via the side chain of the non-naturally encoded amino acid. The present disclosure provides a highly efficient method for the selective modification of proteins with PEG derivatives or cytotoxic tubulin inhibitor-linker derivatives, which involves selectively incorporating non-genetically encoded amino acids, including, but not limited to, amino acids containing functional groups or substituents not found in the 20 naturally incorporated amino acids, such as, but not limited to, ketone, azide, or acetylene moieties, into proteins in response to a selector codon, followed by modification of these amino acids with an appropriately reactive PEG derivative. Once incorporated, the amino acid side chain can then be modified to suit the particular functional group or substituent present in the non-naturally encoded amino acid by utilizing chemical methodologies known to those of skill in the art. A wide variety of known chemical methodologies are suitable for use in the present disclosure for incorporating water-soluble polymers into proteins. Such methodologies include, but are not limited to, the Huisgen [3 + 2] cycloaddition reaction, such as with acetylene or azide derivatives, respectively (see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991) Ed. Trost, BM, Pergamon, Oxford, pp. 1069-1109; and Huisgen, R. in 1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, New York, pp. 1-176).

[0223] The Huisgen [3 + 2] cycloaddition method involves cycloaddition rather than nucleophilic substitution, allowing for highly selective modification of proteins. This reaction can be carried out at room temperature under aqueous conditions with excellent regioselectivity (1,4 > 1,5) by adding a catalytic amount of Cu(I) salt to the reaction mixture. See, for example, Tornoe, et al., (2002) J. Org. Chem. 67:3057-3064; and Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599; and WO 03 / 101972. Molecules that can be added to proteins of the present disclosure via [3 + 2] cycloaddition include virtually any molecule with an appropriate functional group or substituent, including, but not limited to, azide or acetylene derivatives. These molecules may be added to unnatural amino acids bearing an acetylene group, including but not limited to, p-propargyloxyphenylalanine, or an azide group, including but not limited to, p-azidophenylalanine, respectively.

[0224] The five-membered ring resulting from the Huisgen [3 + 2] cycloaddition is generally not reversible in reducing environments and is stable to hydrolysis in aqueous environments for extended periods. As a result, the physical and chemical properties of a wide variety of materials can be modified with the disclosed active PEG derivatives or cytotoxic tubulin inhibitor-linker derivatives under harsh aqueous conditions. Even more importantly, because the azide and acetylene moieties are specific for each other (and do not react with, for example, any of the 20 common genetically encoded amino acids), proteins can be modified at one or more specific sites with very high selectivity.

[0225] The present disclosure also provides water-soluble and hydrolytically stable derivatives of PEG derivatives or cytotoxic tubulin inhibitor-linker derivatives, as well as related hydrophilic polymers containing one or more acetylene or azide moieties. PEG polymer derivatives containing acetylene moieties are highly selective for coupling with azide moieties selectively introduced into proteins in response to a selector codon. Similarly, PEG polymer derivatives containing azide moieties are highly selective for coupling with acetylene moieties selectively introduced into proteins in response to a selector codon. More specifically, azide moieties include, but are not limited to, alkyl azides, aryl azides, and derivatives of these azides. The alkyl and aryl azide derivatives may contain other substituents as long as the inherent reactivity of the acetylene is maintained. The acetylene moiety includes alkyl and aryl acetylenes and their respective derivatives. The alkyl and aryl acetylene derivatives may contain other substituents as long as the inherent reactivity of the azide is maintained.

[0226] The present disclosure relates to substances having a wide variety of functional groups, substituents, or moieties, including, but not limited to, labels; dyes; polymers; water-soluble polymers; derivatives of polyethylene glycol; photocrosslinkers; radionuclides; cytotoxic compounds; drugs; affinity labels; photoaffinity labels; reactive compounds; resins; second proteins or polypeptides or polypeptide analogs; antibodies or antibody fragments; metal chelators; cofactors; fatty acids; carbohydrates; polynucleotides, DNA, RNA, antisense polynucleotides; sugars; water-soluble dendrimers; cyclodextrins; inhibitory ribonucleic acids; biomaterials; nanoparticles; spin labels; fluorophores; metal-containing moieties; radioactive moieties; novel functional groups; and those capable of covalently or reactively bonding to other molecules. Conjugates with other substances, including non-covalently interacting groups, photocaged moieties, actinically excitable moieties, photoisomerizable moieties, biotin, biotin derivatives, biotin analogs, moieties incorporating heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox-active agents, aminothioacids, toxic moieties, isotope-labeled moieties, biophysical probes, phosphorescent groups, chemiluminescent groups, electron-dense groups, magnetic groups, intercalating groups, chromophores, energy transfer agents, biologically active substances, detectable labels, small molecules, quantum dots, nanotransmitters, radionucleotides, radiation transmitters, neutron capture agents, or any combination of the above, or any other desired compound or substance, are provided. The present disclosure also includes conjugates of substances having an azide or acetylene moiety with PEG polymer derivatives having the corresponding acetylene or azide moieties. For example, a PEG polymer containing an azide moiety can be coupled to a biologically active molecule at a position in a protein that contains a non-genetically encoded amino acid bearing an acetylene functionality. Linkages by which PEG and biologically active molecules are coupled include, but are not limited to, Huisgen [3+2] cycloaddition products.

[0227] It is well established in the art that PEG can be used to modify the surface of biomaterials (see, e.g., U.S. Pat. No. 6,610,281; Mehvar, R., J. Pharm Sci., 3(1):125-136 (2000), incorporated herein by reference). The present disclosure also includes biomaterials comprising a surface bearing one or more reactive azide or acetylene moieties and one or more of the azide- or acetylene-containing polymers of the present disclosure coupled to the surface via a Huisgen [3+2] cycloaddition linkage. Biomaterials and other substances can be coupled to azide- or acetylene-activated polymer derivatives via linkages other than azide or acetylene linkages, for example, via linkages containing carboxylic acid, amine, alcohol, or thiol moieties, leaving the azide or acetylene moiety available for subsequent reaction.

[0228] The present disclosure includes methods for synthesizing the azide- and acetylene-containing polymers of the present disclosure. In the case of azide-containing PEG derivatives, the azide can be directly bonded to a carbon atom of the polymer. Alternatively, azide-containing PEG derivatives can be prepared by attaching a linking agent having an azide moiety at one end to a conventional activated polymer, such that the resulting polymer has an azide moiety at its terminus. In the case of acetylene-containing PEG derivatives, the acetylene can be directly bonded to a carbon atom of the polymer. Alternatively, acetylene-containing PEG derivatives can be prepared by attaching a linking agent having an acetylene moiety at one end to a conventional activated polymer, such that the resulting polymer has an acetylene moiety at its terminus.

[0229] More specifically, in the case of azide-containing PEG derivatives, a water-soluble polymer having at least one activated hydroxyl moiety undergoes a reaction to produce a substituted polymer bearing a more reactive moiety thereon, such as a mesylate, tresylate, tosylate, or halogen leaving group. The preparation and use of PEG derivatives or cytotoxic tubulin inhibitor-linker derivatives containing sulfonyl acid halides, halogen atoms, and other leaving groups are known to those skilled in the art. The resulting substituted polymer then undergoes a reaction to replace the more reactive moiety at the end of the polymer with the azide moiety. Alternatively, a water-soluble polymer having at least one activated nucleophilic or electrophilic moiety can be reacted with a linking agent having an azide at one end, forming a covalent bond between the PEG polymer and the linking agent, with the azide moiety being located at the end of the polymer. Nucleophilic and electrophilic moieties, including amines, thiols, hydrazides, hydrazines, alcohols, carboxylates, aldehydes, ketones, thioesters, and the like, are known to those skilled in the art.

[0230] More specifically, in the case of acetylene-containing PEG derivatives, a water-soluble polymer having at least one activated hydroxyl moiety is reacted to displace a halogen or other activated leaving group from a precursor containing an acetylene moiety. Alternatively, a water-soluble polymer having at least one activated nucleophilic or electrophilic moiety is reacted with a linking agent having an acetylene moiety at one end, forming a covalent bond between the PEG polymer and the linking agent, with the acetylene moiety being located at the end of the polymer. The use of halogen moieties, activated leaving groups, nucleophilic and electrophilic moieties in the context of organic synthesis, and the preparation and use of PEG derivatives or cytotoxic tubulin inhibitor-linker derivatives, are well established in the art.

[0231] The present disclosure also provides methods for selectively modifying proteins to attach other substances to the modified protein, including, but not limited to, water-soluble polymers such as PEG and PEG derivatives or cytotoxic tubulin inhibitor-linker derivatives, linkers, or other ADC polypeptides containing azide or acetylene moieties. The azide and acetylene-containing PEG derivatives or cytotoxic tubulin inhibitor-linker derivatives can be used to modify surface and molecular properties where biocompatibility, stability, solubility, and lack of immunogenicity are important, while at the same time providing a means of attaching PEG derivatives or cytotoxic tubulin inhibitor-linker derivatives to proteins more selectively than those previously known in the art.

[0232] General Recombinant Nucleic Acid Methods for Use in This Disclosure In many embodiments of the present disclosure, nucleic acids encoding the targeting polypeptide of the ADC of interest are isolated, cloned, and often modified using recombinant methods. Such embodiments are used, by way of example and not limitation, for protein expression or during the production of variants, derivatives, expression cassettes, or other sequences derived from the targeting polypeptide of the ADC. In some embodiments, the sequence encoding the polypeptide of the present disclosure is operably linked to a heterologous promoter.

[0233] Nucleotide sequences encoding targeting polypeptides of ADCs containing unnatural amino acids may be synthesized based on the amino acid sequence of the parent polypeptide, and the nucleotide sequence may then be altered to introduce (i.e., incorporate or substitute) or remove (i.e., delete or substitute) the relevant amino acid residue(s). The nucleotide sequence may be conveniently modified by site-directed mutagenesis according to conventional methods. Alternatively, the nucleotide sequence may be prepared by chemical synthesis, including, but not limited to, using an oligonucleotide synthesizer, in which oligonucleotides are designed based on the amino acid sequence of the desired polypeptide, preferably by selecting codons suitable for the host cell in which the recombinant polypeptide will be produced. For example, several small oligonucleotides encoding portions of the desired polypeptide may be synthesized and assembled by PCR, ligation, or ligation chain reaction. See, e.g., Barany, et al., Proc. Natl. Acad. Sci. 88:189-193 (1991); U.S. Patent No. 6,521,427 (incorporated herein by reference).

[0234] This disclosure makes use of conventional techniques in the field of recombinant genetics. Basic documents that disclose the general methods used in this disclosure include Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994).

[0235] The present disclosure also relates to eukaryotic host cells, non-eukaryotic host cells, and organisms for in vivo incorporation of unnatural amino acids via orthogonal tRNA / RS pairs, where the host cells have been genetically engineered (including, but not limited to, transformed, transduced, or transfected) with a polynucleotide of the present disclosure or a construct comprising a polynucleotide of the present disclosure, including, but not limited to, a vector of the present disclosure, which may be, for example, a cloning vector or an expression vector.

[0236] Several well-known methods for introducing target nucleic acids into cells are available, any of which may be used in the present disclosure. These include fusion of recipient cells with bacterial protoplasts containing DNA, electroporation, projectile bombardment, and infection with viral vectors (discussed further below). Bacterial cells may be used to amplify the number of plasmids containing the DNA constructs of the present disclosure. Bacteria are grown to logarithmic phase, and the plasmids within the bacteria can be isolated by various methods known in the art (see, e.g., Sambrook). In addition, kits for purifying plasmids from bacteria are commercially available (see, e.g., EasyPrep™, FlexiPrep™ (both from Pharmacia Biotech); StrataClean™ (from Stratagene); and QIAPrep™ (from Qiagen)). The isolated and purified plasmids can then be further manipulated to produce other plasmids, used to transfect cells, or incorporated into related vectors to infect organisms. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulating the expression of specific target nucleic acids. Vectors optionally contain a general expression cassette containing at least one independent terminator sequence, sequences that enable replication of the cassette in eukaryotes, prokaryotes, or both (including, but not limited to, shuttle vectors), and selectable markers for both prokaryotic and eukaryotic systems. Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or both. See Gillam & Smith, Gene 8:81 (1979); Roberts, et al., Nature, 328:731 (1987); Schneider, E., et al., Protein Expr. Purif. 6(1):10-14 (1995); Ausubel, Sambrook, Berger (all supra).Catalogs of bacteria and bacteriophages useful for cloning are provided, for example, by the ATCC, for example, in The ATCC Catalogue of Bacteria and Bacteriophage (1992) Gherna et al. (eds), published by the ATCC. Additional basic procedures for sequencing, cloning, and other aspects of molecular biology, as well as underlying theoretical considerations, can also be found in Watson et al. (1992) Recombinant DNA Second Edition, Scientific American Books, NY. In addition, virtually any nucleic acid (and virtually any labeled nucleic acid, whether standard or not) can be custom-ordered or standard-ordered from any of a variety of commercial sources, such as Midland Certified Reagent Company (Midland, TX, available on the World Wide Web at mcrc.com), The Great American Gene Company (Ramona, CA, available on the World Wide Web at genco.com), ExpressGen Inc. (Chicago, IL, available on the World Wide Web at expressgen.com), Operon Technologies Inc. (Alameda, CA), and others.

[0237] Selector codon The selector codons of the present disclosure expand the genetic codon framework of the protein biosynthetic machinery. For example, selector codons include, but are not limited to, unique three-base codons, nonsense codons, such as stop codons, including, but not limited to, the amber codon (UAG), ochre codon, or opal codon (UGA), unnatural codons, four or more base codons, rare codons, etc. It will be readily apparent to one of skill in the art that there is a wide range in the number of selector codons that can be introduced into a desired gene or polynucleotide, including, but not limited to, one or more, two or more, three or more, four, five, six, seven, eight, nine, ten, or more in a single polynucleotide encoding at least a portion of an ADC.

[0238] In one embodiment, the method involves the use of a selector codon that is a stop codon to incorporate one or more unnatural amino acids in vivo. For example, an O-tRNA is generated that recognizes a stop codon, including but not limited to, UAG, and is aminoacylated with an O-RS bearing a desired unnatural amino acid. This O-tRNA is not recognized by naturally occurring host aminoacyl-tRNA synthetases. Conventional site-directed mutagenesis can be used to introduce a stop codon, including but not limited to, TAG, at a desired site in a polypeptide of interest. See, e.g., Sayers, JR, et al. (1988), 5'-3' Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res, 16:791-802. When the O-RS, O-tRNA, and nucleic acid encoding the polypeptide of interest are combined in vivo, the unnatural amino acid is incorporated in response to the UAG codon to yield a polypeptide containing the unnatural amino acid at the specified position.

[0239] In vivo incorporation of unnatural amino acids can be performed without significant perturbation of the eukaryotic host cell. For example, because the efficiency of suppression for the UAG codon depends on competition between an O-tRNA, including but not limited to, an amber suppressor tRNA, and a eukaryotic release factor (including but not limited to, an eRF), which binds to the stop codon and initiates release of the growing peptide from the ribosome, suppression efficiency can be modulated, by way of example, but not limitation, by increasing the expression levels of the O-tRNA and / or the suppressor tRNA.

[0240] Unnatural amino acids may be encoded by rare codons. For example, when the arginine concentration in an in vitro protein synthesis reaction is reduced, the rare arginine codon, AGG, has been found to be effective for the insertion of Ala using a synthetic tRNA acylated with alanine. See, e.g., Ma et al., Biochemistry, 32:7939 (1993). In this case, the synthetic tRNA competes with the naturally occurring tRNA Arg, which exists as a minor species in Escherichia coli. Some organisms do not use all triplet codons. The unassigned codon AGA in Micrococcus luteus has been utilized for the insertion of amino acids in in vitro transcription / translation extracts. See, e.g., Kowal and Oliver, Nucl. Acid. Res., 25:4685 (1997). Components of the present disclosure can be generated to use these rare codons in vivo.

[0241] Selector codons also include, but are not limited to, extended codons containing four or more bases, for example, four, five, six, or more bases. Examples of four-base codons include, but are not limited to, AGGA, CUAG, UAGA, and CCCU. Examples of five-base codons include, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU, and UAGGC. A feature of the present disclosure is the use of extended codons based on frameshift suppression. Four or more base codons can, for example, but are not limited to, insert one or more unnatural amino acids into the same protein. For example, in the presence of mutant O-tRNAs, including, but not limited to, special frameshift suppressor tRNAs with anticodon loops, e.g., with an anticodon loop of at least 8-10 nt, four or more base codons are read as a single amino acid. In other embodiments, the anticodon loop can decode a codon of, for example, but not limited to, at least a four-base codon, at least a five-base codon, or at least a six-base codon, or more bases. Because there are 256 possible four-base codons, four or more base codons can be used to encode multiple unnatural amino acids in the same cell. See Anderson et al., (2002) Exploring the Limits of Codon and Anticodon Size, Chemistry and Biology, 9:237-244; Magliery, (2001) Expanding the Genetic Code: Selection of Efficient Suppressors of Four-Base Codons and Identification of "Shifty" Four-Base Codons with a Library Approach in Escherichia coli, J. Mol. Biol. 307:755-769.

[0242] For example, four-base codons have been used to incorporate unnatural amino acids into proteins using in vitro biosynthetic methods. See, e.g., Ma et al., (1993) Biochemistry, 32:7939; and Hohsaka et al., (1999) J. Am. Chem. Soc., 121:34. Two frameshift suppressor tRNAs chemically acylated with CGGG and AGGU were used to simultaneously incorporate 2-naphthylalanine and an NBD derivative of lysine into streptavidin in vitro. See, e.g., Hohsaka et al., (1999) J. Am. Chem. Soc., 121:12194. Moore et al. investigated the ability of tRNALeu derivatives bearing an NCUA anticodon to suppress the UAGN codon (where N can be U, A, G, or C) in in vivo studies and found that the UAGA quadruplet could be decoded by tRNALeu bearing a UCUA anticodon with 13-26% efficiency, with little decoding in the 0 or -1 frame. See Moore et al., (2000) J. Mol. Biol., 298:195. In one embodiment, extended codons based on rare or nonsense codons may be used in the present disclosure, which may reduce missense readthrough and frameshift suppression at otherwise undesirable sites.

[0243] For a given system, the selector codon can comprise one of the natural three-base codons, where the endogenous system does not use (or rarely uses) the natural three-base codon. For example, this includes systems that lack a tRNA that recognizes the natural three-base codon and / or systems in which the three-base codon is a rare codon.

[0244] The selector codon optionally contains an unnatural base pair. These unnatural base pairs further expand the existing genetic alphabet. One extra base pair increases the number of triplet codons from 64 to 125. The properties of the third base pair include stable and selective base pairing, efficient enzymatic incorporation into DNA by polymerases with high fidelity, and efficient continued primer extension after synthesis of the nascent unnatural base pair. For descriptions of unnatural base pairs that can be adapted for methods and compositions, see, for example, Hirao, et al., (2002) An unnatural base pair for incorporating amino acid analogues into protein, Nature Biotechnology, 20:177-182. See also Wu, Y., et al., (2002) J. Am. Chem. Soc. 124:14626-14630. Other relevant publications are listed below.

[0245] For in vivo applications, unnatural nucleosides are membrane-permeable and can be phosphorylated to form the corresponding triphosphate. Furthermore, the augmented genetic information is stable and not destroyed by cellular enzymes. Previous efforts by Benner et al. utilized hydrogen-bonding patterns different from those of standard Watson-Crick pairs, the most notable example of which is the iso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc., 111:8322; and Piccirilli et al., (1990) Nature, 343:33; Kool, (2000) Curr. Opin. Chem. Biol., 4:602. These bases generally mispair to some degree with natural bases and cannot be enzymatically replicated. Kool and coworkers demonstrated that hydrophobic packing interactions between bases can replace hydrogen bonding to promote base pair formation. See Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and Guckian and Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort to develop an unnatural base pair that meets all of the above requirements, Schultz, Romesberg, and coworkers systematically synthesized and studied a series of unnatural hydrophobic bases. The PICS:PICS self-pair was found to be more stable than natural base pairs and can be efficiently incorporated into DNA by the Klenow fragment of Escherichia coli DNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem. Soc., 121:11585-6; and Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274. The 3MN:3MN self-pair can be synthesized by KF with sufficient efficiency and selectivity for biological function. See, for example, Ogawa et al., (2000) J. Am. Chem. Soc., 122:8803. However, both bases act as chain terminators for further replication. A mutant DNA polymerase that can be used to replicate the PICS self-pair has recently been evolved. In addition, the 7AI self-pair may be replicated.See, e.g., Tae et al., (2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py, has also been developed that forms a stable pair upon Cu(II) binding. See Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Because extended and unnatural codons are inherently orthogonal to natural codons, the disclosed methods can take advantage of this property to generate orthogonal tRNAs for them.

[0246] Unnatural amino acids can also be incorporated into a desired polypeptide using translational bypassing systems, in which a large sequence is incorporated into a gene but is not translated into protein. This sequence contains a structure that serves as a cue to guide the ribosome to move past the sequence and resume translation downstream of the insertion.

[0247] Nucleic acid molecules encoding proteins of interest, such as targeting polypeptides of ADCs, can be easily mutated to introduce cysteines at any desired position in the polypeptide. Cysteines are widely used to introduce reactive molecules, water-soluble polymers, proteins, or various other molecules into proteins of interest. Suitable methods for incorporating cysteines at desired positions in polypeptides, such as those described in U.S. Pat. No. 6,608,183, incorporated herein by reference, and standard mutagenesis techniques, are known to those skilled in the art.

[0248] Unnaturally Encoded Amino Acids

[0249] A wide variety of non-naturally encoded amino acids are suitable for use in the present disclosure. Any number of non-naturally encoded amino acids can be introduced into ADCs. Generally, the introduced non-naturally encoded amino acids are substantially chemically inert toward the 20 common genetically encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). In some embodiments, the non-naturally encoded amino acids include side chain functional groups that react efficiently and selectively with functional groups not found in the 20 common amino acids (including, but not limited to, azide groups, ketone groups, aldehyde groups, and aminooxy groups) to form stable conjugates. For example, a targeting polypeptide of an ADC containing a non-naturally encoded amino acid containing an azide functional group can be reacted with a polymer (including, but not limited to, poly(ethylene glycol) or alternatively, a second polypeptide or linker containing an alkyne moiety) to form a stable conjugate resulting from the selective reaction of the azide with the alkyne functional group to form a Huisgen [3+2] cycloaddition product.

[0250] The general structure of an alpha-amino acid is shown below (Formula I): [ka]

[0251] Non-naturally encoded amino acids are typically any structure having the formula above, in which the R group is any substituent other than those used in the 20 naturally occurring amino acids, and may be suitable for use in the present disclosure. The non-naturally encoded amino acids of the present disclosure typically differ from the naturally occurring amino acids only in the structure of their side chains, such that non-naturally encoded amino acids form amide bonds with other amino acids, including, but not limited to, natural or non-naturally encoded amino acids, in the same manner as they do in naturally occurring polypeptides. However, non-naturally encoded amino acids have side chain groups that distinguish them from natural amino acids. For example, R optionally includes alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether, thiol, seleno, sulfonyl, borate, boronate, phospho, phosphono, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino group, etc., or any combination thereof. Other non-naturally occurring amino acids of interest that may be suitable for use in the present disclosure include, but are not limited to, amino acids comprising a photoactivatable crosslinker, spin-labeled amino acids, fluorescent amino acids, metal-binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or non-covalently interact with other molecules, photocaged amino acids and / or photoisomerizable amino acids, amino acids comprising biotin or biotin analogs, glycosylated amino acids, e.g., sugar-substituted serines, other carbohydrate-modified amino acids, keto-containing amino acids, amino acids comprising polyethylene glycol or polyethers, heavy atom-substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids having elongated side chains relative to naturally occurring amino acids, including, but not limited to, polyethers or long-chain hydrocarbons, including, but not limited to, greater than about 5 carbons or greater than about 10 carbons, carbon-linked sugar-containing amino acids, redox-active amino acids, aminothioacid-containing amino acids, and amino acids comprising one or more toxic moieties.

[0252] Exemplary non-naturally encoded amino acids that may be suitable for use in the present disclosure and that are useful for reacting with water-soluble polymers include, but are not limited to, those having a carbonyl-reactive group, an aminooxy-reactive group, a hydrazine-reactive group, a hydrazide-reactive group, a semicarbazide-reactive group, an azide-reactive group, and an alkyne-reactive group. In some embodiments, the non-naturally encoded amino acid comprises a sugar moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine. Examples of such amino acids also include those in which the naturally occurring N- or O-bond between the amino acid and the sugar is replaced with a covalent bond not normally found in nature, including, but not limited to, an alkene, oxime, thioether, amide, etc. Examples of such amino acids also include sugars not normally found in naturally occurring proteins, such as 2-deoxyglucose and 2-deoxygalactose.

[0253] Many of the non-naturally encoded amino acids provided herein are commercially available, for example, from Sigma-Aldrich (St. Louis, MO, USA), Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), or Peptech (Burlington, MA, USA). Amino acids that are not commercially available are optionally synthesized as described herein or using standard methods known to those of skill in the art. For organic synthesis techniques, see, for example, Organic Chemistry by Fessendon and Fessendon, (1982, Second Edition, Willard Grant Press, Boston Mass.); Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistry by Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York). See also U.S. Patent Nos. 7,045,337 and 7,083,970, which are incorporated herein by reference. In addition to unnatural amino acids comprising novel side chains, unnatural amino acids that may be suitable for use in the present disclosure also optionally include modified backbone structures, such as, but not limited to, those shown by the structures of Formulas II and III. [ka] (wherein Z typically comprises OH, NH, SH, NH-R', or S-R'; X and Y, which may be the same or different, typically comprise S or O, with R and R', which are optionally the same or different, typically selected from the same list of R group components described above for unnatural amino acids having formula I and hydrogen.) For example, unnatural amino acids of the disclosure optionally comprise substitutions at the amino or carboxyl group, as shown by formulas II and III. Unnatural amino acids of this type include, but are not limited to, α-hydroxy acids, α-thioacids, α-aminothiocarboxylates, which have side chains corresponding to the 20 common natural amino acids or unnatural side chains. Additionally, substitutions at the α-carbon optionally include, but are not limited to, L, D, or α-α-disubstituted amino acids, such as D-glutamic acid, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Other structural alternatives include cyclic amino acids, such as proline analogs and 3-, 4-, 6-, 7-, 8-, and 9-membered ring proline analogs, β and γ amino acids, such as substituted β-alanine and γ-aminobutyric acid.

[0254] gamma Many unnatural amino acids are based on natural amino acids, such as tyrosine, glutamine, phenylalanine, and are suitable for use in the present disclosure. Tyrosine analogs include, but are not limited to, para-substituted tyrosines, ortho-substituted tyrosines, and meta-substituted tyrosines, including, but not limited to, keto groups (including, but not limited to, acetyl groups), benzoyl groups, amino groups, hydrazine, hydroxylamine, thiol groups, carboxy groups, isopropyl groups, methyl groups, C6-C8 20These include straight-chain or branched hydrocarbons, saturated or unsaturated hydrocarbons, O-methyl groups, polyether groups, nitro groups, alkynyl groups, and the like. Additionally, multiple substituted aryl rings are also contemplated. Glutamine analogs that may be suitable for use in the present disclosure include, but are not limited to, α-hydroxy derivatives, γ-substituted derivatives, cyclic derivatives, and amide-substituted glutamine derivatives. Exemplary phenylalanine analogs that may be suitable for use in the present disclosure include, but are not limited to, para-substituted phenylalanines, ortho-substituted phenylalanines, and meta-substituted phenylalanines, where the substituents include, but are not limited to, hydroxy groups, methoxy groups, methyl groups, allyl groups, aldehyde, azido, iodo, bromo, keto groups (including, but not limited to, acetyl groups), benzoyl, alkynyl groups, and the like. Illustrative examples of unnatural amino acids that may be suitable for use in the present disclosure include, but are not limited to, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, and p-propargyloxy-phenylalanine. Examples of structures of various unnatural amino acids that may be suitable for use in the present disclosure are provided, for example, in WO2002 / 085923, entitled "In vivo incorporation of unnatural amino acids."For additional methionine analogs, see also Kiick et al., (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation, PNAS 99:19-24, incorporated herein by reference. International Application No. PCT / US06 / 47822, entitled "Compositions Containing, Methods Involving, and Uses of Non-natural Amino Acids and Polypeptides," incorporated herein by reference, describes reductive alkylation of aromatic amine moieties, including, but not limited to, p-amino-phenylalanine and reductive amination.

[0255] In another embodiment of the present disclosure, ADC polypeptides are covalently modified with one or more non-naturally encoded amino acids. Selective chemical reactions orthogonal to the diverse functionalities of biological systems are recognized as important tools in chemical biology. As relevant newcomers to the repertoire of synthetic chemistry, these bioorthogonal reactions have inspired new strategies for compound library synthesis, protein engineering, functional proteomics, and chemical remodeling of cell surfaces. Azides have secured a prominent role as unique chemical handles for bioconjugation. The Staudinger ligation has been used with phosphines to tag azido sugars that are metabolically introduced into cellular glycoconjugates. Although the Staudinger ligation can be performed in living animals without physiological injury, the Staudinger reaction is not obligatory. The necessary phosphines are prone to air oxidation, and their optimization for improved water solubility and increased reaction rate has proven synthetically challenging.

[0256] Azide groups possess an alternative mode of bioorthogonal reactivity: the [3 + 2] cycloaddition with alkynes described by Huisgen. In its traditional form, this reaction requires high temperatures (or pressures) for reasonable reaction rates, limiting its applicability in biological systems. Sharpless and coworkers overcame this obstacle by developing a copper(I)-catalyzed version, termed "click chemistry," that proceeds readily at physiological temperatures and in fully functionalized biological environments. This discovery enabled the selective modification of virus particles, nucleic acids, and proteins from complex tissue lysates. Unfortunately, the required copper catalyst is toxic to both bacterial and mammalian cells, thus precluding applications where cell viability must be maintained. The uncatalyzed Huisgen cycloaddition of alkynes activated by electron-withdrawing substituents has been reported to occur at ambient temperatures. However, these compounds undergo Michael reactions with biological nucleophiles.

[0257] In one embodiment, a composition of a targeting polypeptide for an ADC is provided that includes an unnatural amino acid (e.g., p-(propargyloxy)-phenylalanine). Various compositions are also provided that include q-(propargyloxy)-phenylalanine and, including, but not limited to, proteins and / or cells. In one aspect, a composition that includes the p-(propargyloxy)-phenylalanine unnatural amino acid further includes an orthogonal tRNA. The unnatural amino acid can be attached (including, but not limited to, covalently) to the orthogonal tRNA, including, but not limited to, covalently attached to the orthogonal tRNA via an amino-acyl bond, covalently attached to the 3' OH or 2' OH of the terminal ribose sugar of the orthogonal tRNA, etc.

[0258] Chemical moieties that can be incorporated into proteins via unnatural amino acids offer a variety of advantages and manipulations of proteins. For example, the unique reactivity of the keto functional group allows for the selective modification of proteins in vitro and in vivo with either multiple hydrazine- or hydroxylamine-containing reagents. Heavy atom unnatural amino acids can be useful, for example, for phasing X-ray structural data. Site-specific introduction of heavy atoms using unnatural amino acids also provides selectivity and flexibility in selecting the location of the heavy atom. Photoreactive unnatural amino acids (including, but not limited to, amino acids with benzophenone and aryl azide (including, but not limited to, phenyl azide) side chains) enable, for example, efficient photocrosslinking of proteins in vivo and in vitro. Examples of photoreactive unnatural amino acids include, but are not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine. Proteins with photoreactive unnatural amino acids can optionally be crosslinked by time-controlled excitation of the provided photoreactive groups. In one example, the methyl group of the unnatural amino can be replaced with an isotopic label, including but not limited to, the methyl group, as a probe of local structure and dynamics, including but not limited to, using nuclear magnetic resonance and vibrational spectroscopy. Alkynyl or azido functional groups allow for the selective modification of proteins with molecules, for example, via [3+2] cycloaddition reactions.

[0259] Unnatural amino acids incorporated into polypeptides at the amino terminus may be composed of an R group that is any substituent other than those used in the 20 natural amino acids, and a second reactive group that is different from the NH group normally present in alpha-amino acids. Similar unnatural amino acids may be incorporated at the C-terminus with a second reactive group that is different from the COOH group normally present in alpha-amino acids.

[0260] The unnatural amino acids of the disclosure can be selected or designed to provide additional properties not available with the 20 naturally occurring amino acids. For example, unnatural amino acids can optionally be designed or selected to, e.g., modify the biological properties of the protein into which they are incorporated. For example, the following properties can be optionally modified by including an unnatural amino acid in a protein: toxicity, biodistribution, solubility, stability (e.g., resistance to heat, hydrolysis, oxidation, enzymatic degradation, etc.), ease of purification and processing, structural properties, spectroscopic properties, chemical and / or photochemical properties, catalytic activity, redox potential, half-life, ability to react (e.g., covalently or non-covalently) with other molecules, etc.

[0261] In some embodiments, the present disclosure provides ADCs conjugated to water-soluble polymers, e.g., PEG, via an oxime bond. Many types of non-naturally encoded amino acids are suitable for oxime bond formation. These include, but are not limited to, non-naturally encoded amino acids containing a carbonyl, dicarbonyl, or hydroxylamine group. Such amino acids are described in U.S. Patent Publication Nos. 2006 / 0194256, 2006 / 0217532, and 2006 / 0217289, and WO 2006 / 069246 (entitled "Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides"), which are incorporated herein by reference in their entireties. Non-naturally encoded amino acids are also described in U.S. Patent Nos. 7,083,970 and 7,045,337, which are incorporated herein by reference in their entireties.

[0262] Some embodiments of the present disclosure utilize ADC polypeptides substituted at one or more positions with a para-acetylphenylalanine amino acid. The synthesis of p-acetyl-(+ / -)-phenylalanine and m-acetyl-(+ / -)-phenylalanine is described in Zhang, Z., et al., Biochemistry 42:6735-6746 (2003), which is incorporated by reference. Other carbonyl- or dicarbonyl-containing amino acids can be similarly prepared by one of skill in the art. Additionally, non-limiting exemplary syntheses of unnatural amino acids included herein are provided in U.S. Pat. No. 7,083,970, which is incorporated by reference in its entirety.

[0263] Amino acids having electrophilic reactive groups allow for a variety of reactions, particularly for linking molecules via nucleophilic addition reactions. Such electrophilic reactive groups include carbonyl groups (including keto and dicarbonyl groups), carbonyl-like groups (which have reactivity similar to and are structurally similar to carbonyl groups (including keto and dicarbonyl groups)), masked carbonyl groups (which can be easily converted to carbonyl groups (including keto and dicarbonyl groups)), or protected carbonyl groups (which have reactivity similar to carbonyl groups (including keto and dicarbonyl groups) upon deprotection). Such amino acids include amino acids having the structure of formula (IV): [ka] (In the formula: A is optionally, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, if present, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (wherein k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; J is [ka] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; each R" is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R" group is present, two R" optionally form a heterocycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or heterocycloalkyl; or -ABJR groups taken together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl containing at least one carbonyl group (including a dicarbonyl group), protected carbonyl group (including a protected dicarbonyl group), or masked carbonyl group (including a masked dicarbonyl group); or the -JR groups taken together form a monocyclic or bicyclic cycloalkyl or heterocycloalkyl containing at least one carbonyl group (including a dicarbonyl group), protected carbonyl group (including a protected dicarbonyl group), or masked carbonyl group (including a masked dicarbonyl group); provided that when A is phenylene and each R is H, then B is present; when A is -(CH)- and each R is H, then B is not -NHC(O)(CHCH)-; and when A and B are absent and each R is H, then R is not methyl).

[0264] Also included are those having the structure of formula (V): [ka] (In the formula, A is optionally, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, if present, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (wherein k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; provided that when A is phenylene, then B is present; when A is -(CH2)4-, then B is not -NHC(O)(CH2CH2)-; and when A and B are not present, then R is not methyl).

[0265] Additionally included are amino acids having the structure of formula (VI): [ka] (In the formula: B is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (wherein k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each Ra is independently H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) k R', where each R' is independently selected from the group consisting of H, alkyl, or substituted alkyl.

[0266] Additionally, it contains the following amino acids: [ka] wherein such compounds are optionally amino-protecting groups, carboxyl-protecting groups or salts thereof. Additionally, any of the following unnatural amino acids may be incorporated into the unnatural amino acid polypeptide:

[0267] Additionally included are the following amino acids having the structure of formula (VII): [ka] (In the formula, B is optional and, if present, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (wherein k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) kN(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each R a are independently H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) k R' (each R' is independently H, alkyl, or substituted alkyl); and n is 0 to 8; However, when A is -(CH2)4-, B is not -NHC(O)(CH2CH2)-.

[0268] Additionally, it contains the following amino acids: [ka] wherein such compounds are optionally amino-protected, optionally carboxyl-protected, optionally amino-protected and carboxyl-protected, or salts thereof. Additionally, these unnatural amino acids, as well as any of the following unnatural amino acids, may be incorporated into unnatural amino acid polypeptides.

[0269] Additionally included are the following amino acids having the structure of formula (VIII): [ka] wherein A is optional and, if present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, if present, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (wherein k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, if present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide).

[0270] Additionally included are the following amino acids having the structure of formula (IX): [ka] (B is optional and, if present, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (wherein k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; In the formula, each R a are independently H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) k R', where each R' is independently selected from the group consisting of H, alkyl, or substituted alkyl.

[0271] Additionally, it contains the following amino acids: [ka] Such compounds are optionally amino-protected, optionally carboxyl-protected, optionally amino-protected and carboxyl-protected, or salts thereof. Additionally, these unnatural amino acids, as well as any of the following unnatural amino acids, can be incorporated into unnatural amino acid polypeptides.

[0272] Additionally included are the following amino acids having the structure of formula (X): [ka] (wherein B is optional and, if present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (wherein k is 1, 2, or 3), -S(O) k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each R a are independently H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) k R' (each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8).

[0273] Additionally, it contains the following amino acids: [ka] Such compounds are optionally amino-protected, optionally carboxyl-protected, optionally amino-protected and carboxyl-protected, or salts thereof. Additionally, these unnatural amino acids, as well as any of the following unnatural amino acids, can be incorporated into unnatural amino acid polypeptides.

[0274] In addition to monocarbonyl structures, the unnatural amino acids described herein can contain groups such as dicarbonyl groups, dicarbonyl-like groups, masked dicarbonyl groups, and protected dicarbonyl groups.

[0275] For example, the following amino acids having the structure of formula (XI) are included: [ka] wherein A is optional and, if present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, if present, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (wherein k is 1, 2, or 3), -S(O) k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, if present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide).

[0276] Additionally included are the following amino acids having the structure of formula (XII): [ka] (B is optional and, if present, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (wherein k is 1, 2, or 3), -S(O) k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; In the formula, each R a are independently H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) k R', where each R' is independently selected from the group consisting of H, alkyl, or substituted alkyl.

[0277] Additionally, it contains the following amino acids: [ka] Such compounds are optionally amino-protected, optionally carboxyl-protected, optionally amino-protected and carboxyl-protected, or salts thereof. Additionally, these unnatural amino acids, as well as any of the following unnatural amino acids, can be incorporated into unnatural amino acid polypeptides.

[0278] Additionally included are the following amino acids having the structure of formula (XIII): [ka] (wherein B is optional and, if present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (wherein k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each R a are independently H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) k R' (each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8).

[0279] Additionally, it contains the following amino acids: [ka] wherein such compounds are optionally amino-protected, optionally carboxyl-protected, optionally amino-protected and carboxyl-protected, or salts thereof. Additionally, these unnatural amino acids, as well as any of the following unnatural amino acids, may be incorporated into unnatural amino acid polypeptides.

[0280] Additionally included are the following amino acids having the structure of formula (XIV): [ka] (In the formula, A is optionally, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; X1 is C, S, or S(O); and L is alkylene, substituted alkylene, N(R')(alkylene), or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0281] Additionally included are the following amino acids having the structure of formula (XIV-A): [ka] (In the formula: A is optionally, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; L is alkylene, substituted alkylene, N(R')(alkylene) or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0282] Additionally included are the following amino acids having the structure of formula (XIV-B): [ka] (In the formula: A is optionally, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; L is alkylene, substituted alkylene, N(R')(alkylene) or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.

[0283] Additionally included are the following amino acids having the structure of formula (XV): [ka] (In the formula: A is optionally, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; X1 is C, S, or S(O); n is 0, 1, 2, 3, 4, or 5, and each CR 8 R 9 Each R on the group 8 and R 9 are independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 and R 9 are taken together to form ═O or cycloalkyl, or adjacent R 8 Any of the groups may be taken together to form a cycloalkyl.

[0284] Additionally included are the following amino acids having the structure of formula (XV-A): [ka] (In the formula: A is optionally, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; n is 0, 1, 2, 3, 4, or 5; and each CR 8 R 9 Each R on the group 8 and R 9 are independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 and R 9 are taken together to form ═O or cycloalkyl, or adjacent R 8 Any of the groups may be taken together to form a cycloalkyl.

[0285] Additionally included are the following amino acids having the structure of formula (XV-B): [ka] (In the formula: A is optionally, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; n is 0, 1, 2, 3, 4, or 5; and each CR 8 R 9 Each R on the group 8 and R 9 are independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 and R 9 are taken together to form ═O or cycloalkyl, or adjacent R 8 Any of the groups may be taken together to form a cycloalkyl.

[0286] Additionally included are the following amino acids having the structure of formula (XVI): [ka] (In the formula: A is optionally, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; X1 is C, S, or S(O); and L is alkylene, substituted alkylene, N(R')(alkylene), or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0287] Additionally included are the following amino acids having the structure of formula (XVI-A): [ka] (In the formula: A is optionally, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; L is alkylene, substituted alkylene, N(R')(alkylene) or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0288] Additionally included are the following amino acids having the structure of formula (XVI-B): [ka] (In the formula: A is optionally, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; L is alkylene, substituted alkylene, N(R')(alkylene) or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.

[0289] Additionally included are amino acids having the structure of formula (XVII): [ka] (In the formula: A is optionally, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; M is [ka] wherein (a) indicates a bond to the A group, and (b) indicates that the bonds to each carbonyl group R3 and R4 are independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, or R3 and R4 or two R3 groups or two R4 groups optionally form a cycloalkyl or heterocycloalkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; T3 represents a bond, C(R)(R), O, or S, where R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, if present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide).

[0290] Additionally included are amino acids having the structure of formula (XVIII): [ka] (In the formula: M is [ka] wherein (a) indicates a bond to the A group, and (b) indicates that the bonds to each carbonyl group R3 and R4 are independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, or R3 and R4 or two R3 groups or two R4 groups optionally form a cycloalkyl or heterocycloalkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; T3 represents a bond, C(R)(R), O, or S, where R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each R a are independently H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) k R', where each R' is independently selected from the group consisting of H, alkyl, or substituted alkyl.

[0291] Additionally included are amino acids having the structure of formula (XIX): [ka] (In the formula: R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; and T3 is O or S).

[0292] Additionally included are amino acids having the structure of formula (XX): [ka] (In the formula: R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0293] Additionally included are the following amino acids having the structure of formula (XXI): [ka]

[0294] In some embodiments, polypeptides containing unnatural amino acids are chemically modified to generate reactive carbonyl or dicarbonyl functional groups. For example, aldehyde functional groups useful for conjugation reactions can be generated from functional groups bearing adjacent amino and hydroxyl groups. When the biologically active molecule is a polypeptide, for example, an N-terminal serine or threonine (which may be normally present or may be exposed by chemical or enzymatic digestion) can be used to generate aldehyde functional groups under mild oxidative cleavage conditions using periodate. See, e.g., Gaertner, et al., Bioconjug. Chem. 3:262-268 (1992); Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertner et al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known in the art are restricted to the N-terminal amino acid of a peptide or protein.

[0295] In the present disclosure, unnatural amino acids bearing adjacent hydroxyl and amino groups may be incorporated into polypeptides as "masked" aldehyde functional groups. For example, 5-hydroxylysine has a hydroxyl group adjacent to the epsilon amine. Reaction conditions for generating the aldehyde typically involve the addition of a molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other sites within the polypeptide. The pH of the oxidation reaction is typically about 7.0. A typical reaction involves adding about a 1.5 molar excess of sodium metaperiodate to a buffered solution of the polypeptide, followed by incubation in the dark for about 10 minutes. See, e.g., U.S. Patent No. 6,423,685.

[0296] Carbonyl or dicarbonyl functional groups can selectively react with hydroxylamine-containing reagents under mild conditions in aqueous solution to form the corresponding oxime bond that is stable under physiological conditions. See, for example, Jencks, WP, J. Am. Chem. Soc. 81, 475-481 (1959); Shao, J. and Tam, JP, J. Am. Chem. Soc. 117: 3893-3899 (1995). Furthermore, the inherent reactivity of carbonyl or dicarbonyl groups allows for selective modification in the presence of other amino acid side chains. See, e.g., Cornish, VW, et al., J. Am. Chem. Soc. 118:8150-8151 (1996); Geoghegan, KF & Stroh, JG, Bioconjug. Chem. 3:138-146 (1992); Mahal, LK, et al., Science 276:1125-1128 (1997).

[0297] A. Carbonyl-Reactive Groups Amino acids with carbonyl reactive groups allow for a variety of reactions to link molecules (including, but not limited to, PEG or other water-soluble molecules) via, among other things, nucleophilic addition or aldol condensation reactions.

[0298] Exemplary carbonyl-containing amino acids can be represented as follows: [ka] wherein n is 0-10, R1 is alkyl, aryl, substituted alkyl, or substituted aryl, R2 is H, alkyl, aryl, substituted alkyl, and substituted aryl; and R3 is H, an amino acid, a polypeptide, or an amino-terminus modification group, and R4 is H, an amino acid, a polypeptide, or a carboxy-terminus modification group. In some embodiments, n is 1, R1 is phenyl, and R2 is a simple alkyl (i.e., methyl, ethyl, or propyl), and the ketone moiety is positioned para to the alkyl side chain. In some embodiments, n is 1, R1 is phenyl, and R2 is a simple alkyl (i.e., methyl, ethyl, or propyl), and the ketone moiety is positioned meta to the alkyl side chain.

[0299] The synthesis of p-acetyl-(+ / -)-phenylalanine and m-acetyl-(+ / -)-phenylalanine is described in Zhang, Z., et al., Biochemistry 42:6735-6746 (2003), which is incorporated herein by reference. Other carbonyl-containing amino acids can be similarly prepared by one skilled in the art.

[0300] In some embodiments, polypeptides containing non-naturally encoded amino acids are chemically modified to generate reactive carbonyl functional groups. For example, aldehyde functional groups useful for conjugation reactions may be generated from functional groups bearing adjacent amino and hydroxyl groups. When the biologically active molecule is a polypeptide, for example, an N-terminal serine or threonine (which may be normally present or may be exposed by chemical or enzymatic digestion) may be used to generate aldehyde functional groups under mild oxidative cleavage conditions using periodate. See, e.g., Gaertner, et al., Bioconjug. Chem. 3:262-268 (1992); Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertner et al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known in the art are limited to the N-terminal amino acid of a peptide or protein.

[0301] In the present disclosure, non-naturally encoded amino acids bearing adjacent hydroxyl and amino groups may be incorporated into polypeptides as "masked" aldehyde functionalities. For example, 5-hydroxylysine has a hydroxyl group adjacent to the epsilon amine. Reaction conditions for generating the aldehyde typically involve the addition of a molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other sites within the polypeptide. The pH of the oxidation reaction is typically about 7.0. A typical reaction involves adding about a 1.5 molar excess of sodium metaperiodate to a buffered solution of the polypeptide, followed by incubation in the dark for about 10 minutes. See, e.g., U.S. Patent No. 6,423,685, incorporated herein by reference.

[0302] The carbonyl functional group can be selectively reacted with hydrazine-, hydrazide-, hydroxylamine-, or semicarbazide-containing reagents under mild conditions in aqueous solution to form the corresponding hydrazone, oxime, or semicarbazone bond, respectively, which is stable under physiological conditions. See, for example, Jencks, WP, J. Am. Chem. Soc. 81, 475-481 (1959); Shao, J. and Tam, JP, J. Am. Chem. Soc. 117: 3893-3899 (1995). Furthermore, the inherent reactivity of the carbonyl group allows for selective modification in the presence of other amino acid side chains. See, e.g., Cornish, VW, et al., J. Am. Chem. Soc. 118:8150-8151 (1996); Geoghegan, KF & Stroh, JG, Bioconjug. Chem. 3:138-146 (1992); Mahal, LK, et al., Science 276:1125-1128 (1997).

[0303] B. Hydrazine, hydrazide, or semicarbazide reactive groups Non-naturally encoded amino acids containing a nucleophilic group, such as hydrazine, hydrazide, or semicarbazide, are capable of reacting with a variety of electrophilic groups to form conjugates, including, but not limited to, conjugates with PEG or other water-soluble polymers.

[0304] Exemplary hydrazine, hydrazide, or semicarbazide-containing amino acids can be represented as follows: [ka] where n is 0-10; R1 is alkyl, aryl, substituted alkyl, or substituted aryl, or is absent; X is O, N, or S, or is absent; R2 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.

[0305] In some embodiments, n is 4, R1 is not present, and X is N. In some embodiments, n is 2, R1 is not present, and X is not present. In some embodiments, n is 1, R1 is phenyl, X is O, and the oxygen atom is positioned para to the aliphatic group on the aryl ring.

[0306] Hydrazide-, hydrazine-, and semicarbazide-containing amino acids are obtained from commercial sources. For example, L-glutamic acid-γ-hydrazide is available from Sigma Chemical (St. Louis, MO). Other amino acids that are not commercially available can be prepared by those skilled in the art. See, for example, U.S. Patent No. 6,281,211, incorporated herein by reference.

[0307] Polypeptides containing non-naturally encoded amino acids bearing hydrazide, hydrazine, or semicarbazide functional groups can be reacted efficiently and selectively with a variety of molecules containing aldehydes or other functional groups with similar chemical reactivity. See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995). The inherent reactivity of hydrazide, hydrazine, and semicarbazide functional groups makes these functional groups significantly more reactive toward aldehydes, ketones, and other electrophilic groups than the nucleophilic groups present in the 20 common amino acids (including, but not limited to, the hydroxyl groups of serine or threonine, or the amino groups of lysine and the N-terminus).

[0308] C. Aminoxy-containing amino acids Non-naturally encoded amino acids containing an aminooxy (also called hydroxylamine) group can react with various electrophilic groups to form conjugates, including, but not limited to, conjugates with PEG or other water-soluble polymers. Similar to hydrazines, hydrazides, and semicarbazides, the enhanced nucleophilicity of the aminooxy group allows it to react efficiently and selectively with a variety of molecules containing aldehydes or other functional groups with similar chemical reactivity. See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34:727-736 (2001). However, while reaction with a hydrazine group generates the corresponding hydrazone, oximes are generally generated by the reaction of an aminooxy group with a carbonyl-containing group, such as a ketone.

[0309] Exemplary amino acids containing an aminooxy group can be represented as follows: [ka] wherein n is 0-10; R1 is alkyl, aryl, substituted alkyl, or substituted aryl, or not present; X is O, N, S, or not present; m is 0-10; Y=C(O) or not present; R2 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group. In some embodiments, n is 1, R1 is phenyl, X is O, m is 1, and Y is present. In some embodiments, n is 2, R1 and X are not present, m is 0, and Y is not present.

[0310] Aminoxy-containing amino acids can be prepared from readily available amino acid precursors (homoserine, serine, and threonine). See, e.g., M. Carrasco and R. Brown, J. Org. Chem. 68:8853-8858 (2003). Certain aminooxy-containing amino acids, such as L-2-amino-4-(aminooxy)butyric acid, have been isolated from natural sources (Rosenthal, G., Life Sci. 60:1635-1641 (1997)). Other aminooxy-containing amino acids can be prepared by those skilled in the art.

[0311] D. Azide and Alkyne Reactive Groups The inherent reactivity of azide and alkyne functional groups makes them highly useful for the selective modification of polypeptides and other biological molecules. Organic azides, particularly aliphatic azides and alkynes, are generally stable to common reactive chemical conditions. In particular, both azide and alkyne functional groups are inert toward the side chains (i.e., R groups) of the 20 common amino acids found in naturally occurring polypeptides. However, when brought into close proximity, the "spring-loaded" nature of the azide and alkyne groups becomes apparent, and they react selectively and efficiently via a Huisgen [3 + 2] cycloaddition reaction to generate the corresponding triazoles. See, for example, Chin J., et al., Science 301:964-7(2003); Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193(2003); Chin, JW, et al., J. Am. Chem. Soc. 124:9026-9027(2002).

[0312] Because the Huisgen cycloaddition reaction involves a selective cycloaddition reaction rather than a nucleophilic substitution (see, e.g., Padwa, A., Comprehensive Organic Synthesis, Vol. 4, (ed. Trost, BM, 1991), pp. 1069-1109; Huisgen, R., in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), pp. 1-176), the incorporation of non-naturally encoded amino acids with azide- and alkyne-containing side chains allows the resulting polypeptide to be selectively modified at the non-naturally encoded amino acid position. Cycloaddition reactions involving azide- or alkyne-containing ADCs can be carried out under aqueous conditions at room temperature by the addition of Cu(II) (including, but not limited to, in the form of a catalytic amount of CuSO) in situ in the presence of a reducing agent to reduce Cu(II) to Cu(I). See, e.g., Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Tornoe, CW, et al., J. Org. Chem. 67:3057-3064 (2002); Rostovtsev, et al., Angew. Chem. Int. Ed. 41:2596-2599 (2002). Exemplary reducing agents include, but are not limited to, ascorbate, metallic copper, quinine, hydroquinone, vitamin K, glutathione, cysteine, Fe 2+ , Co 2+ , and the applied potential.

[0313] In some cases, where a Huisgen [3+2] cycloaddition reaction between an azide and an alkyne is desired, the ADC comprises a non-naturally encoded amino acid containing an alkyne moiety and the water-soluble polymer attached to the amino acid contains an azide moiety. Alternatively, the reverse reaction (i.e., with the azide moiety on the amino acid and the alkyne moiety present on the water-soluble polymer) can be performed.

[0314] The azide functional group can also be selectively reacted with water-soluble polymers containing aryl esters, optionally functionalized with aryl phosphine moieties, to form amide bonds. The aryl phosphine group reduces the azide in situ, and the resulting amine then reacts efficiently with the proximal ester bond to form the corresponding amide. See, for example, E. Saxon and C. Bertozzi, Science 287, 2007-2010 (2000). The azide-containing amino acid can be either an alkyl azide (including, but not limited to, 2-amino-6-azido-1-hexanoic acid) or an aryl azide (p-azido-phenylalanine).

[0315] Exemplary water-soluble polymers containing aryl ester and phosphine moieties can be represented as follows: [ka] wherein X can be O, N, S, or absent; Ph is phenyl; W is a water-soluble polymer; and R can be H, alkyl, aryl, substituted alkyl, and substituted aryl groups. Exemplary R groups include, but are not limited to, —CH, —C(CH), —OR′, —NR′R″, —SR′, -halogen, —C(O)R′, —CONR′R″, —S(O)R′, —S(O)NR′R″, —CN, and —NO. R′, R″, R′″, and R′″ each independently represent hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, including, but not limited to, aryl substituted with 1 to 3 halogens, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compound of the present disclosure includes more than one R group, for example, each R group is independently selected, as are each of the R', R", R'", and R"" groups when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they may be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the discussion of the substituents above, those of skill in the art will understand that the term "alkyl" is meant to include groups containing carbon atoms bonded to groups other than hydrogen groups, for example, haloalkyl (including, but not limited to, -CF3 and -CH2CF3) and acyl (including, but not limited to, -C(O)CH3, -C(O)CF3, -C(O)CHOCH3, etc.).

[0316] The azide functional group can also be selectively reacted with a water-soluble polymer containing a thioester, optionally functionalized with an aryl phosphine moiety, to form an amide bond. The aryl phosphine group reduces the azide in situ, and the resulting amine then reacts efficiently with the thioester bond to form the corresponding amide. Exemplary water-soluble polymers containing thioester and phosphine moieties can be represented as follows: [ka] where n is 1-10; X is O, N, S, or absent; Ph is phenyl; and W is a water-soluble polymer.

[0317] Exemplary alkyne-containing amino acids can be represented as follows: [ka] wherein n is 0-10; R1 is alkyl, aryl, substituted alkyl, or substituted aryl or not present; X is O, N, S, or not present; m is 0-10, R2 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group. In some embodiments, n is 1, R1 is phenyl, X is not present, m is 0, and the acetylene moiety is positioned in the para position relative to the alkyl side chain. In some embodiments, n is 1, R1 is phenyl, X is O, m is 1, and the propargyloxy group is positioned in the para position relative to the alkyl side chain (i.e., O-propargyl-tyrosine). In some embodiments, n is 1, R1 and X are not present, and m is 0 (i.e., propargylglycine).

[0318] Alkyne-containing amino acids are commercially available. For example, propargylglycine is commercially available from Peptech (Burlington, MA). Alternatively, alkyne-containing amino acids can be prepared according to standard methods. For example, p-propargyloxyphenylalanine can be synthesized as described in, for example, Deiters, A., et al., J. Am. Chem. Soc. 125:11782-11783 (2003), and 4-alkynyl-L-phenylalanine can be synthesized as described in, for example, Kayser, B., et al., Tetrahedron 53(7):2475-2484 (1997). Other alkyne-containing amino acids can be prepared by those skilled in the art.

[0319] Exemplary azide-containing amino acids can be represented as follows: [ka] wherein n is 0-10; R1 is alkyl, aryl, substituted alkyl, substituted aryl, or not present; X is O, N, S, or not present; m is 0-10; R2 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group. In some embodiments, n is 1, R1 is phenyl, X is not present, m is 0, and the azide moiety is positioned para to the alkyl side chain. In some embodiments, n is 0-4, R1 and X are not present, and m=0. In some embodiments, n is 1, R1 is phenyl, X is O, m is 2, and the azidoethoxy moiety is positioned para to the alkyl side chain.

[0320] Azide-containing amino acids are available from commercial sources. For example, 4-azidophenylalanine can be obtained from Chem-Impex International, Inc. (Wood Dale, IL). For azide-containing amino acids that are not commercially available, the azide group can be relatively easily prepared using standard methods known to those skilled in the art, for example, but not limited to, by displacement of an appropriate leaving group (including, but not limited to, halide, mesylate, tosylate) or by cleavage of a suitably protected lactone. See, for example, Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York).

[0321] E. Aminothiol-Reactive Groups The unique reactivity of beta-substituted aminothiol functional groups makes them highly useful for the selective modification of polypeptides and other biological molecules containing aldehyde groups via thiazolidine formation. See, e.g., J. Shao and J. Tam, J. Am. Chem. Soc. 1995, 117(14) 3893-3899. In some embodiments, beta-substituted aminothiol amino acids can be incorporated into ADC polypeptides and then reacted with water-soluble polymers containing aldehyde functional groups. In some embodiments, water-soluble polymers, drug conjugates, or other payloads can be coupled to the targeting polypeptide of an ADC containing a beta-substituted aminothiol amino acid via thiazolidine formation.

[0322] F. Additional Reactive Groups

[0323] Additional reactive groups and non-naturally encoded amino acids (including, but not limited to, for example, para-amino-phenylalanine) that can be incorporated into the ADC polypeptides of the disclosure are described in the following patent applications, all of which are incorporated by reference in their entirety: U.S. Patent Publication No. 2006 / 0194256, U.S. Patent Publication No. 2006 / 0217532, U.S. Patent Publication No. 2006 / 0217289, U.S. Provisional Patent No. Nos. 60 / 755,338; U.S. Provisional Patent No. 60 / 755,711; U.S. Provisional Patent No. 60 / 755,018; International Patent Application No. PCT / US06 / 49397; WO2006 / 069246; U.S. Provisional Patent No. 60 / 743,041; U.S. Provisional Patent No. 60 / 743,040; International Patent Application No. PCT / US06 / 47822; U.S. Provisional Patent No. 60 / 882,819; U.S. Provisional Patent No. 60 / 882,500; and U.S. Provisional Patent No. 60 / 870,594. These applications also discuss reactive groups that may be present on PEG or other polymers, including, but not limited to, hydroxylamine (aminooxy) groups for conjugation.

[0324] Position of unnatural amino acids in ADC polypeptides The methods and compositions described herein include incorporating one or more unnatural amino acids into a targeting polypeptide to generate an ADC of the disclosure. The one or more unnatural amino acids may be incorporated at one or more specific positions that do not interfere with the activity of the targeting polypeptide. This may be achieved by making "conservative" substitutions, including, but not limited to, substituting a hydrophobic amino acid for a non-natural or natural hydrophobic amino acid, a bulky amino acid for a non-natural or natural bulky amino acid, or a hydrophilic amino acid for a non-natural or natural hydrophilic amino acid, and / or by inserting the unnatural amino acid into a position not required for activity.

[0325] A variety of biochemical and structural approaches can be used to select a desired site for substitution with an unnatural amino acid within a targeting polypeptide of an ADC. In some embodiments, the unnatural amino acid is linked at the C-terminus of the cytotoxic tubulin inhibitor derivative. In other embodiments, the unnatural amino acid is linked at the N-terminus of the cytotoxic tubulin inhibitor derivative. Any position of the targeting polypeptide of an ADC is suitable for selection for incorporating an unnatural amino acid, and selection can be based on rational design for any or specific desired purpose, or by random selection. Selection of a desired site can be based on generating a unnatural amino acid polypeptide (which can be further modified or remain unmodified) with any desired property or activity, including, but not limited to, receptor binding modulator, receptor activity modulator, modulator of binding to a binder partner, modulator of binding partner activity, modulator of binding partner conformation, formation of dimers or multimers, no change in activity or property compared to the native molecule, or manipulation of any physical or chemical property of the polypeptide, such as solubility, aggregation, or stability. Alternatively, sites identified as important for biological activity may also be good candidates for substitution with an unnatural amino acid, depending on the desired activity sought for the polypeptide. Another option is to simply make serial substitutions at each position along the polypeptide chain with an unnatural amino acid and observe the effect on the activity of the polypeptide. Any means, technique, or method for selecting positions in a given polypeptide to be substituted with an unnatural amino acid is suitable for use in the methods, techniques, and compositions described herein.

[0326] The structure and activity of naturally occurring variants of the polypeptide, including deletions, may also be examined to determine regions of the protein that are likely to tolerate substitution with unnatural amino acids. Once residues that are likely to be intolerant of substitution with unnatural amino acids have been eliminated, the impact of proposed substitutions at each remaining position can be tested using methods including, but not limited to, the three-dimensional structure of the relevant polypeptide and any relevant ligands or binding proteins. X-ray crystallographic and NMR structures of many polypeptides are available in the Protein Data Bank (PDB, World Wide Web, rcsb.org), a centralized database containing three-dimensional structural data for large protein and nucleic acid molecules, which can be used to identify amino acid positions that can be substituted with unnatural amino acids. In addition, if three-dimensional structural data is not available, models can be created that examine the secondary and tertiary structure of the polypeptide. This can readily provide the identity of amino acid positions that can be substituted with unnatural amino acids.

[0327] Exemplary sites for incorporation of unnatural amino acids include, but are not limited to, those excluded from potential receptor binding regions or regions for binding to binding proteins or ligands, which may be fully or partially solvent exposed, have minimal or no hydrogen bonding interactions with nearby residues, have minimal exposure to nearby reactive residues, and / or are in regions of high flexibility predicted in the three-dimensional crystal structure of a particular polypeptide with its associated receptor, ligand, or binding protein.

[0328] A wide variety of unnatural amino acids may be substituted for or incorporated into a given position within a polypeptide. By way of example, a particular unnatural amino acid may be selected for incorporation based on examination of the three-dimensional crystal structure of the polypeptide and its associated ligand, receptor, and / or binding protein, and the desirability of a conservative substitution.

[0329] In one embodiment, a method described herein comprises incorporating an unnatural amino acid into a targeting polypeptide of an ADC, where the targeting polypeptide of the ADC comprises a first reactive group, and contacting the targeting polypeptide of the ADC with a molecule (including, but not limited to, a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; and any combination thereof) comprising a second reactive group. In certain embodiments, the first reactive group is a hydroxylamine moiety and the second reactive group is a carbonyl or dicarbonyl moiety, thereby forming an oxime bond. In certain embodiments, the first reactive group is a carbonyl or dicarbonyl moiety and the second reactive group is a hydroxylamine moiety, thereby forming an oxime bond. In certain embodiments, the first reactive group is a carbonyl or dicarbonyl moiety and the second reactive group is an oxime moiety, thereby forming an oxime exchange reaction. In certain embodiments, the first reactive group is an oxime moiety and the second reactive group is a carbonyl or dicarbonyl moiety, thereby resulting in an oxime exchange reaction.

[0330] In some cases, the targeting polypeptide for ADC incorporation(s) of unnatural amino acids is combined with other additions, substitutions, or deletions within the polypeptide to affect other chemical, physical, pharmacological, and / or biological properties. In some cases, the other additions, substitutions, or deletions may increase the stability of the polypeptide (including, but not limited to, resistance to proteolytic degradation) or increase the affinity of the polypeptide for its appropriate receptor, ligand, and / or binding protein. In some cases, the other additions, substitutions, or deletions may increase the solubility of the polypeptide (including, but not limited to, when expressed in E. coli or other host cells). In some embodiments, a site for substitution with a naturally encoded or unnatural amino acid is selected in addition to another site for introduction of the unnatural amino acid in order to increase the solubility of the polypeptide after expression in E. coli or other recombinant host cells. In some embodiments, the polypeptide contains other additions, substitutions, or deletions that modulate affinity for associated ligands, binding proteins, and / or receptors, modulate (including, but not limited to, increasing or decreasing) receptor dimerization, stabilize receptor dimers, modulate circulating half-life, modulate release or bioavailability, facilitate purification, or improve or alter a particular route of administration. Similarly, non-natural amino acid polypeptides may contain chemical or enzymatic cleavage sequences, protease cleavage sequences, reactive groups, antibody binding domains (including, but not limited to, FLAG or poly-His), or other affinity-based sequences (including, but not limited to, FLAG, poly-His, GST, etc.) or linking molecules (including, but not limited to, biotin) that improve detection (including, but not limited to, GFP), purification, transport through tissue or cell membranes, prodrug release or activation, size reduction, or other traits of the polypeptide.

[0331] Anti-HER2 antibodies as examples for targeting moieties The methods, compositions, strategies, and techniques described herein are not limited to a particular type, class, or family of targeting moiety polypeptide or protein. Indeed, virtually any targeting moiety polypeptide can be designed or modified to include at least one "modified or unmodified" unnatural amino acid, including the targeting polypeptides of the ADCs described herein. By way of example only, targeting moieties can be selected from a wide variety of targeting moieties, including alpha-1 antitrypsin, angiostatin, antihemolytic factors, antibodies, antibody fragments, monoclonal antibodies (e.g., bevacizumab, cetuximab, panitumumab, infliximab, adalimumab, basiliximab, daclizumab, omalizumab, ustekinumab, ethanercept, gemtuzumab, alemtuzumab, rituximab, trastuzumab, nimotuzumab, palivizumab, and abciximab), apolipoproteins, and the like. Lipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, CXC chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit ligand, CC chemokine, monocyte chemoattractant protein 1, monocyte chemoattractant protein 2, monocyte chemoattractant protein 3, monocyte inflammatory protein- 1 alpha, monocyte inflammatory protein-i beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, collagen, colony-stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil-activating peptide-78, MIP-16, MCP-1, epidermal growth factor (EGF), epidermal neutrophil-activating peptide, erythropoietin (EPO), exfoliation Toxins, Factor IX, Factor VII, Factor VIII, Factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helix bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropins, growth factors, growth factor receptors, growth hormone-releasing factors, hedgehog proteins, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1,ICAM-1 receptor, LFA-1, LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, Luciferase, neurturin, neutrophil inhibitory factor (NIF), oncostatin M, bone morphogenetic protein, oncogene product, paracitonin, parathyroid hormone (PTH), PD-ECGF, PDGF, peptide hormone, pleiotrophin, protein A, protein G, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, peptide YY (PYY), relaxin, renin, SCF, small biosynthetic protein, soluble complement receptor I, soluble The antibody may be homologous to a therapeutic protein selected from the group consisting of soluble I-CAM1, soluble interleukin receptor, soluble TNF receptor, somatomedin, somatostatin, somatotropin, streptokinase, superantigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen activator, transforming growth factor (TGF), tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, tumor necrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone.

[0332] In one embodiment, there is a method of treating a solid tumor that overexpresses HER-2, has a HER-2 mutation, or has HER-2 gene amplification, selected from the group consisting of breast cancer, small cell lung cancer, ovarian cancer, endometrial cancer, bladder cancer, head and neck cancer, prostate cancer, gastric cancer, cervical cancer, uterine cancer, esophageal cancer, and colorectal cancer. In another embodiment, the solid tumor is breast cancer. In a further embodiment, the solid tumor is ovarian cancer. In a further embodiment, the solid tumor is gastric cancer.

[0333] Therefore, the following description of trastuzumab is provided merely as an example for illustrative purposes and is not intended to limit the scope of the methods, compositions, strategies, and techniques described herein. Furthermore, reference to trastuzumab in this application is intended to be a general term for any antibody. Thus, it is understood that the modifications and chemical entities described herein with respect to trastuzumab can be equally applied to any antibody or monoclonal antibody, including those specifically listed herein.

[0334] Trastuzumab is a humanized monoclonal antibody that binds to domain IV of the extracellular segment of the HER2 / neu receptor. The HER2 gene (also known as the HER2 / neu and ErbB2 genes) is amplified in 20-30% of early-stage breast cancers, leading to overexpression. In cancer, HER2 can transmit signals without mitogens reaching and binding to either receptor, leading to overactivity.

[0335] HER2 spans the cell membrane, transmitting signals from the outside to the inside of the cell. In healthy individuals, signaling compounds called mitogens reach the cell membrane and bind to the outer portions of other members of the HER family of receptors. These bound receptors then bind (dimerize) with and activate HER2. HER2 then transmits a signal to the interior of the cell. This signal passes through various biochemical pathways, including the PI3K / Akt pathway and the MAPK pathway. These signals promote cell invasion, survival, and growth of blood vessels (angiogenesis).

[0336] Trastuzumab-treated cells arrest in the G1 phase of the cell cycle, resulting in suppressed proliferation. It has been suggested that trastuzumab exerts its effects in part through downregulation of HER2 / neu, leading to disruption of receptor dimerization and downstream signaling through the PI3K cascade. P27Kip1 is then unphosphorylated and can enter the nucleus, inhibiting cdk2 activity and causing cell cycle arrest. Trastuzumab also inhibits angiogenesis by both inducing antiangiogenic factors and suppressing proangiogenic factors. It is thought that its contribution to the unregulated growth observed in cancer may be due to proteolytic cleavage of HER2 / neu, which results in release of the extracellular domain. Trastuzumab has been shown to inhibit HER2 / neu ectodomain cleavage in breast cancer cells.

[0337] Expression in non-eukaryotes and eukaryotes To obtain high-level expression of a cloned ADC polynucleotide, a polynucleotide encoding a targeting polypeptide of the disclosed ADC polypeptide is typically subcloned into an expression vector containing a strong promoter to direct transcription, a transcription / translation terminator, and, in the case of a protein-encoding nucleic acid, a ribosome binding site for translation initiation. Suitable bacterial promoters are known to those of skill in the art and are described, for example, in Sambrook et al. and Ausubel et al.

[0338] Bacterial expression systems for expressing the ADC polypeptides of the disclosure are available, including, but not limited to, E. coli, Bacillus sp., Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, and Salmonella (Palva et al., Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are known to those of skill in the art and are commercially available. When orthogonal tRNAs and aminoacyl-tRNA synthetases (described above) are used to express the ADC polypeptides of the disclosure, host cells for expression are selected based on their ability to use the orthogonal components. Exemplary host cells include Gram-positive bacteria (including, but not limited to, B. brevis, B. subtilis, or Streptomyces) and Gram-negative bacteria (E. coli, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida), as well as yeast and other eukaryotic cells. Cells containing an O-tRNA / O-RS pair can be used as described herein.

[0339] The eukaryotic or non-eukaryotic host cells of the disclosure provide the ability to synthesize proteins that include large, effective amounts of unnatural amino acids. In one aspect, the composition optionally includes, but is not limited to, at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10 milligrams, at least 100 milligrams, at least 1 gram, or more of protein (including the unnatural amino acid), or an amount achievable by in vivo protein production methods (details of recombinant protein production and purification are provided herein). In another embodiment, the protein is optionally present in a composition at a concentration of, but not limited to, at least 10 micrograms of protein per liter, at least 50 micrograms of protein per liter, at least 75 micrograms of protein per liter, at least 100 micrograms of protein per liter, at least 200 micrograms of protein per liter, at least 250 micrograms of protein per liter, at least 500 micrograms of protein per liter, at least 1 milligram of protein per liter, or at least 10 milligrams of protein per liter or more, including but not limited to, a cell lysate, buffer, pharmaceutical buffer, or other liquid suspension (including but not limited to, any volume from about 1 nL to about 100 L or more). Large-scale production of proteins in eukaryotic cells containing at least one unnatural amino acid (greater than is typically possible by other methods, including but not limited to, in vitro translation) is a feature of the present disclosure.

[0340] The nucleotide sequence encoding the targeting polypeptide of an ADC polypeptide may or may not include a sequence encoding a signal peptide. A signal peptide is present if the polypeptide is to be secreted from the cells in which it is expressed. Such a signal peptide may be any sequence. The signal peptide may be prokaryotic or eukaryotic. Coloma, M. (1992) J. Imm. Methods 152:89 104) describes a signal peptide (mouse Ig kappa light chain signal peptide) for use in mammalian cells. Other signal peptides include, but are not limited to, the alpha-factor signal peptide from S. cerevisiae (U.S. Pat. No. 4,870,008, incorporated herein by reference), the signal peptide of mouse salivary amylase (O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646), a modified carboxypeptidase signal peptide (LAValls et al., Cell 48, 1987, pp. 887-897), the yeast BAR1 signal peptide (WO 87 / 02670, incorporated herein by reference), and the yeast aspartic protease 3 (YAP3) signal peptide (see M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137).

[0341] Examples of suitable mammalian host cells are known to those of skill in the art. Such host cells may be Chinese hamster ovary (CHO) cells (e.g., CHO-K1; ATCC CCL-61), Savannah monkey cells (COS) (e.g., COS1 (ATCC CRL-1650), COS7 (ATCC CRL-1651)); mouse cells (e.g., NS / O), baby hamster kidney (BHK) cell lines (e.g., ATCC CRL-1632 or ATCC CCL-10), and human cells (e.g., HEK 293 (ATCC CRL-1573)), as well as plant cells in tissue culture. These cell lines and others are available from public depositories such as the American Type Culture Collection, Rockville, Md. To improve glycosylation of ADC polypeptides, mammalian host cells can be modified to express sialyltransferases, e.g., 1,6-sialyltransferases, e.g., as described, e.g., in U.S. Pat. No. 5,047,335, incorporated herein by reference.

[0342] Methods for introducing exogenous DNA into mammalian host cells include, but are not limited to, calcium phosphate-mediated transfection, electroporation, DEAE-dextran-mediated transfection, liposome-mediated transfection, viral vectors, and the transfection methods described by Life Technologies Ltd, Paisley, UK, using Lipofectamin 2000, and Roche Diagnostics Corporation, Indianapolis, USA, using FuGENE 6. These methods are well known in the art and are described by Ausbel et al. (eds.), 1996, Current Protocols in Molecular Biology, John Wiley & Sons, New York, USA. Mammalian cell culture can be carried out according to established methods, for example as disclosed in (Animal Cell Biotechnology, Methods and Protocols, edited by Nigel Jenkins, 1999, Human Press Inc. Totowa, NJ, USA and Harrison Mass. and Rae IF, General Techniques of Cell Culture, Cambridge University Press 1997).

[0343] E. coli, Pseudomonas, and other prokaryotes

[0344] Bacterial expression techniques are known to those skilled in the art. A wide variety of vectors are available for use in bacterial hosts. Vectors may be single copy or low or high multicopy vectors. Vectors may be useful for cloning and / or expression. In view of the extensive literature on vectors, the commercial availability of numerous vectors, and even manuals describing vectors and their constraint maps and properties, a detailed discussion is not necessary here. As is well known, vectors usually contain a marker that allows selection, which may provide resistance to cytotoxic agents, prototrophy, or immunity. Often, there are multiple markers that provide various properties.

[0345] A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating downstream (3') transcription of a coding sequence (e.g., a structural gene) into mRNA. A promoter has a transcription initiation region, usually located near the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. Bacterial promoters may also have a second domain called an operator, which may overlap with an adjacent RNA polymerase binding site at which RNA synthesis is initiated. Operators allow for negatively regulated (inducible) transcription, as gene repressor proteins can bind to the operator and inhibit transcription of a particular gene. Constitutive expression can occur in the absence of negative regulatory elements such as operators. Additionally, positive regulation can be achieved by gene activator protein binding sequences, which, if present, are usually proximal (5') to the RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli) (see Raibaud et al., ANNU. REV. GENET. (1984) 18:173). Thus, regulated expression can be either positive or negative, thereby promoting or reducing transcription.

[0346] The term "bacterial host" or "bacterial host cell" refers to bacteria that can be or have been used as a recipient of a recombinant vector or other transfer DNA. This term includes the progeny of the original transfected bacterial host cell. It is understood that the progeny of a single parent cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of a parent cell that is sufficiently similar to the parent to be characterized by relevant properties, such as the presence of a nucleotide sequence encoding an ADC polypeptide, are included in the progeny intended by this definition.

[0347] The selection of suitable host bacteria for expression of ADC polypeptides is well known to those of skill in the art. When selecting a bacterial host for expression, suitable hosts may include those shown to have, among other things, good inclusion body formation ability, low proteolytic activity, and overall robustness. Bacterial hosts are generally available from a variety of sources, including, but not limited to, the Bacterial Genetic Stock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA); and the American Type Culture Collection ("ATCC") (Manassas, VA). Industrial...

Claims

1. An antibody drug conjugate (ADC) comprising: A cytotoxic tubulin analog having the structure 【Chemistry 1】 And an anti-HER2 antibody or antibody fragment comprising SEQ ID NO:2 and SEQ ID NO:3; wherein 【Chemistry 2】 indicates a single or double bond and # indicates binding to said anti-HER2 antibody or antibody fragment.

2. 2. The ADC of claim 1, wherein the anti-HER2 antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains of the ADC.

3. An antibody drug conjugate (ADC) comprising: A cytotoxic tubulin analog having the structure 【Chemistry 3】 and an anti-HER2 antibody or antibody fragment comprising SEQ ID NO:2; wherein the anti-HER2 antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains, 【Chemistry 4】 indicates a single or double bond, where # indicates binding to said anti-HER2 antibody or antibody fragment.

4. The ADC of any one of claims 1 to 3, wherein the anti-HER2 antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain. 【Request 5】 【Chemistry 5】 represents a double bond, and said tubulin analog is covalently attached to said anti-HER2 antibody via a double bond; 4. The ADC of any one of claims 1 to 3, optionally wherein the double bond is between the tubulin analog and one or more members of a non-naturally encoded amino acid.

6. The ADC comprises two heavy chains, wherein each heavy chain comprises SEQ ID NO:2, and wherein each heavy chain is individually conjugated to a different tubulin analog having the structure: 【Chemistry 6】 The ADC of any one of claims 1 to 3, wherein # indicates binding to the anti-HER2 antibody or antibody fragment.

7. (a) the anti-HER2 antibody or antibody fragment is humanized; (b) the one or more unnatural amino acids is para-acetylphenylalanine; and / or (c) the ADC of any one of claims 1 to 3, wherein the light chain of the anti-HER2 antibody or antibody fragment is any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13.

8. A composition comprising the antibody-drug conjugate of any one of claims 1 to 3.

9. and optionally, said additional therapeutic agent is (a) an immunotherapeutic, chemotherapeutic, hormonal, anti-tumor, immunostimulatory, or immunomodulatory agent, or a combination thereof; (b) is a HER2 targeted therapeutic; or 9. The composition of claim 8, wherein (c) the therapeutic agent is a checkpoint inhibitor, a HER2 kinase inhibitor, a cyclin dependent kinase inhibitor, a tyrosine kinase inhibitor, a small molecule kinase inhibitor, or a platinum-based therapeutic agent, or a combination thereof.

10. Use of the ADC according to any one of claims 1 to 3 in a method for killing a cell, the method comprising contacting the cell with the ADC (except for use in humans).

11. The use according to claim 10, wherein the cell is a tumor or cancer cell.

12. A pharmaceutical composition comprising the ADC of any one of claims 1 to 3.

13. 13. The pharmaceutical composition of claim 12, further comprising a pharma- ceutically acceptable excipient.

14. Use of the ADC according to any one of claims 1 to 3 in the manufacture of a medicament in the following method: A method of treating a tumor or cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of an ADC of any one of claims 1 to 3.

15. (a) the effective amount is about 0.22, 0.33, 0.44, 0.55, 0.66, 0.77, 0.88, 0.99, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 milligrams per kilogram (mg / kg) of body weight of the human subject, and optionally about 0.33, 0.66, 0.88, 1.1, 1.3, 1.5, 1.6, 1.7, or 1.8 mg / kg of body weight of the human subject; (b) the administration occurs once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks; (c) the administration continues for two or more 1, 2, 3, 4, 5, 6, 7, or 8 week cycles; (d) said administration continues for at least 4 weeks; or said administration continues for at least 3 weeks; (e) the administration is administered two or more times within a 1, 2, 3, 4, 5, 6, 7, or 8 week cycle, and optionally (i) the administration occurs two or more times within a four week cycle; or (ii) the administration occurs two or more times within a three week cycle; (f) the administered dose is the same on different administration days, or the administered dose is different on different administration days; (g) the ADC is administered once every two weeks for more than eight weeks; or The ADC is administered once every 4 weeks for more than 8 weeks; and / or (h) The use of claim 14, wherein the tumor or cancer is breast cancer, ovarian cancer, gastric cancer, gastroesophageal junction adenocarcinoma, cervical cancer, uterine cancer, endometrial cancer, testicular cancer, prostate cancer, colon cancer, esophageal cancer, bladder cancer, non-small cell lung cancer (NSCLC), urothelial carcinoma, cholangiocarcinoma, colon biliary tract cancer, pancreatic cancer, or a solid tumor, optionally wherein the tumor or cancer is breast cancer, ovarian cancer, or gastric cancer.

16. (a) the administration comprises an ADC at a dose of about 1.8 mg / kg on day 1 of a first 4-week cycle, and optionally, the administration further comprises an ADC at a dose of about 1.7 mg / kg, 1.5 mg / kg, 1.3 mg / kg or less on day 1 of a second 4-week cycle; (b) said administering comprises ADC at a dose of about 1.7 mg / kg on day 1 of a first 4 week cycle, and optionally, said administering further comprises ADC at a dose of about 1.5 mg / kg, 1.3 mg / kg or less on day 1 of a second 4 week cycle; (c) said administering comprises ADC at a dose of about 1.6 mg / kg on day 1 of a first 4 week cycle, and optionally, said administering further comprises ADC at a dose of about 1.5 mg / kg, 1.3 mg / kg, 1.1 mg / kg or less on day 1 of a second 4 week cycle; or (d) The use of (c)-(e)(i) and (f)-(h) of claim 15, wherein the administration comprises ADC at a dose of about 1.5 mg / kg on day 1 of a first 4-week cycle, and optionally, the administration further comprises ADC at a dose of about 1.3 mg / kg, 1.1 mg / kg or less on day 1 of a second 4-week cycle.

17. The administration continues for two or more three-week cycles, and optionally (a) said administering comprises ADC at a dose of about 1.8 mg / kg on day 1 of a first 3-weekly cycle; (b) said administration comprises an ADC at a dose of about 1.7 mg / kg on day 1 of a first 3 week cycle; (c) said administering comprises ADC at a dose of about 1.5 mg / kg on day 1 of a first 3 week cycle; (d) said administering comprises ADC at a dose of about 1.3 mg / kg on day 1 of a first 3 week cycle; (e) said administering comprises ADC at a dose of about 1.1 mg / kg on day 1 of a first 3 week cycle; (f) said administering comprises ADC at a dose of about 0.88 mg / kg on day 1 of a first 3-week cycle; or (g) The use of (c)-(e) and (e)(ii)-(h) of claim 15, wherein the administration comprises an ADC at a dose of about 0.66 mg / kg on day 1 of a first 3-week cycle.

18. and administering to said human subject an effective amount of an additional therapeutic agent, optionally comprising: (a) a chemotherapeutic, hormonal, anti-tumor, immunostimulatory, immunomodulatory, or immunotherapeutic agent, or a combination thereof; (b) a checkpoint inhibitor, a HER2 kinase inhibitor, a cyclin dependent kinase inhibitor, a tyrosine kinase inhibitor, a small molecule kinase inhibitor, or a platinum-based therapeutic, or a combination thereof; or (c) The use according to claim 14 which is a HER2 targeted therapeutic agent.

19. 15. The use of claim 14, which improves or optimizes cancer cell killing or delays the progression or recurrence of said tumor or cancer.

20. (a) the administration is oral, intradermal, intratumoral, intravenous, or subcutaneous; (b) the tumor or cancer is a HER-2-expressing cancer; (c) the cancer is a HER-2 low expressing cancer, a HER2 intermediate expressing cancer, or a HER2 high expressing cancer; and / or (d) The use according to claim 14, wherein the subject to be treated has a HER-2 expressing cancer and / or cancer metastasis from the same or a different cancer.

21. 11. A formulation comprising: (i) about 20 mg / mL of an ADC of any one of claims 1-3; (ii) about 5 mM histidine buffer at about pH 6.0; (iii) about 6% (w / v) trehalose; and (iv) about 0.02% (w / v) polysorbate 80.

22. 22. The formulation of claim 21, wherein the histidine is L-histidine.

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