Antibodies comprising site-specific non-natural amino acid residues, methods of preparation and uses thereof

Abstract

Provided herein are antibodies comprising one or more non-natural amino acid residues at one or more site-specific positions, antibody conjugates comprising the antibodies, compositions comprising the antibodies and antibody conjugates, and methods of their production and use. The antibodies, antibody conjugates, and compositions are useful for methods of treatment and prevention of diseases, methods of detection and methods of diagnosis.

Description

Attorney Docket No. : 108843.00484(SU0451WO)ANTIBODIES COMPRISING SITE-SPECIFIC NON-NATURAL AMINO ACID RESIDUES, METHODS OF PREPARATION AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONThis application claims the benefit of, and priority to, U.S. Provisional Application No. 63 / 684,596, filed August 19, 2024. The entirety of the above-identified application is hereby fully incorporated herein by reference.FIELD

[0001] Provided herein are antibodies comprising non-natural amino acid residues at sitespecific positions, conjugates thereof, compositions comprising the antibodies or conjugates, methods of their production and use.BACKGROUND

[0002] Antibodies are biological molecules with remarkable affinities for their target antigens. Nature provides antibodies as part of a defense system in certain vertebrates for the elimination or destruction of foreign proteins, cells and organisms. If a certain vertebrate is presented with a foreign protein on, for example, an infected cell or an infectious bacterium, an antibody can bind its target foreign protein to direct the foreign entity to its elimination or destruction.

[0003] The selective affinity of antibodies can be used by man to target nearly any antigen desired. The antigen can be a protein on an infected cell or infectious microorganism. It can also be, for example, a protein on a cancer cell, a protein on a cell of a target tissue, a protein in the bloodstream, a protein on an inflamed or inflammatory cell or any other protein whose selective binding is useful. Antibodies have thus found use in therapy for conditions such as cancer, inflammatory diseases, autoimmune diseases and transplant rejection. The antibody can signal the immune system to destroy or eliminate a diseased cell, or an engineered antibody can carry a molecular payload to destroy the target. In certain applications therapeutic antibodies are linked to molecular shields to increase their lifetime within an organism. Antibodies have also found use as diagnostics. These antibodies can carry a label to indicate the presence of a target antigen on a cell or in a tissue. These labels are typically linked to the antibodies by covalent bonds.

[0004] To date, techniques for linking antibodies molecular entities such as molecular payloads, molecular shields and labels have been limited by their heterogeneity in degree andAttorney Docket No. : 108843.00484(SU0451WO) location of linking to the antibodies, by their low yields and by losses in activity. Typical conjugation sites include random locations on antibody chains, e.g. random amines on amino acid side chains, and the N-terminus of certain antibody chains. In such techniques, some antibodies might be linked to the conjugate at one location while some antibodies are linked to the same conjugate at another location, and some antibodies might not be linked at all.

[0005] There is a need for antibodies modified at site-specific positions optimized for uniformity, yield and / or activity to further the promising use of antibodies in, for example, therapy and diagnostics.BRIEF DESCRIPTION OF THE FIGURES

[0006] FIG. 1 shows TAb concentration - time profiles in mouse plasma following single IV administration of antibody conjugates.

[0007] FIG. 2A shows MC38-hHER2 tumor growth curves in response to treatment with a single i.v. dose of 20 mg / kg a reference anti-HER2 antibody conjugate (Enhertu®) or 3, 10, or 20 mg / kg Conjugate 45. FIG. 2B shows a scatter plot of individual tumor volumes on day 21 post treatment. Statistical analysis was performed on tumor volumes on day 21 using a Welch’s ANOVA with Dunnett’s T3 multiple comparisons test versus the vehicle group. A probability of less than 5% (p<0.05) was considered significant. ** = p<0.01; *** = p<0.001. All graphs are presented as individual values or mean ± SEM.

[0008] FIG. 3A shows MC38-hHER2 tumor growth curves in response to treatment with repeat doses of 10 mg / kg anti-PD-1 (q3-4dx4) and / or 1 mg / kg Conjugate 45 (q7dx3). FIG. 3B shows a scatter plot of individual tumor volumes on day 10 post treatment. Statistical analysis was performed on tumor volumes on day 10 using a one-way ANOVA with Dunnett’ s multiple comparisons test versus the vehicle group. A probability of less than 5% (p<0.05) was considered significant. ** = p<0.01; *** = p<0.001; **** = p<0.0001. All graphs are presented as individual values or mean ± SEM.SUMMARY

[0009] Provided herein are antibodies modified at one or more site-specific positions with non-natural amino acid residues. These site-specific positions are optimal for substitution of a natural amino acid residue with a non-natural amino acid residue. In certain embodiments, substitution at these site-specific positions yields antibodies that are uniform in substitution, i.e., that are substantially modified in the selected position. In certain embodiments, an antibody substituted at these site-specific positions has advantageous production yield,Attorney Docket No. : 108843.00484(SU0451WO) advantageous solubility, advantageous binding and / or advantageous activity. The properties of these antibodies and antibody conjugates are described in detail in the sections below.

[0010] In one aspect, the present disclosure provides an antibody comprising one or more non-natural amino acid residues at one or more positions selected from the group consisting of HC-S113 according to Kabat numbering, HC-A118, HC-T120, HC-K121, HC-G138, HC- A140, HC-T155, HC-S160, HC-A162, HC-L163, HC-Q196, HC-I199, HC-N203, HC-T209, HC-K210, HC-K246, HC-E258, HC-K290, HC-E294, HC-Q295, HC-Y296, HC-N297, HC- Y300, HC-V303, HC-V305, HC-E318, HC-A330, HC-T335, HC-I336, HC-S337, HC-R344, HC-E345, HC-R355, HC-M358, HC-Q362, HC-E382, HC-E388, HC-N390, HC-Y391, HC- K392, HC-T393, HC-T411, HC- N421, HC-S424, HC-Q438, HC-L443 according to EU index of Kabat, LC-T109, LC-V110, LC-A111, LC-A112, LC-D122, LC-Q124, LC-K126, LC- S127, LC-T129, LC-N138, LC-R142, LC-E143, LC-Q147, LC-K149, LC-D151, LC-A153, LC-L154, LC-Q155, LC-S159, LC-E165, LC-Q166, LC-S168, LC-K169, LC-S171, LC-T172, LC-T180, LC-A184, LC-E187, LC-H189, LC-K190, LC-V191, LC-E195, LC-L201, LC- S202, LC-S203, LC-V205, and LC-T206 according to Kabat numbering, or a post- translationally modified variant thereof.

[0011] In some embodiments, the present disclosure provides an antibody comprising one or more non-natural amino acid residues at one or more positions selected from the group consisting of HC-S113 according to Kabat numbering, HC-A118, HC-T120, HC-K121, HC- G138, HC-A140, HC-T155, HC-S160, HC-A162, HC-L163, HC-Q196, HC-I199, HC-N203, HC-T209, HC-K210, HC-E258, HC-K290, HC-Y296, HC-T335, HC-I336, HC-S337, HC- R344, HC-E345, HC-R355, HC-M358, HC-Q362, HC-E382, HC-E388, HC-N390, HC-Y391, HC-K392, HC-T393, HC-T411, HC- N421, HC-S424, HC-Q438, HC-L443 according to EU index of Kabat, LC-T109, LC-V110, LC-A111, LC-A112, LC-D122, LC-Q124, LC-K126, LC-S127, LC-T129, LC-N138, LC-R142, LC-E143, LC-Q147, LC-K149, LC-D151, LC- A153, LC-L154, LC-Q155, LC-S159, LC-E165, LC-Q166, LC-S168, LC-K169, LC-S171, LC-T172, LC-T180, LC-A184, LC-E187, LC-H189, LC-K190, LC-V191, LC-E195, LC- L201, LC-S202, LC-S203, LC-V205, and LC-T206 according to Kabat numbering, or a post- translationally modified variant thereof.

[0012] In some embodiments, the antibody comprises one or more non-natural amino acid residues at one or more positions selected from the group consisting of HC-Q196, HC-Y391, and HC-S337 according to EU index of Kabat, or a post-translationally modified variantAttorney Docket No. : 108843.00484(SU0451WO) thereof. In some embodiments, the antibody comprises a non-natural amino acid residue at HC- Y391 according to EU index of Kabat, or a post-translationally modified variant thereof.

[0013] In some embodiments, the antibody further comprises one or more additional nonnatural amino acid residues at one or more additional positions selected from the group consisting of HC-F404, HC-K121, HC-Y180, HC-F241, HC-221, LC-T22, LC-S7, LC-N152, LC-K42, LC-E161, LC-D170, HC-S136, HC-S25, HC-A40, HC-S119, HC-S190, HC-K222, HC-R19, HC-Y52, and HC-S70 according to EU index of Kabat, or a post-translationally modified variant thereof. In some embodiments, the one or more additional non-natural amino acid residues is at one or more positions selected from the group consisting of HC-Y180, HC- F241, HC-F404 HC, and LC-K42 according to EU index of Kabat, or a post-translationally modified variant thereof.

[0014] In some embodiments, the present disclosure provides an antibody comprising one or more non-natural amino acid residues at one or more positions selected from the group consisting of HC-S113, HC-A118, HC-T120, HC-K121, HC-G138, HC-A140, HC-T155, HC- S160, HC-A162, HC-L163, HC-Q196, HC-I199, HC-N203, HC-T209, HC-K210, HC-K246, HC-E258, HC-K290, HC-E294, HC-Q295, HC-Y296, HC-N297, HC-Y300, HC-V303, HC- V305, HC-E318, HC-A330, HC-T335, HC-I336, HC-S337, HC-R344, HC-E345, HC-R355, HC-M358, HC-Q362, HC-E382, HC-E388, HC-N390, HC-Y391, HC-K392, HC-T393, HC- T411, HC- N421, HC-S424, HC-Q438, HC-L443, LC-T109, LC-V110, LC-A111, LC-A112, LC-D122, LC-Q124, LC-K126, LC-S127, LC-T129, LC-N138, LC-R142, LC-E143, LC- Q147, LC-K149, LC-D151, LC-A153, LC-L154, LC-Q155, LC-S159, LC-E165, LC-Q166, LC-S168, LC-K169, LC-S171, LC-T172, LC-T180, LC-A184, LC-E187, LC-H189, LC-K190, LC-V191, LC-E195, LC-L201, LC-S202, LC-S203, LC-V205, and LC-T206 according to the Kabat, Chothia, or EU numbering scheme, or a post-translationally modified variant thereof.

[0015] In some embodiments, the antibody comprises one or more non-natural amino acid residues at one or more positions selected from the group consisting of HC-Q196, HC-Y391, and HC-S337 according to the Kabat, Chothia, or EU numbering scheme, or a post- translationally modified variant thereof. In some embodiments, the antibody comprises a non- natural amino acid residue at HC-Y391 according to the Kabat, Chothia, or EU numbering scheme, or a post-translationally modified variant thereof.

[0016] In some embodiments, the antibody further comprises one or more additional non- natural amino acid residues at one or more additional positions selected from the group consisting of HC-F404, HC-K121, HC-Y180, HC-F241, HC-221, LC-T22, LC-S7, LC-N152,Attorney Docket No. : 108843.00484(SU0451WO)LC-K42, LC-E161, LC-D170, HC-S136, HC-S25, HC-A40, HC-S119, HC-S190, HC-K222, HC-R19, HC-Y52, and HC-S70 according to the Kabat, Chothia, or EU numbering scheme, or a post-translationally modified variant thereof. In some embodiments, the one or more additional non-natural amino acid residues is at one or more positions selected from the group consisting of HC-Y180, HC-F241, HC-F404 HC, andLC-K42 according to the Kabat, Chothia, or EU numbering scheme, or a post-translationally modified variant thereof.

[0017] In some embodiments, the antibody comprises a heavy chain of a type selected from the group consisting of a, y, 5, s, and p. In some embodiments, the antibody comprises a light chain of a type selected from the group consisting of and K. In some embodiments, the light chain is of type K. In some embodiments, the antibody is of a class or subclass selected from the group consisting of IgA, IgA, IgA2, IgD, IgE, IgG, and IgM. In some embodiments, the antibody is an IgG antibody. In some embodiments, the IgG antibody is a IgGl, IgG2, or IgG3 antibody.

[0018] In some embodiments, the antibody is in a form selected from the group consisting of Fv, Fc, Fab, (Fab')2, single chain Fv (scFv) and full-length antibody.

[0019] In some embodiments, at least one of the one or more non-natural amino acid residues comprises a moiety selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semi carb azido, sulfanyl, azido, alkynyl, and tetrazine. In some embodiments, at least one of the one or more non-natural amino acid residues comprises an azide moiety. In some embodiments, at least one of the one or more non-natural amino acid residues comprises an acetyl moiety. In some embodiments, at least one of the one or more non-natural amino acid residues comprises a tetrazine moiety. In some embodiments, one or more non-natural amino acid residue is according to the formulawherein each L is independently a divalent linker; each R is independently a functional group; and each wavy line indicates a bond that connects to remainder of a polypeptide chain of the antibody. In some embodiments, R is a reactive group, a therapeutic moiety, or a labeling moiety. In some embodiments, R is a reactive group selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, alkynyl, and tetrazine. In some embodiments, L is a divalent linker selected from the group consisting of a bond, alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene,Attorney Docket No. : 108843.00484(SU0451WO) substituted arylene, heteroarlyene and substituted heteroarylene. In some embodiments, at least one of the one or more non-natural amino acid residue is selected from the group consisting of ortho-substituted tyrosine, meta substituted tyrosine, para-substituted phenylalanine, orthosubstituted phenylalanine, and meta-substituted phenylalanine. In some embodiments, at least one of the one or more non-natural amino acid residue is selected from the group consisting of / ?-acetyl-L-phenylalanine, O-methyl-L-tyrosine, 3-methyl-phenylalanine, O-4-allyl-L- tyrosine, 4-propyl-L-tyrosine, fluorinated phenylalanine, isopropyl-L-phenylalanine, / ?-azido- L-phenylalanine, / ?-acyl-L-phenylalanine, -A benzoyl-L-phenylalanine, / ’-iodo- phenylalanine, / ?-bromophenylalanine, / ?-amino-L-phenylalanine, isopropyl-L-phenylalanine, -propargyloxy -phenylalanine, and / ?-azidomethyl-L-phenyl alanine. In some embodiments, at least one of the one or more non-natural amino acid residue is / ?-azidomethyl-L-phenylalanine. In some embodiments, at least one of the one or more non-natural amino acid residue is p- acety 1 -L-pheny 1 al anine .

[0020] In some embodiments, at least one of the one or more additional non-natural amino acid residues comprises a moiety selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semi carb azido, sulfanyl, azido, alkynyl, and tetrazine. In some embodiments, at least one of the one or more additional non-natural amino acid residues comprises an azide moiety. In some embodiments, at least one of the one or more additional non-natural amino acid residues comprises an acetyl moiety. In some embodiments, at least one of the one or more additional non-natural amino acid residues comprises a tetrazine moiety.

[0021] In some embodiments, the antibody is aglycosylated. In some embodiments, the antibody is glycosylated.

[0022] In another aspect, the present disclosure provides an antibody conjugate comprising the antibody herein linked to one or more payloads via the one or more non-natural amino acid residues.

[0023] In some embodiments, the one or more payloads comprises one or more therapeutic moieties. In some embodiments, one or more therapeutic moieties comprises an immunomodulatory agent, an alkylating agent, a DNA-crosslinking agent, an anti-tumor antibiotic, an anti-metabolite, an anti-mitotic agent, a histone-deacetylase (HD AC) inhibitor, a telomerase inhibitor, an immunogenic cell death agent, a tubulin inhibitor, or a topoisomerase inhibitor. In some embodiments, one or more therapeutic moieties comprises a topoisomerase I inhibitor and the topoisomerase I inhibitor is selected from the group consisting of camptothecin, irinotecan, SN-38, topotecan, and exatecan. In some embodiments, theAttorney Docket No. : 108843.00484(SU0451WO) topoisomerase I inhibitor is exatecan. In some embodiments, the one or more therapeutic moieties comprises a hemiasterlin. In some embodiments, the one or more therapeutic moieties comprises an immunomodulatory agent. In some embodiments, the immunomodulatory agent is a STING agonist. In some embodiments, the immunomodulatory agent is a Toll-like receptor (TLR) agonist. In some embodiments, the TLR agonist is a TLR 7 / 8 agonist. In some embodiments, the one or more payloads comprises one or more labeling moieties.

[0024] In some embodiments, the antibody is linked to the one or more payloads via one or more linkers. In some embodiments, the linker is hydrolytically stable. In some embodiments, the linker is cleavable. In some embodiments, the linker is a protease cleavable linker, an enzyme cleavable linker, a pH-sensitive linker, or a non-cleavable linker.

[0025] In some embodiments, the antibody conjugate is represented by the structure of Formula (I):Formula (I) or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, regioisomer, or mixture of regioisomers thereof; wherein Ab is an anti-HER2 antibody or antigen-binding fragment thereof;Lais a first linker;Lbis a second linker;CY is independently a residue of a cytotoxic agent;STING is independently a residue of a STING agonist; subscript n is an integer selected from 1 to 10; and subscript m is an integer selected from I to KT

[0026] In another aspect, the present disclosure provides a pharmaceutical composition comprising the antibody or the antibody conjugate herein, and a pharmaceutically acceptable carrier.

[0027] In another aspect, the present disclosure provides a method of treating or preventing a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of an antibody conjugate herein or pharmaceutical composition herein. In some embodiments, the disease is lung cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, uterine cervix cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular cancer, colon cancer, rectal cancer,Attorney Docket No. : 108843.00484(SU0451WO) colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulval cancer, thyroid cancer, penis cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma. In some embodiments, the method further comprises administering to the subject one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents comprises an anti-PD-1 antibody.

[0028] In another aspect, the present disclosure provides a method of detecting an antigen, comprising: contacting a sample comprising the antigen with an antibody conjugate herein, or a pharmaceutical composition comprising the antibody conjugate, at least one of the nonnatural amino acids comprising a labeling moiety or is linked to a payload comprising a labeling moiety; and detecting a complex formed between the antibody and the antigen.DETAILED DESCRIPTION OF EMBODIMENTS

[0029] Provided herein are antibodies having non-natural amino acids at one or more sitespecific positions, compositions comprising the antibodies, methods of making the antibodies, and methods of their use.Definitions

[0030] When referring to the antibodies provided herein, the following terms have the following meanings unless indicated otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

[0031] As used herein, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise. Thus, for example, reference to an “antibody” is a reference to one or more such antibodies, etc.

[0032] The term “about” indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” indicates the designated value ± 10%, ± 5%, or ± 1%. In certain embodiments, the term “about” indicates the designated value ± one standard deviation of that value. In certain embodiments, for example, logarithmic scales (e.g., pH), the term “about” indicates the designated value ± 0.3, ±0.2, or ± 0.1.

[0033] The term “isolated” refers to an antibody that is substantially or essentially free of components that normally accompany or interact with the antibody as found in its naturally occurring environment or in its production environment, or both. Isolated antibody preparations have less than about 30%, less than about 25%, less than about 20%, less than about 15%, lessAttorney Docket No. : 108843.00484(SU0451WO) than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2% or less than about 1% of contaminating protein by weight, e.g. dry weight.

[0034] The term “HER2” refers to, unless specified otherwise, any variants, isoforms and species homologs of human epidermal growth factor receptor 2 (may also be referred to as neu or ErbB-2) that are naturally expressed by cells, or that are expressed by cells transfected with a HER2 gene. HER2 is a transmembrane receptor belonging to the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases together with HER1 (EGFR or ErbB-1), HER3 (ErbB-3), and HER4 (ErbB-4).

[0035] The term “antibody” refers to any macromolecule that would be recognized as an antibody by those of skill in the art. Antibodies share common properties including binding to an antigen and a structure comprising at least one polypeptide chain that is substantially identical to a polypeptide chain that can be encoded by any of the immunoglobulin genes recognized by those of skill in the art. The immunoglobulin genes include, but are not limited to, the K, , a, y (IgGl, IgG2, IgG3, and IgG4), 5, a and p constant region genes, as well as the immunoglobulin variable region genes (e.g., IGHV, IGHD, IGHJ, IGLV, IGKV, IGLJ, and IGKJ genes). The term includes full-length antibodies and antibody fragments recognized by those of skill in the art, and variants thereof.

[0036] The light chain from any vertebrate species can be assigned to one of two types, called kappa (K) and lambda (X), based on the sequence of the constant domain.

[0037] The heavy chain from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated a, 5, a, y, and p, respectively. The IgG and IgA classes are further divided into subclasses on the basis of differences in sequence and function. Humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

[0038] The term “antibody fragment” refers to any form of an antibody other than the full- length form. Antibody fragments herein include antibodies that are smaller components that exist within full-length antibodies, and antibodies that have been engineered. Antibody fragments include but are not limited to Fv, Fc, Fab, and (Fab')2, single chain Fv (scFv), domain antibodies (dAbs), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDR's, variable regions, framework regions, constant regions, and the like (Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9:395-402).Attorney Docket No. : 108843.00484(SU0451WO)

[0039] The term “immunoglobulin (Ig)” refers to a protein consisting of one or more polypeptides substantially encoded by one or more of the immunoglobulin genes, or a protein substantially identical thereto in amino acid sequence. Immunoglobulins include but are not limited to antibodies. Immunoglobulins may have a number of structural forms, including but not limited to full-length antibodies, antibody fragments, and individual immunoglobulin domains including but not limited to VH, DH, JH, CH (e.g., Cyl, Cy2, Cy3), VL, JL, and CL (e.g., VK and V ).

[0040] The term “immunoglobulin (Ig) domain” refers to a protein domain consisting of a polypeptide substantially encoded by an immunoglobulin gene. Ig domains include but are not limited to VH, DH, JH, CH (e.g., Cyl, Cy2, Cy3), VL, JL, and CL (e.g., VK and V ).

[0041] The term “variable region” of an antibody refers to a polypeptide or polypeptides composed of the VH immunoglobulin domain, the VL immunoglobulin domains, or the VH and VL immunoglobulin domains. Variable region may refer to this or these polypeptides in isolation, or as a fragment (e.g., as an Fv fragment or as an scFv fragment), as this region in the context of a larger antibody fragment, or as this region in the context of a full-length antibody or an alternative, non-antibody scaffold molecule.

[0042] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are responsible for the binding specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three segments called Complementarity Determining Regions (CDRs). Three of the CDRs are located in the light chain variable domain and three of the CDRs are located in the heavy chain variable domain. The more highly conserved portions of the variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a P-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the P-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0043] The constant domains are not typically involved directly in binding an antibody to an antigen, but exhibit various effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins can be assigned toAttorney Docket No. : 108843.00484(SU0451WO) different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgGl, IgG2, IgG3, and IgG4; IgAl and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called a, 5, a, y and p, respectively. Of the various human immunoglobulin classes, only human IgGl, IgG2, IgG3 and IgM are known to activate complement.

[0044] The term “variant protein sequence” refers to a protein sequence that has one or more residues that differ in amino acid identity from another similar protein sequence. Said similar protein sequence may be the natural wild type protein sequence, or another variant of the wild type sequence. Variants include proteins that have one or more amino acid insertions, deletions or substitutions. Variants also include proteins that have one or more post- translationally non-natural amino acids.

[0045] The term “parent antibody” refers to any antibody known to those of skill in the art that is modified according to the description provided herein. The modification can be physical, i.e., chemically or biochemically replacing or modifying one or more amino acids of the parent antibody to yield an antibody within the scope of the present description. The modification can also be conceptual, i.e., using the sequence of one or more polypeptide chains of the parent antibody to design an antibody comprising one or more site-specific non-natural amino acids according to the present description. Parent antibodies can be naturally occurring antibodies or antibodies designed or developed in a laboratory. Parent antibodies can also be artificial or engineered antibodies, e.g., chimeric or humanized antibodies.

[0001] The amino acid position can be identified using any number of a known numbering schemes, including those described by Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD, (“Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Pliickthun, J. Mol. Biol., 2001, 309:657-70 (“AHo” numbering scheme), each of which is incorporated by reference in its entirety.

[0002] The “EU numbering scheme” is generally used when referring to a residue in an antibody heavy chain constant region (e.g., as reported in Kabat et al., supra). Unless stated otherwise, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.Attorney Docket No. : 108843.00484(SU0451WO)

[0046] A “conservative substitution” or a “conservative amino acid substitution,” refers to the substitution of an amino acid with a chemically or functionally similar amino acid. Conservative substitution tables providing similar amino acids are well known in the art. Polypeptide sequences having such substitutions are known as “conservatively modified variants.” Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. By way of example, the groups of amino acids provided in Tables 1-3 are, in some embodiments, considered conservative substitutions for one another.Table 1. Selected groups of amino acids that are considered conservative substitutions for one another, in certain embodiments.Table 2. Additional selected groups of amino acids that are considered conservative substitutions for one another, in certain embodiments.Table 3. Further selected groups of amino acids that are considered conservative substitutions for one another, in certain embodiments.Attorney Docket No. : 108843.00484(SU0451WO)

[0047] Additional conservative substitutions may be found, for example, in Creighton, Proteins: Structures and Molecular Properties 2nd ed. (1993) W. H. Freeman & Co., New York, NY. An antibody generated by making one or more conservative substitutions of amino acid residues in a parent antibody is referred to as a “conservatively modified variant.”

[0048] The terms “identical” or “identity,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same. Sequences are “substantially identical” if they have a percentage of amino acid residues or nucleotides that are the same (z.e., about 60% identity, optionally about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 99.9% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. The identity can exist over a region that is at least about 50 amino acid residues or nucleotides in length, over a region that is about 10-17 residues in length (e.g., the approximate length of CDRL1), over a region that is about 7 residues in length (e.g., the approximate length of CDRL2), over a region that is about 7-11 residues in length (e.g., the approximate length of CDRL3), over a region that is about 10-12 residues in length (e.g., the approximate length of CDRH1), over a region that is about 16-19 residues in length (e.g., the approximate length of CDRH2), over a region that is about 3-35 residues in length (e.g., the approximate length of CDRH3), or over a region that is 75-100 amino acid residues or nucleotides in length, or, where not specified, across the entire sequence or a polypeptide. In the case of antibodies, identity can be measured outside the variable CDRs. Identity can also be measured within the entirety of the heavy or light chains, or within the variable regions of the heavy or light chains. Optimal alignment of sequences for comparison can be conducted, including but not limited to, by the local homology algorithm of Smith and Waterman (1970) Adv. AppL Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'L Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575Attorney Docket No. : 108843.00484(SU0451WO)Science Dr., Madison, Wis.); or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0049] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Awe. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm is typically performed with the “low complexity” filter turned off.

[0050] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873- 5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0051] The term “amino acid” refers to the twenty common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gin; Q), Glycine (Gly; G); histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Vai; V), and the less common pyrrolysine and selenocysteine. Natural amino acids also include citrulline. Naturally encoded amino acids include post-translational variants of the 22 naturally occurring amino acids such as prenylated amino acids, isoprenylated amino acids, myrisoylated amino acids, palmitoylated amino acids, N-linked glycosylated amino acids, O-linked glycosylated amino acids, phosphorylated amino acids, and acylated amino acids.Attorney Docket No. : 108843.00484(SU0451WO)

[0052] The term “non-natural amino acid” or “nnAA” refers to are a, 0, y, or 6 amino acids, and includes but is not limited to, amino acids found in proteins, i.e., glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine. In certain embodiments, the amino acid is in the L-configuration. Alternatively, the amino acid can be a derivative of alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl, 0-alanyl, 0-valinyl, 0-leucinyl, 0- isoleuccinyl, 0-prolinyl, 0-phenylalaninyl, 0 -tryptophanyl, 0-methioninyl, 0-glycinyl, 0- serinyl, 0-threoninyl, 0-cysteinyl, 0-tyrosinyl, 0-asparaginyl, 0-glutaminyl, 0-aspartoyl, 0- glutaroyl, 0-lysinyl, 0-argininyl or 0-histidinyl. Unnatural amino acids are not proteinogenic amino acids, or post-translationally modified variants thereof that either occur naturally or are chemically synthesized. In particular, the term non-natural amino acid refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine, or post- translationally modified variants thereof. Non-limiting examples of non-natural amino acids include sulfoalanine, hydroxyproline (Hyp), beta-alanine, citrulline (Cit), ornithine (Orn), norleucine (Nle), 3 -nitrotyrosine, nitroarginine, pyroglutamic acid (Pyr), naphtylalanine (Nal), 2,4-diaminobutyric acid (DAB), methionine sulfoxide, and methionine sulfone.

[0053] “pAMF” or “p AzMeF” mutation refers to a variant phenylalanine residue, i. e. , para- azidomethyl-L-phenylalanine, added or substituted into a polypeptide.

[0054] “pAcF,” “pAcPhe” or “pAcetylF” mutation refers to a variant phenylalanine residue, i.e., para-acetyl-L-phenylalanine, added or substituted into a polypeptide.

[0055] A “functional Releasing Factor 1 (RF1) protein” refers to RF1 that retains activity equal to or substantially similar to wild-type or unmodified RF1 protein. Functional RF1 activity can be tested, for example, by measuring the growth rate of bacteria expressing the modified RF1 protein, and comparing the growth rate to bacteria expressing wild-type or unmodified RF1. Functional RF1 activity can also be tested, for example, by the ability of the modified RF1 protein to reduce orthogonal tRNA incorporation of a nnAA at a specified position in an mRNA encoding a target protein, thereby increasing the amount of premature chain termination (i.e., increasing the amount of truncated protein).

[0056] An “attenuated Releasing Factor 1 (RF1) protein” refers to a modified RF1 that has reduced activity relative to wild-type or unmodified RF1 protein. RF1 activity can be tested, for example, by the ability of the modified RF1 protein to reduce orthogonal tRNAAttorney Docket No. : 108843.00484(SU0451WO) incorporation of a nnAA at a specified position in an mRNA encoding a target protein, thereby increasing the amount of premature chain termination (i.e., increasing the amount of truncated protein). In some embodiments, the attenuated RF1 protein comprises transcriptional modifications; for example, the expression level of the RF1 protein (wild type or modified) can be reduced to achieve attenuation. The reduction can also be achieved by using RNAi technologies. In some embodiments, the attenuated RF1 protein comprises translational modifications; for example, the amount of the synthesized RF 1 protein (wild type or modified) can be reduced to achieve attenuation, e.g., by increasing the rate at which the protein is digested by protease via insertion of protease-specific sequence into the RF1 sequence.

[0057] The term “strained alkene” refers to a molecule comprising an alkene moiety that is capable of reacting with tetrazine in a tetrazine ligation. Exemplary tetrazine ligations are described in Blackman et al., 2008, J. Am. Chem. Soc. 130: 13518-13519. Examples include / ra / / .s-cyclooctenes and norbomenes. Useful compounds include, but are not limited to, trans- cyclooctene, (E)-cyclooct-4-enol, (E)-cyclooct-4-enyl 2,5-dioxo-l-pyrrolidinyl carbonate, 5- norbomene-2-acetic acid succinimidyl ester, and 5-norbornene-2-endo-acetic acid.

[0058] The term “conjugate” or “antibody conjugate” refers to an antibody linked to one or more payload moieties. The antibody can be any antibody described herein. The payload can be any payload such as cytotoxic agents and immunomodulatory agents described herein. The antibody can be directly linked to the payload via a covalent bond, or the antibody can be linked to the payload indirectly via a linker. Typically, the linker is covalently bonded to the antibody and also covalently bonded to the payload. The term “antibody drug conjugate” or “ADC” refers to a conjugate wherein at least one payload is a therapeutic moiety such as a drug. In some embodiments, an antibody conjugate refers to an antibody, directly or indirectly, covalently bound via a linker to at least one cytotoxic agent and at least one immunomodulatory agent, such as a STING agonist. In some embodiments, an antibody conjugate refers to an antibody, directly or indirectly, covalently bound via a linker to at least one immunomodulatory agent, such as a STING agonist.

[0059] The term “site-specific” refers to a modification of a polypeptide at a predetermined sequence location in the polypeptide. The modification is at a single, predictable residue of the polypeptide with little or no variation. In particular embodiments, a non-natural amino acid is introduced at that sequence location, for instance recombinantly or synthetically. Similarly, a moiety can be “site-specifically” linked to a residue at a particular sequence location in theAttorney Docket No. : 108843.00484(SU0451WO) polypeptide. In certain embodiments, a polypeptide can comprise more than one site-specific modification.

[0060] The term “payload” refers to a molecular moiety that can be conjugated to an antibody. In particular embodiments, payloads are selected from the group consisting of therapeutic moieties, for example, the immunomodulatory agents and cytotoxic agents described herein. In one embodiment, the immunomodulatory agent is a STING agonist.

[0061] The term “therapeutically effective amount” or “effective amount” refers to an amount of an antibody or composition that when administered to a subject is effective to treat a disease or disorder. In some embodiments, a therapeutically effective amount or effective amount refers to an amount of an antibody or composition that when administered to a subject is effective to prevent or ameliorate a disease or the progression of the disease or result in amelioration of symptoms. A “therapeutically effective amount” can vary depending on, inter alia, the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated.

[0062] The term “treating” or “treatment” of any disease or disorder refers, in certain embodiments, to ameliorating a disease or disorder that exists in a subject. In another embodiment, “treating” or “treatment” includes ameliorating at least one physical parameter, which may be indiscernible by the subject. In yet another embodiment, “treating” or “treatment” includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. In yet another embodiment, “treating” or “treatment” includes delaying or preventing the onset of the disease or disorder or delaying or preventing recurrence of the disease or disorder. In yet another embodiment, “treating” or “treatment” includes the reduction or elimination of either the disease or disorder, or to retard the progression of the disease or disorder or of one or more symptoms of the disease or disorder, or to reduce the severity of the disease or disorder or of one or more symptoms of the disease or disorder.

[0063] The term “alkyl,” as used herein, unless otherwise specified, refers to a saturated straight or branched hydrocarbon. In certain embodiments, the alkyl group is a primary, secondary, or tertiary hydrocarbon. In certain embodiments, the alkyl group includes one to ten carbon atoms (i.e., Ci to Cio alkyl). In certain embodiments, the alkyl is a lower alkyl, for example, Ci-ealkyl, and the like. In certain embodiments, the alkyl group is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, ec-butyl, / -butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, 3 -methylpentyl, 2,2-dimethylbutyl, and 2,3-Attorney Docket No. : 108843.00484(SU0451WO) dimethylbutyl. In certain embodiments, “substituted alkyl” refers to an alkyl substituted with, for example, one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, -CN, -NO2, amido, -C(O)-, -C(S)-, ester, carbamate, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, dialkylamino, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy. In some embodiments, alkyl is unsubstituted. In one embodiment, alkyl is methyl or ethyl.

[0064] The term “alkylene,” as used herein, unless otherwise specified, refers to a divalent alkyl group, as defined herein. “Substituted alkylene” refers to an alkylene group substituted as described herein for alkyl. In some embodiments, alkylene is unsubstituted. In one embodiment, alkylene is Ce alkylene.

[0065] “Alkenyl” refers to an olefinically unsaturated hydrocarbon group, in certain embodiments, having up to about eleven carbon atoms or from two to six carbon atoms (e.g., “lower alkenyl”), which can be straight-chained or branched, and having at least one or from one to two sites of olefinic unsaturation. “Substituted alkenyl” refers to an alkenyl group substituted as described herein for alkyl.

[0066] “Alkenylene” refers to a divalent alkenyl as defined herein. Lower alkenylene is, for example, C2-Ce-alkenylene. In one embodiment, alkylene is C2 alkenylene.

[0067] “Alkynyl” refers to acetylenically unsaturated hydrocarbon groups, in certain embodiments, having up to about eleven carbon atoms or from two to six carbon atoms (e.g., “lower alkynyl”), which can be straight-chained or branched, and having at least one or from one to two sites of acetylenic unsaturation. Non-limiting examples of alkynyl groups include acetylene (-C=CH), propargyl (-CH2OCH), and the like. “Substituted alkynyl” refers to an alkynyl group substituted as described herein for alkyl.

[0068] “Alkynylene” refers to a divalent alkynyl as defined herein. Lower alkynylene is, for example, C2-Ce-alkynylene.

[0069] “Amino” refers to -NH2.

[0070] The term “aminoalkyl,” as used herein, and unless otherwise specified, refers to the an alkyl group, as defined herein, which is substituted with one or more amino groups. In some embodiments, the aminoalkyl is an alkyl group substituted with one -NH2 group (e.g., -R'(NH2) wherein R' is alkyl as defined herein).

[0071] The term “alkylamino,” as used herein, and unless otherwise specified, refers to the group -NHR" where R" is, for example, Ci-ioalkyl, C2-10 alkenyl, C2-10 alkynyl, C3-12Attorney Docket No. : 108843.00484(SU0451WO) carbocycle, 3- to 12-membered heterocycle, Ci-io haloalkyl, and the like as defined herein. In certain embodiments, alkylamino is Ci-ealkylamino.

[0072] The term “dialkylamino,” as used herein, and unless otherwise specified, refers to the group -NR''R" where each R" is independently Ci-ioalkyl, as defined herein. In certain embodiments, dialkylamino is, for example, di-Ci-ealkylamino, C2-10 alkenyl, C2-10 alkynyl, C3- 12 carbocycle, 3- to 12-membered heterocycle, C1-10 haloalkyl, and the like.

[0073] The term “aryl,” as used herein, and unless otherwise specified, refers to phenyl, biphenyl, or naphthyl. The term includes both substituted and unsubstituted moieties. An aryl group can be substituted with any described moiety including, but not limited to, one or more moieties (e.g., in some embodiments one, two, or three moieties) selected from the group consisting of halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, and phosphonate, wherein each moiety is independently either unprotected, or protected as necessary, as would be appreciated by those skilled in the art (see, e.g., Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991); and wherein the aryl in the arylamino and aryloxy substituents are not further substituted.

[0074] The term “arylamino,” as used herein, and unless otherwise specified, refers to an - NR'R" group where R' is hydrogen or Ci-Ce-alkyl; and R" is aryl, as defined herein.

[0075] The term “arylene,” as used herein, and unless otherwise specified, refers to a divalent aryl group, as defined herein.

[0076] The term “aryloxy,” as used herein, and unless otherwise specified, refers to an - OR group where R is aryl, as defined herein.

[0077] “Alkarylene” refers to an arylene group, as defined herein, wherein the aryl ring is substituted with one or two alkyl groups. “Substituted alkarylene” refers to an alkarylene, as defined herein, where the arylene group is further substituted, as defined herein for aryl.

[0078] “Aralkylene” refers to a -CEE-arylene-, -arylene-CEE-, or -CEE-arylene-CEE- group, where arylene is as defined herein. “Substituted aralkylene” refers to an aralkylene, as defined herein, where the aralkylene group is substituted, as defined herein for aryl.

[0079] “Carboxyl” or “carboxy” refers to -C(O)OH or -COOH.”Attorney Docket No. : 108843.00484(SU0451WO)

[0080] “Divalent hydrophilic group;” as used herein, and unless otherwise specified, refers to PEG, for example of the formula, wherein e is an integer between 1 and 50, inclusive; and eachis the point of attachment to the rest of the formula.

[0081] “Polyethylene glycoloxy (PEG)” as used herein, and unless otherwise specified, refers to the monovalent or divalent residue of polyethylene glycol (PEG), for example of the formula, respectively, wherein e or f is an integer between 1 and 50, inclusive; and eachis the point of attachment to the rest of the formula. In one embodiment, e is an integer between 1 and 10. In one embodiment, e is an integer between 5 and 10. In one embodiment, f is an integer between 11 and 20.

[0082] “Methoxypolyethylene glycoloxy” as used herein, and unless otherwise specified, refers to the monovalent residue of poly(ethylene glycol) monomethyl ether (mPEG) and is ofH3C(o^ °y the formulafwherein f is an integer between 1 and 50, inclusive; and each is the point of attachment to the rest of the formula. In one embodiment, f is an integer between 1 and 20. In one embodiment, f is an integer between 11 and 20.

[0083] “Methoxypolyethylene glycoloxy-CEECEE-C O)-” as used herein, and unless otherwise specified, refers to the monovalent residue of mPEG as described herein further substituted with -CH2CH2-C(O)- (mPEG-CH2CH2-C(O)-) and is of the formulaH3cfo / S'°^yrA' 4 II° wherein g is an integer between 1 and 50, inclusive; and each is the point of attachment to the rest of the formula. In one embodiment, g is an integer between 1 and 20. In one embodiment, g is an integer between 11.

[0084] “Polyethylene glycoloxy-CH2CH2C(O)-” as used herein, and unless otherwise specified, refers to the monovalent residue of PEG as described herein further substituted with-CH2CH2-C(O)- (PEG-CH2CH2-C(O)-) and is of the formulaAttorney Docket No. : 108843.00484(SU0451WO) wherein h is an integer between 1 and 50, inclusive; and each / Ms the point of attachment to the rest of the formula. In one embodiment, h is an integer between 1 and 20. In one embodiment, h is an integer between 11 and 20.

[0085] The term “carbocycle” as used herein, unless otherwise specified, refer to a saturated, unsaturated, or aromatic ring in which all atoms of the ring are carbon. In certain embodiments, the “carbocycle” group may be saturated, and / or bridged, and / or non-bridged, and / or a fused bicyclic group, and / or a spirocyclic bicyclic group. In certain embodiments, the “carbocycle” group includes three to ten carbon atoms (i.e., C3 to C10 cycloalkyl). In some embodiments, the “carbocycle” has from three to fifteen carbons (C3-15), from three to ten carbons (C3-10), from three to seven carbons (C3-7), or from three to six carbons (C3-C6) (i.e., “lower cycloalkyl”). In certain embodiments, the “carbocycle” group is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexylmethyl, cycloheptyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, decalinyl, or adamantyl Exemplary “carbocycles” include cyclopentyl, cyclohexyl, cyclohexenyl, adamantyl phenyl, indanyl, and naphthyl. “Carbocycle” includes 3- to 10-membered monocyclic rings, 6- to 12-membered bicyclic rings, and 6- to 12-membered bridged rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated, and aromatic rings. A bicyclic carbocycle includes any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits. A bicyclic carbocycle includes any combination of ring sizes such as 4-5 fused ring systems, 5-5 fused ring systems, 5-6 fused ring systems, 6-6 fused ring systems, 5-7 fused ring systems, 6-7 fused ring systems, 5-8 fused ring systems, and 6-8 fused ring systems. Non-limiting examples of bridged bicyclic carbocycle groups include, but are not limited to, bicyclo[l. l.l]pentyl, bicyclo[2.1.1]hexyl, bicyclo[2.1.1]hexyl, bicyclo[3.1.1]heptyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl, bicyclo[3.3.2]decyl, and 2-oxabicyclo[2.2.2]octyl. Non-limiting examples of spirocyclic carbocycle groups include, but are not limited to, spiro[3.3]heptyl, spiro[3.4]octyl, spiro[3.5]nonyl, spiro[3.6]decyl, spiro[4.4]nonyl, spiro[4.5]decyl, spiro[5.5]undecyl, spiro[5.6]dodecyl, and spiro[5.7]tridecyl.

[0086] “Carbocyclene” refers to a divalent carbocycle as defined herein.

[0087] The term “bicyclic ring system” includes 6-12 (e.g., 8-12 or 9-, 10-, or 11-) membered structures that form two rings, wherein the two rings have at least one atom in common (e.g., two atoms in common). Bicyclic rings can be fused, bridged, or spirocyclic. Bicyclic ring systems include bicycloaliphatics (e.g., bicycloalkyl or bicycloalkenyl), bicycloheteroaliphatics, bicyclic aryls, and bicyclic heteroaryls.Attorney Docket No. : 108843.00484 (SU0451WO)

[0088] The term “bridged bicyclic ring system” refers to a bicyclic heterocyclicalipahtic ring system or bicyclic cycloaliphatic ring system in which the rings are bridged. Examples of bridged bicyclic ring systems include, but are not limited to, adamantanyl, norbomenyl, bicyclo[l.l.l]pentyl, bicyclo[2.1.1]hexyl, bicyclo[2.1.1]hexyl, bicyclo[3.1.1]heptyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl, bicyclo[3.3.2]decyl, 2-oxabicyclo[2.2.2]octyl, 6-azabicyclo[3.1.1]heptyl, 6- azabicyclo[3.1.1]heptyl, l-azabicyclo[2.2.1]heptyl, 2-azabicyclo[2.2.1]heptyl, 7- azabicyclo[2.2.1]heptyl, l-azabicyclo[2.2.2]octyl, 3-azabicyclo[3.2.1]octyl, and 2- oxabicyclo[3.1.1]heptyl, 2,6-dioxa-tricyclo[3.3.1.03’7]nonyl. A bridged bicyclic ring system can be optionally substituted with one or more substituents such as alkyl (including carboxyalkyl, hydroxyalkyl, and haloalkyl such as trifluoromethyl), alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, heteroaryl, alkoxy, cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, aroyl, heteroaroyl, nitro, carboxy, alkoxycarbonyl, alkylcarbonyloxy, aminocarbonyl, alkylcarbonylamino, cycloalkylcarbonylamino,(cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino,(heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino, heteroarylcarbonylamino, heteroaralkylcarbonylamino, cyano, halo, hydroxy, acyl, mercapto, alkylsulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, or carbamoyl.

[0089] The term “spiro bicyclic ring system” refers to a bicyclic heterocyclicalipahtic ring system or bicyclic cycloaliphatic ring system in which 2 or 3 rings are linked together by one common atom. Spiro compounds depicted with overlapping rings indicate that the rings canN bond at any vertex. For instance, in the spiro group, the two rings can bond at any of the three available vertex atoms in either ring, in some embodiments, a spiro bicyclic ring is a 3- to 12- membered spirocyclic bicyclic heterocycle comprising two nitrogen atoms and one oxygen atom. Non-limiting examples of a spirocyclic bicyclic heterocycle include a 10- membered spirocyclic bicyclic heterocycle, a 9- membered spirocyclic bicyclic heterocycle, and a 8- membered spirocyclic bicyclic heterocycle. The 3- to 12- membered spirocyclic bicyclic heterocycle include, but are not limited to, nitrogen (N), oxygen (O), and sulfur (S) atoms, for example two nitrogen atoms and one oxygen atom. For example, a 5-oxa- 2,8-diazaspiro[3.5]nonane is a compound in which a 4 membered heterocyclic ring and a 6 membered heterocyclic ring are bonded through a single carbon atom wherein an oxygen atom is in the 6 membered heterocyclic ring.Attorney Docket No. : 108843.00484(SU0451WO)

[0090] The term “cycloalkylene,” as used herein refers to a divalent cycloalkyl group, as defined herein. In certain embodiments, the cycloalkylene group is cyclopropylenec , cyclopentylene, cyclohexylene, cycloheptylene, an the like. Lower cycloalkylene refers to a Cs-Ce-cycloalkylene.

[0091] The term “cycloalkylalkyl,” as used herein, unless otherwise specified, refers to an alkyl group, as defined herein, substituted with one or two cycloalkyl, as defined herein.

[0092] The term “ester,” as used herein, refers to -C(O)OR or -COOR where R is alkyl, as defined herein.

[0093] The term “fluorene” as used herein refers to, wherein any one or more carbons bearing one or more hydrogens can be substituted with a chemical functional group as described herein.

[0094] The term “haloalkyl” refers to an alkyl group, as defined herein, substituted with one or more halogen atoms (e.g., in some embodiments one, two, three, four, or five) which are independently selected.

[0095] The term “heteroalkyl” refers to an alkyl, as defined herein, in which one or more carbon atoms are replaced by heteroatoms. As used herein, “heteroalkenyl” refers to an alkenyl, as defined herein, in which one or more carbon atoms are replaced by heteroatoms. As used herein, “heteroalkynyl” refers to an alkynyl, as defined herein, in which one or more carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen (N), oxygen (O), and sulfur (S) atoms. Heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted. Examples of heteroalkyl moieties include, but are not limited to, aminoalkyl, sulfonylalkyl, and sulfinylalkyl. Examples of heteroalkyl moieties also include, but are not limited to, methylamino, methylsulfonyl, and methyl sulfinyl. “Substituted heteroalkyl” refers to heteroalkyl substituted with one, two, or three groups independently selected from halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy. In some embodiments, a heteroalkyl group may comprise one, two, three, or four heteroatoms. Those of skill in the art will recognize that a 4- membered heteroalkyl may generally comprise one or two heteroatoms, a 5- or 6-memberedAttorney Docket No. : 108843.00484(SU0451WO) heteroalkyl may generally comprise one, two, or three heteroatoms, and a 7- to 10-membered heteroalkyl may generally comprise one, two, three, or four heteroatoms.

[0096] The term “heteroalkylene,” as used herein, refers to a divalent heteroalkyl, as defined herein. “Substituted heteroalkylene” refers to a divalent heteroalkyl, as defined herein, substituted as described for heteroalkyl.

[0097] The term “heterocycle” refers to a saturated, unsaturated or aromatic ring comprising one or more heteroatoms. Exemplary heteroatoms include N, O, Si, P, B, and S atoms where the nitrogen or sulfur atoms may be optionally oxidized, and the nitrogen atoms may be optionally quaternized and the remaining ring atoms of the non-aromatic ring are carbon atoms. A “heterocycle” includes 3- to 10-membered monocyclic rings, 6- to 12- membered bicyclic rings, and 6- to 12-membered bridged rings. In certain embodiments, “heterocycle” is a monovalent, monocyclic, or multicyclic fully -saturated ring system. In certain embodiments, the “heterocycle” group may be unsaturated, and / or bridged, and / or nonbridged, and / or a fused bicyclic group, and / or a spirocyclic bicyclic group. A bicyclic “heterocycle” includes any combination of ring sizes such as 4-5 fused ring systems, 5-5 fused ring systems, 5-6 fused ring systems, 6-6 fused ring systems, 5-7 fused ring systems, 6-7 fused ring systems, 5-8 fused ring systems, and 6-8 fused ring systems. In certain embodiments, the “heterocycle” group has from three to twenty, from three to fifteen, from three to twelve from three to ten, from three to eight, from four to seven, from four to eleven, or from five to six ring atoms. The “heterocycle” may be attached to a core structure at any heteroatom or carbon atom which results in the creation of a stable compound. In certain embodiments, the “heterocycle” is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include a fused or bridged or spirocyclic ring system and in which the nitrogen or sulfur atoms may be optionally oxidized, and / or the nitrogen atoms may be optionally quaternized. In some embodiments, “heterocycle” radicals include, but are not limited to, 2,5- diazabicyclo[2.2.2]octanyl, decahydroisoquinolinyl, dihydrobenzisoxazinyl, dihydrofuryl, dihydroisoindolyl, dihydropyranyl, dihydropyrazolyl, dihydropyrazinyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dioxolanyl, 1,4-dithianyl, furanonyl, imidazolidinyl, imidazolinyl, indolinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, oxazolidinonyl, oxazolidinyl, oxiranyl, piperazinyl, piperidinyl, 4- piperidonyl, pyrazolidinyl, pyrazolinyl, pyrrolidinyl, azetidinyl, pyrrolinyl, quinuclidinyl, tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydrothienyl, thiamorpholinyl, thiazolidinyl, tetrahydroquinolinyl, and 1,3,5-trithianyl. Non-limitingAttorney Docket No. : 108843.00484(SU0451WO) examples of bridged heterocycle groups include, but are not limited to, 6- azabicy clo[3.1.1 ]heptyl, 6-azabicy clo[3.1.1 ]heptyl, 1 -azabicy clo[2.2. l]heptyl, 2- azabicyclo[2.2.1]heptyl, 7-azabicyclo[2.2.1 ]heptyl, 1 -azabicy clo[2.2.2]octyl, 3- azabicy clo[3.2.1 ]octyl, and 2-oxabicyclo[3.1.1]heptyl, 2,6-dioxa-tricyclo[3.3.1.03’7]nonyl.Non-limiting examples of spirocyclic heterocycle groups include, but are not limited to, 2,8- diazaspiro[4.5]decyl; 2,7-diazaspiro[3.5]nonyl; 3,9-diazaspiro[5.5]undecyl; 3- azaspiro[5.5]undecyl; 2-oxa-6-azaspiro[3.4]octyl; 2-oxa-9-azaspiro[5.5]undecyl; 3-oxa-9- azaspiro[5.5]undecyl; 7-azaspiro[3.5]nonyl; 2-azaspiro[3.5]nonyl; 7-oxaspiro[3.5]nonyl; and 2-oxaspiro[3.5]nonyl. In certain embodiments, “heterocycle” may also be optionally substituted as described herein. In certain embodiments, “heterocycle” is substituted with one, two, or three groups independently selected from halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy. In some embodiments, a “heterocycle” group may comprise one, two, three, or four heteroatoms. Those of skill in the art will recognize that a 4-membered “heterocycle” may generally comprise one or two heteroatoms, a 5- or 6-membered “heterocycle” may generally comprise one, two, or three heteroatoms, and a 7- to 10-membered “heterocycle” may generally comprise one, two, three, or four heteroatoms.

[0098] The term “heterocycloalkylene” refers to a divalent heterocycloalkyl as defined herein.

[0099] The term “heteroaryl” refers to a monovalent, monocyclic aromatic group and / or multicyclic aromatic group, wherein at least one aromatic ring contains one or more heteroatoms independently selected from oxygen, sulfur, and nitrogen within the ring. Each ring of a heteroaryl group can contain one or two oxygen atoms, one or two sulfur atoms, and / or one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less and each ring contains at least one carbon atom. In certain embodiments, the heteroaryl has from five to twenty, from five to fifteen, or from five to ten ring atoms. A heteroaryl may be attached to the rest of the molecule via a nitrogen or a carbon atom. In some embodiments, monocyclic heteroaryl groups include, but are not limited to, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, triazolyl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl, and triazinyl. Examples of bicyclic heteroaryl groups include, but are not limited to, benzofuranyl, benzimidazolyl, benzoisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothienyl, benzotri azolyl, benzoxazolyl, furopyridyl, imidazopyridinyl, imidazothiazolyl, indolizinyl,Attorney Docket No. : 108843.00484(SU0451WO) indolyl, indazolyl, isobenzofuranyl, isobenzothienyl, isoindolyl, isoquinolinyl, naphthyridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl, pyridopyridyl, pyrrolopyridyl, quinolinyl, quinoxalinyl, quinazolinyl, thiadiazol opyrimidyl, and thi enopyridyl. Examples of tricyclic heteroaryl groups include, but are not limited to, acridinyl, benzindolyl, carbazolyl, dibenzofuranyl, perimidinyl, phenanthrolinyl, phenanthridinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxazinyl, and xanthenyl. In certain embodiments, heteroaryl may also be optionally substituted as described herein. “Substituted heteroaryl” is a heteroaryl substituted as defined for aryl.

[0100] The term “heteroarylene” refers to a divalent heteroaryl group, as defined herein. “Substituted heteroarylene” is a heteroarylene substituted as defined for aryl.

[0101] “Pharmaceutically acceptable salt” refers to any salt of a compound provided herein which retains its biological properties and which is not toxic or otherwise undesirable for pharmaceutical use. Such salts may be derived from a variety of organic and inorganic counterions well known in the art. Such salts include, but are not limited to: (1) acid addition salts formed with organic or inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic, trifluoroacetic, trichloroacetic, propionic, hexanoic, cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic, succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric, benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic, phthalic, lauric, methanesulfonic, ethanesulfonic, 1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic, camphoric, camphorsulfonic, 4-methylbicyclo[2.2.2]-oct-2-ene-l- carboxylic, glucoheptonic, 3 -phenylpropionic, trimethylacetic, tert-butyl acetic, lauryl sulfuric, gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic, cyclohexylsulfamic, quinic, muconic acid and the like acids; or (2) salts formed when an acidic proton present in the parent compound either (a) is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion or an aluminum ion, or alkali metal or alkaline earth metal hydroxides, such as sodium, potassium, calcium, magnesium, aluminum, lithium, zinc, and barium hydroxide, ammonia or (b) coordinates with an organic base, such as aliphatic, alicyclic, or aromatic organic amines, such as ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N'- dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine, N- benzylphenethylamine, / ' / -methylglucamine piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and the like.Attorney Docket No. : 108843.00484(SU0451WO)

[0102] Pharmaceutically acceptable salts further include, by way of example only and without limitation, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium and the like, and when the compound contains a basic functionality, salts of non-toxic organic or inorganic acids, such as hydrohalides, e.g. hydrochloride and hydrobromide, sulfate, phosphate, sulfamate, nitrate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate, malonate, succinate, sorbate, ascorbate, malate, maleate, fumarate, tartarate, citrate, benzoate, 3-(4- hydroxybenzoyl)benzoate, picrate, cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate), ethanesulfonate, 1,2-ethane-disulfonate, 2 -hydroxy ethanesulfonate, benzenesulfonate (besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4- toluenesulfonate, camphorate, camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-l- carboxylate, glucoheptonate, 3 -phenylpropionate, trimethylacetate, tert-butyl acetate, lauryl sulfate, gluconate, benzoate, glutamate, hydroxynaphthoate, salicylate, stearate, cyclohexylsulfamate, quinate, muconate and the like.

[0103] The term “substantially free of’ or “substantially in the absence of’ with respect to a composition refers to a composition that includes at least 85 or 90% by weight, in certain embodiments 95%, 98 %, 99% or 100% by weight, of the designated enantiomer of that compound. In certain embodiments, in the methods and compounds provided herein, the compounds are substantially free of enantiomers.

[0104] Similarly, the term “isolated” with respect to a composition refers to a composition that includes at least 85, 90%, 95%, 98%, and 99% to 100% by weight, of the compound, the remainder comprising other chemical species or enantiomers.

[0105] Solvate” refers to a compound provided herein or a salt thereof that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is a hydrate.

[0106] “Isotopic composition” refers to the amount of each isotope present for a given atom, and “natural isotopic composition” refers to the naturally occurring isotopic composition or abundance for a given atom. Atoms containing their natural isotopic composition may also be referred to herein as “non-enriched” atoms. Unless otherwise designated, the atoms of the compounds recited herein are meant to represent any stable isotope of that atom. For example, unless otherwise stated, when a position is designated specifically as “H” or “hydrogen,” the position is understood to have hydrogen at its natural isotopic composition.Attorney Docket No. : 108843.00484(SU0451WO)

[0107] “Isotopic enrichment” refers to the percentage of incorporation of an amount of a specific isotope at a given atom in a molecule in the place of that atom’s natural isotopic abundance. For example, deuterium enrichment of 1% at a given position means that 1% of the molecules in a given sample contain deuterium at the specified position. Because the naturally occurring distribution of deuterium is about 0.0156%, deuterium enrichment at any position in a compound synthesized using non-enriched starting materials is about 0.0156%. The isotopic enrichment of the compounds provided herein can be determined using conventional analytical methods known to one of ordinary skill in the art, including mass spectrometry and nuclear magnetic resonance spectroscopy.

[0108] “Isotopically enriched” refers to an atom having an isotopic composition other than the natural isotopic composition of that atom. “Isotopically enriched” may also refer to a compound containing at least one atom having an isotopic composition other than the natural isotopic composition of that atom.

[0109] As used herein, “alkyl,” “alkylene,” “alkylamino,” “dialkylamino,” “cycloalkyl,” “aryl,” “alkoxy,” “alkoxycarbonyl,” “amino,” “carboxyl,” “heterocyclyl,” “heterocycloalkyl,” “heteroaryl,” “partially saturated heteroaryl,” “carboxyl,” and “amino acid” groups optionally comprise deuterium at one or more positions where hydrogen atoms are present, and wherein the deuterium composition of the atom or atoms is other than the natural isotopic composition.

[0110] Also as used herein, “alkyl,” “alkylamino,” “dialkylamino,” “cycloalkyl,” “aryl,” “alkoxy,” “alkoxycarbonyl,” “amino,” “carboxyl,” “heterocyclyl,” “heteroaryl,” “carboxyl” and “amino acid” groups optionally comprise carbon- 13 at an amount other than the natural isotopic composition.[OHl] In some chemical structures illustrated herein, certain substituents, chemical groups, and atoms are depicted with a curvy / wavy line (e.g., ) that intersects a bond or bonds to indicate the atom through which the substituents, chemical groups, and atoms are bonded.For example, in some structures, such as but not limited to,curvy / wavy lines indicates the atoms in the backbone of a conjugate structure to which the illustrated chemical entity is bonded. In some structures, such as but not limited to,Attorney Docket No. : 108843.00484(SU0451WO), the curvy / wavy lines indicate the atoms in the antibody or antibody fragment as well as the atoms in the backbone of a conjugate or linker-payload structure to which the illustrated chemical entity is bonded.

[0112] As used herein, illustrations showing substituents bonded to a cyclic group (e.g., aromatic, heteroaromatic, fused ring, and saturated or unsaturated cycloalkyl or heterocycloalkyl) through a bond between ring atoms are meant to indicate, unless specified otherwise, that the cyclic group may be substituted with that substituent at any ring position in the cyclic group or on any ring in the fused ring group, according to techniques set forth herein or which are known in the field to which the instant disclosure pertains. For example, the group,, wherein subscripts z and y are integers and in which the positions of substituents -O-Su and RAis described generically, z.e., not directly attached to any vertex of the bond line structure, z.e., specific ring carbon atom, includes the following, non-limiting examples of groups in which the substituent RAand Osu is bonded to a specific ring carbon atom:Attorney Docket No. : 108843.00484(SU0451WO)Antibodies

[0113] Provided herein are antibodies comprising one or more non-natural amino acid residues at one or more site-specific positions in the amino acid sequence of, collectively, one or more polypeptide chains. These positions are selected based on advantageous properties of the antibodies having non-natural amino acids at these positions. The advantageous properties can relate to production yield, conjugation, solubility, binding and / or advantageous activity.

[0114] In certain embodiments, the antibody comprises one non-natural amino acids at a site-specific position. In certain embodiments, the antibody comprises two or more non-natural amino acids at site-specific positions. In certain embodiments, the antibody comprises three or more non-natural amino acids at site-specific positions. In certain embodiments, the antibody comprises four or more non-natural amino acids at site-specific positions. In certain embodiments, the antibody comprises five or more non-natural amino acids at site-specific positions. In certain embodiments, the antibody comprises six or more non-natural amino acids at site-specific positions. In certain embodiments, the antibody comprises seven or more non- natural amino acids at site-specific positions. In certain embodiments, the antibody comprises eight or more non-natural amino acids at site-specific positions. In certain embodiments, the antibody comprises nine or more non-natural amino acids at site-specific positions. In certain embodiments, the antibody comprises ten or more non-natural amino acids at site-specific positions. In certain embodiments, the antibody comprises eleven or more non-natural amino acids at site-specific positions. In certain embodiments, the antibody comprises twelve or more non-natural amino acids at site-specific positions.

[0115] In certain embodiments, each non-natural amino acid residue is independently at a specific site selected from the group consisting of optimally substitutable positions of anyAttorney Docket No. : 108843.00484(SU0451WO) polypeptide chain of said antibody. These optimally substitutable positions are described in detail below. Exemplary optimally substitutable positions are also described below.

[0116] In certain embodiments, the antibody comprises one to twelve non-natural amino acids at site-specific positions. In certain embodiments, the antibody comprises three to twelve non-natural amino acids at site-specific positions. In certain embodiments, the antibody comprises four to twelve non-natural amino acids at site-specific positions. In certain embodiments, the antibody comprises five to twelve non-natural amino acids at site-specific positions. In certain embodiments, the antibody comprises six to twelve non-natural amino acids at site-specific positions. In certain embodiments, the antibody comprises seven to twelve non-natural amino acids at site-specific positions. In certain embodiments, the antibody comprises eight to twelve non-natural amino acids at site-specific positions. In certain embodiments, the antibody comprises nine to twelve non-natural amino acids at site-specific positions. In certain embodiments, the antibody comprises ten to twelve non-natural amino acids at site-specific positions. In certain embodiments, the antibody comprises eleven to twelve non-natural amino acids at site-specific positions.

[0117] In certain embodiments, the antibody comprises one site-specific non-natural amino acid residue in a single light chain polypeptide. In certain embodiments, the antibody comprises two or more site-specific non-natural amino acid residues in a single light chain polypeptide. In certain embodiments, the antibody comprises three or more site-specific non-natural amino acid residues in a single light chain polypeptide. In certain embodiments, the antibody comprises four or more site-specific non-natural amino acid residues in a single light chain polypeptide. In certain embodiments, the antibody comprises five or more site-specific non- natural amino acid residues in a single light chain polypeptide. In certain embodiments, the antibody comprises six or more site-specific non-natural amino acid residues in a single light chain polypeptide. In certain embodiments, the antibody comprises seven or more site-specific non-natural amino acid residues in a single light chain polypeptide. In certain embodiments, the antibody comprises eight or more site-specific non-natural amino acid residues in a single light chain polypeptide. In certain embodiments, the antibody comprises nine or more sitespecific non-natural amino acid residues in a single light chain polypeptide. In certain embodiments, the antibody comprises ten or more site-specific non-natural amino acid residues in a single light chain polypeptide. In certain embodiments, the antibody comprises eleven or more site-specific non-natural amino acid residues in a single light chain polypeptide. In certainAttorney Docket No. : 108843.00484(SU0451WO) embodiments, the antibody comprises twelve or more site-specific non-natural amino acid residues in a single light chain polypeptide.

[0118] In certain embodiments, the antibody comprises two to twelve site-specific nonnatural amino acid residues in a single light chain polypeptide. In certain embodiments, the antibody comprises three to twelve site-specific non-natural amino acid residues in a single light chain polypeptide. In certain embodiments, the antibody comprises four to twelve sitespecific non-natural amino acid residues in a single light chain polypeptide. In certain embodiments, the antibody comprises five to twelve site-specific non-natural amino acid residues in a single light chain polypeptide. In certain embodiments, the antibody comprises six to twelve site-specific non-natural amino acid residues in a single light chain polypeptide. In certain embodiments, the antibody comprises seven to twelve site-specific non-natural amino acid residues in a single light chain polypeptide. In certain embodiments, the antibody comprises eight to twelve site-specific non-natural amino acid residues in a single light chain polypeptide. In certain embodiments, the antibody comprises nine to twelve site-specific non- natural amino acid residues in a single light chain polypeptide. In certain embodiments, the antibody comprises ten to twelve site-specific non-natural amino acid residues in a single light chain polypeptide. In certain embodiments, the antibody comprises eleven to twelve sitespecific non-natural amino acid residues in a single light chain polypeptide.

[0119] In certain embodiments, the antibody comprises one site-specific non-natural amino acid residue in a single heavy chain polypeptide. In certain embodiments, the antibody comprises two or more site-specific non-natural amino acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises three or more site-specific non- natural amino acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises four or more site-specific non-natural amino acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises five or more site-specific non-natural amino acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises six or more site-specific non-natural amino acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises seven or more sitespecific non-natural amino acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises eight or more site-specific non-natural amino acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises nine or more site-specific non-natural amino acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises ten or more site-specific non-natural aminoAttorney Docket No. : 108843.00484(SU0451WO) acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises eleven or more site-specific non-natural amino acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises twelveor more site-specific nonnatural amino acid residues in a single heavy chain polypeptide.

[0120] In certain embodiments, the antibody comprises two to twelve site-specific nonnatural amino acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises three to twelve site-specific non-natural amino acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises four to twelve sitespecific non-natural amino acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises five to twelve site-specific non-natural amino acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises six to twelve site-specific non-natural amino acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises seven to twelve site-specific non-natural amino acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises eight to twelve site-specific non-natural amino acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises nine to twelve site-specific non- natural amino acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises ten to twelve site-specific non-natural amino acid residues in a single heavy chain polypeptide. In certain embodiments, the antibody comprises eleven to twelve sitespecific non-natural amino acid residues in a single heavy chain polypeptide.

[0121] In certain embodiments, the antibody comprises at least one site-specific non- natural amino acid in a light chain polypeptide and at least one site-specific non-natural amino acid in a heavy chain polypeptide. In certain embodiments, the antibody comprises at least two site-specific non-natural amino acids in a light chain polypeptide and at least two site-specific non-natural amino acids in a heavy chain polypeptide. In certain embodiments, the antibody comprises at least three site-specific non-natural amino acids in a light chain polypeptide and at least three site-specific non-natural amino acids in a heavy chain polypeptide. In certain embodiments, the antibody comprises at least four site-specific non-natural amino acids in a light chain polypeptide and at least four site-specific non-natural amino acids in a heavy chain polypeptide. In certain embodiments, the antibody comprises at least five site-specific non- natural amino acids in a light chain polypeptide and at least five site-specific non-natural amino acids in a heavy chain polypeptide. In certain embodiments, the antibody comprises at least sixAttorney Docket No. : 108843.00484(SU0451WO) site-specific non-natural amino acids in a light chain polypeptide and at least six site-specific non-natural amino acids in a heavy chain polypeptide.

[0122] In certain embodiments, the antibody comprises at least one site-specific non- natural amino acid in each of two light chain polypeptides and at least one site-specific non- natural amino acid in a heavy chain polypeptide. In certain embodiments, the antibody comprises at least one site-specific non-natural amino acid in each of two light chain polypeptides and at least one site-specific non-natural amino acid in each of two heavy chain polypeptides. In certain embodiments, the antibody comprises at least two site-specific non- natural amino acids in each of two light chain polypeptides and at least one site-specific non- natural amino acid in each of two heavy chain polypeptides. In certain embodiments, the antibody comprises at least one site-specific non-natural amino acid in each of two light chain polypeptides and at least two site-specific non-natural amino acids in each of two heavy chain polypeptides. In certain embodiments, the antibody comprises at least two site-specific non- natural amino acids in each of two light chain polypeptides and at least two site-specific non- natural amino acids in each of two heavy chain polypeptides. In certain embodiments, the antibody comprises at least three site-specific non-natural amino acids in each of two light chain polypeptides and at least two site-specific non-natural amino acids in each of two heavy chain polypeptides. In certain embodiments, the antibody comprises at least two site-specific non-natural amino acids in each of two light chain polypeptides and at least three site-specific non-natural amino acids in each of two heavy chain polypeptides. In certain embodiments, the antibody comprises at least three site-specific non-natural amino acids in each of two light chain polypeptides and at least three site-specific non-natural amino acids in each of two heavy chain polypeptides.

[0123] The antibodies provided herein can be of any class or type known to those of skill in the art. In certain embodiments, the antibody can comprise a heavy chain of any type known to those of skill in the art. In certain embodiments, the antibody comprises a heavy chain of a type selected from the group consisting of a, y, 5, a and p. In certain embodiments, the antibody comprises an a heavy chain. In certain embodiments, the antibody comprises an y heavy chain. In certain embodiments, the antibody comprises a 5 heavy chain. In certain embodiments, the antibody comprises a a heavy chain. In certain embodiments, the antibody comprises a p heavy chain.

[0124] In certain embodiments, the antibody can comprise a light chain of any type known to those of skill in the art. In certain embodiments, the antibody comprises a light chain of aAttorney Docket No. : 108843.00484(SU0451WO) type selected from the group consisting of X and K. In certain embodiments, the antibody comprises a X light chain. In certain embodiments, the antibody comprises a K light chain.

[0125] Any of the above antibodies can be of any class known to those of skill in the art. In certain embodiments, the antibody is of a class or subclass selected from the group consisting of IgA, IgAl, IgA2, IgD, IgE, IgG, IgGl, IgG2, IgG3 and IgM. In certain embodiments, the antibody is an IgA antibody. In certain embodiments, the antibody is an IgAl or an IgA2 antibody. In certain embodiments, the antibody is an IgD antibody. In certain embodiments, the antibody is an IgE antibody. In certain embodiments, the antibody is an IgG antibody. In certain embodiments, the antibody is an IgGl, IgG2 or IgG3 antibody. In certain embodiments, the antibody is an IgM antibody.

[0126] The antibody can be of any antibody form known to those of skill in the art. In certain embodiments, the antibody is an antibody fragment recognized by those of skill in the art. In certain embodiments, the antibody is an Fv, Fc, Fab or (Fab')2 antibody. In certain embodiments, the antibody is a single chain Fv antibody (scFv). In certain embodiments, the antibody is in the form of Fv, Fc, Fab, (Fab1)?, single chain Fv (scFv) and / or scFv-Fc.

[0127] The antibody can share high sequence identity with any antibody recognized by those of skill in the art, i.e. a parent antibody. In certain embodiments, the amino acid sequence of the antibody is identical to the amino acid sequence of the parent antibody, other than the non-natural amino acids at site-specific position. In further embodiments, the antibody provided herein can have one or more insertions, deletions or mutations relative to the parent antibody in addition to the one or more non-natural amino acids at the site-specific positions. In certain embodiments, the antibody provided herein can have a unique primary sequence, so long as it would be recognized as an antibody by those of skill in the art.

[0128] The antibody is typically a protein comprising multiple polypeptide chains. In certain embodiments, the antibody is a heterotetramer comprising two identical light (L) chains and two identical heavy (H) chains. Each light chain can be linked to a heavy chain by one covalent disulfide bond. Each heavy chain can be linked to the other heavy chain by one or more covalent disulfide bonds. Each heavy chain and each light chain can also have one or more intrachain disulfide bonds. As is known to those of skill in the art, each heavy chain typically comprises a variable domain (VH) followed by a number of constant domains. Each light chain typically comprises a variable domain at one end (VL) and a constant domain. As is known to those of skill in the art, antibodies typically have selective affinity for their target molecules, i.e. antigens.Attorney Docket No. : 108843.00484(SU0451WO)

[0129] The non-natural amino acids are positioned at select locations in a polypeptide chain of the antibody. These locations were identified as providing optimum sites for substitution with the non-natural amino acids. Each site is capable of bearing a non-natural amino acid with optimum structure, function and / or methods for producing the antibody.

[0130] In certain embodiments, the one or more site-specific positions for substitution herein provides an antibody that is capable of advantageous conjugation. As described below, several non-natural amino acids have side chains or functional groups that facilitate conjugation of the antibody to a second agent, either directly or via a linker. In certain embodiments, the antibody can show enhanced conjugation efficiency compared to an antibody without the same or other non-natural amino acids at other positions. In certain embodiments, the antibody can show enhanced conjugation yield compared to an antibody without the same or other non-natural amino acids at other positions. In certain embodiments, the antibody can show enhanced conjugation specificity compared to an antibody without the same or other non- natural amino acids at other positions.

[0131] In certain embodiments, the one or more site-specific positions for substitution herein provides an antibody that is stable. Stability can be measured by any technique apparent to those of skill in the art. In certain embodiments, the substituted antibody or conjugate has a melting temperature that is within about 5 °C of the corresponding parent antibody, as described herein. In certain embodiments, the substituted antibody or conjugate has a melting temperature that is within about 4 °C of the corresponding parent antibody, as described herein. In certain embodiments, the substituted antibody or conjugate has a melting temperature that is within about 3 °C of the corresponding parent antibody, as described herein. In certain embodiments, the substituted antibody or conjugate has a melting temperature that is within about 2 °C of the corresponding parent antibody, as described herein. In certain embodiments, the substituted antibody or conjugate has a melting temperature that is within about 1 °C of the corresponding parent antibody, as described herein. In certain embodiments, the substituted antibody or conjugate has a melting temperature that is at least about 5 °C greater than the corresponding parent antibody, as described herein. In certain embodiments, the substituted antibody or conjugate has a melting temperature that is at least about 4 °C greater than the corresponding parent antibody, as described herein. In certain embodiments, the substituted antibody or conjugate has a melting temperature that is at least about 3 °C greater than the corresponding parent antibody, as described herein. In certain embodiments, the substituted antibody or conjugate has a melting temperature that is at least about 2 °C greater than theAttorney Docket No. : 108843.00484(SU0451WO) corresponding parent antibody, as described herein. In certain embodiments, the substituted antibody or conjugate has a melting temperature that is at least about 1 °C greater than the corresponding parent antibody, as described herein. The melting temperature can be Tml, Tm2 or both Tml and Tm2 as will be recognized by those of skill in the art.

[0132] In certain embodiments, the one or more site-specific positions for substitution herein provides an antibody that has optimal functional properties. For instance, the antibody can show little or no loss of binding affinity for its target antigen compared to an antibody without the site-specific non-natural amino acid. In certain embodiments, the antibody can show enhanced binding compared to an antibody without the site-specific non-natural amino acid.

[0133] In certain embodiments, the one or more site-specific positions for substitution herein provide an antibody that can be made advantageously. For instance, in certain embodiments, the antibody shows advantageous properties in its methods of synthesis, discussed below. In certain embodiments, the antibody can show little or no loss in yield in production compared to an antibody without the site-specific non-natural amino acid. In certain embodiments, the antibody can show enhanced yield in production compared to an antibody without the site-specific non-natural amino acid. In certain embodiments, the antibody can show little or no loss of tRNA suppression, described below, compared to an antibody without the site-specific non-natural amino acid. In certain embodiments, the antibody can show enhanced tRNA suppression, described below, in production compared to an antibody without the site-specific non-natural amino acid.

[0134] In certain embodiments, the one or more site-specific positions for substitution herein provide an antibody that has advantageous solubility. In certain embodiments, the antibody can show little or no loss in solubility compared to an antibody without the sitespecific non-natural amino acid. In certain embodiments, the antibody can show enhanced solubility compared to an antibody without the site-specific non-natural amino acid.

[0135] In certain embodiments, the one or more site-specific positions for substitution herein provide an antibody that has advantageous expression. In certain embodiments, the antibody can show little or no loss in expression compared to an antibody without the sitespecific non-natural amino acid. In certain embodiments, the antibody can show enhanced expression compared to an antibody without the site-specific non-natural amino acid.

[0136] In certain embodiments, the one or more site-specific positions for substitution herein provide an antibody that has advantageous folding. In certain embodiments, the antibodyAttorney Docket No. : 108843.00484(SU0451WO) can show little or no loss in proper folding compared to an antibody without the site-specific non-natural amino acid. In certain embodiments, the antibody can show enhanced folding compared to an antibody without the site-specific non-natural amino acid.

[0137] The one or more non-natural amino acids are located at selected site-specific positions in at least one polypeptide chain of the antibody. The polypeptide chain can be any polypeptide chain of the antibody without limitation, including either light chain or either heavy chain. The site-specific position can be in any domain of the antibody, including any variable domain and any constant domain.

[0138] In certain embodiments, the antibodies provided herein comprise one non-natural amino acid at a site-specific position. In certain embodiments, the antibodies provided herein comprise two non-natural amino acids at site-specific positions. In certain embodiments, the antibodies provided herein comprise three non-natural amino acids at site-specific positions. In certain embodiments, the antibodies provided herein comprise more than three non-natural amino acids at site-specific positions.

[0139] In some embodiments, provided herein are antibodies comprising one or more non- natural amino acid residues at one or more positions in Table 4A. In some examples, an antibody provided herein comprises one non-natural amino acid residue at one of the positions in Table 4A. In some examples, an antibody provided herein comprises one or more non- natural amino acid residues at two of the positions in Table 4A. In some examples, an antibody provided herein comprises one or more non-natural amino acid residues at three of the positions in Table 4A. In some examples, an antibody provided herein comprises one or more non- natural amino acid residues at four of the positions in Table 4. In some examples, an antibody provided herein comprises one or more non-natural amino acid residues at five of the positions in Table 4A. In some examples, an antibody provided herein comprises one or more non- natural amino acid residues at six of the positions in Table 4A. In some examples, an antibody provided herein comprises one or more non-natural amino acid residues at seven of the positions in Table 4A. In some examples, an antibody provided herein comprises one or more non-natural amino acid residues at eight of the positions in Table 4A.Attorney Docket No. : 108843.00484(SU0451WO)Table 4A Examples of site-specific positions for amino acid substitutionAttorney Docket No. : 108843.00484(SU0451WO)Attorney Docket No. : 108843.00484(SU0451WO)Attorney Docket No. : 108843.00484(SU0451WO)Table 4B. Additional examples of site-specific positions for amino acid substitution

[0140] In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-S113 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-A118 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-T120 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-K121 according to EU index of Kabat. In some embodiments, an antibodyAttorney Docket No. : 108843.00484(SU0451WO) provided herein comprises a non-natural amino acid residue at the position HC-G138 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a nonnatural amino acid residue at the position HC-A140 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-T155 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-S160 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non- natural amino acid residue at the position HC-A162 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-L163 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-Q 196 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non- natural amino acid residue at the position HC-I199 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-N203 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-T209 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non- natural amino acid residue at the position HC-K210 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-K222 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-K246 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non- natural amino acid residue at the position HC-E258 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-K290 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-E294 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non- natural amino acid residue at the position HC-Q295 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-Y296 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-N297 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non- natural amino acid residue at the position HC-Y300 according to EU index of Kabat. In someAttorney Docket No. : 108843.00484(SU0451WO) embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-V303 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-V305 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non- natural amino acid residue at the position HC-E318 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-A330 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-T335 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non- natural amino acid residue at the position HC-I336 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-S337 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-R344 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non- natural amino acid residue at the position HC-E345 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-R355 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-M358 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-Q362 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-E382 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-E388 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non- natural amino acid residue at the position HC-N390 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-Y391 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-K392 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non- natural amino acid residue at the position HC-T393 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-T411 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC- N421Attorney Docket No. : 108843.00484(SU0451WO) according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-S424 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-Q438 according to EU index of Kabat. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position HC-L443 according to EU index of Kabat.

[0141] In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-T109 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-V110 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-A111 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-A112 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-D122 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non- natural amino acid residue at the position LC-Q124 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-K126 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-S127 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-T129 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-N138 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-R142 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-E143 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-Q147 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non- natural amino acid residue at the position LC-K149 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-D151 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-A153 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural aminoAttorney Docket No. : 108843.00484(SU0451WO) acid residue at the position LC-L154 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-Q155 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-S159 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-E165 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-Q166 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non- natural amino acid residue at the position LC-S168 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-K169 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-S171 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-T172 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-T180 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-A184 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-E187 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-H189 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non- natural amino acid residue at the position LC-K190 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-V191 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-E195 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-L201 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-S202 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-S203 according to Kabat numbering. In some embodiments, an antibody provided herein comprises a non-natural amino acid residue at the position LC-V205 according to Kabat numbering. In some embodiments, an antibodyAttorney Docket No. : 108843.00484(SU0451WO) provided herein comprises a non-natural amino acid residue at the position LC-T206 according to Kabat numbering.

[0142] In some embodiments, an antibody herein further comprises one or more nonnatural amino acid at one or more positions selected from HC-F404, HC-K121, HC-Y180, HC- F241, HC-221, LC-T22, LC-S7, LC-N152, LC-K42, LC-E161, LC-D170, HC-S136, HC-S25, HC-A40, HC-S119, HC-S190, HC-K222, HC-R19, HC-Y52, or HC-S70 according to EU index of Kabat.

[0143] In some embodiments, an antibody herein further comprises one or more nonnatural amino acid at one or more positions selected from HC-Y180, HC-F241, HC-F404 HC, or LC-K42 according to EU index of Kabat. In some embodiments, an antibody herein further comprises non-natural amino acids at HC-Y180, HC-F241, HC-F404 HC, and LC-K42 according to EU index of Kabat.

[0144] In some embodiments, an antibody herein further comprises one or more non- natural amino acid at one or more positions selected from HC-Y180, HC-F241, or HC-F404 HC according to EU index of Kabat. In some embodiments, an antibody herein further comprises non-natural amino acids at HC-Y180, HC-F241, and HC-F404 HC according to EU index of Kabat.

[0145] In some embodiments, the antibody herein comprises a mutation at V264, e.g., a V264S mutation.

[0146] In some embodiments, an antibody herein further comprises one or more non- natural amino acids at one or more positions as described in WO2017218698, WO2015006555, W02014004639, and WO2013185115, each of which is incorporated by reference herein in its entirety.

[0147] The site-specific positions can also be identified relative to the amino acid sequences of the polypeptide chains of a reference antibody. For example, the amino acid sequence of a reference heavy chain is provided at SEQ ID NO: 7 and the amino acid sequence of a reference light chain is provided at SEQ ID NO: 12. The site-specific positions in the heavy chain and light chain reference sequences are shown in Table 4.

[0148] In certain embodiments, the antibody comprises a polypeptide chain that can be described by the following formula (I):Xaa1- . . . -(Naap(1))n- . . . -Xaaq(I).

[0149] In Formula (I), each Xaa represents an amino acid in the polypeptide chain of any identity. In other words, each Xaa can be any amino acid, typically any naturally occurringAttorney Docket No. : 108843.00484(SU0451WO) amino acid, or a variant thereof. The superscript to the right of each Xaa represents the position of the amino acid within the primary sequence of the polypeptide chain. Xaa1represents the first, or N-terminal, amino acid in the polypeptide chain, and Xaaqrepresents the last, or C- terminal, amino acid in the polypeptide chain. The variable q is an integer that is equal to the total number of amino acids in the polypeptide chain. In some embodiments, each Naa represents a non-natural amino acid within the polypeptide chain. Examples of useful nonnatural amino acids are described in the sections below. The integer n represents the number of non-natural amino acids in the polypeptide chain, n may be an integer of 1 or greater than 1. In some embodiments, each integer p(i) is greater than 1 and less than q, and the variable i is an integer that varies from 1 to n. In some embodiments, each integer p(i) represents a sitespecific location in the amino acid sequence for the corresponding Naa. In some embodiments, each site-specific location p(i) is optimal for substitution of a naturally occurring amino acid with a non-natural amino acid, such as Naap(1), according to the techniques described herein.

[0150] In certain embodiments, further provided herein are conservatively modified variants of the above antibodies. Conservatively modified variants of an antibody include one or more insertions, deletions or substitutions that do not disrupt the structure and / or function of the antibody when evaluated by one of skill in the art. In certain embodiments, conservatively modified variants include 20 or fewer amino acid insertions, deletions, or substitutions. In certain embodiments, conservatively modified variants include 15 or fewer amino acid insertions, deletions, or substitutions. In certain embodiments, conservatively modified variants include 10 or fewer amino acid insertions, deletions, or substitutions. In certain embodiments, conservatively modified variants include 9 or fewer amino acid insertions, deletions, or substitutions. In certain embodiments, conservatively modified variants include 8 or fewer amino acid insertions, deletions or substitutions. In certain embodiments, conservatively modified variants include 7 or fewer amino acid insertions, deletions, or substitutions. In certain embodiments, conservatively modified variants include 6 or fewer amino acid insertions, deletions, or substitutions. In certain embodiments, conservatively modified variants include 5 or fewer amino acid insertions, deletions, or substitutions. In certain embodiments, conservatively modified variants include 4 or fewer amino acid insertions, deletions, or substitutions. In certain embodiments, conservatively modified variants include 3 or fewer amino acid insertions, deletions, or substitutions. In certain embodiments, conservatively modified variants include 2 or fewer amino acid insertions, deletions, or substitutions. In certain embodiments, conservatively modifiedAttorney Docket No. : 108843.00484(SU0451WO) variants include 1 amino acid insertion, deletion, or substitution. In particular embodiments the substitutions are conservative, substituting an amino acid within the same class, as described above.

[0151] In certain embodiments, the antibodies can be modified to modulate structure, stability and / or activity. In such embodiments, the modifications can be conservative or other than conservative. The modifications need only be suitable to the practitioner carrying out the methods and using the compositions described herein. In certain embodiments, the modifications decrease but do not eliminate antigen binding affinity. In certain embodiments, the modifications increase antigen binding affinity. In certain embodiments, the modifications enhance structure or stability of the antibody. In certain embodiments, the modifications reduce but do not eliminate structure or stability of the antibody. In certain embodiments, modified variants include 20 or fewer amino acid insertions, deletions or substitutions. In certain embodiments, modified variants include 15 or fewer amino acid insertions, deletions or substitutions. In certain embodiments, modified variants include 10 or fewer amino acid insertions, deletions or substitutions. In certain embodiments, modified variants include 9 or fewer amino acid insertions, deletions or substitutions. In certain embodiments, modified variants include 8 or fewer amino acid insertions, deletions or substitutions. In certain embodiments, modified variants include 7 or fewer amino acid insertions, deletions or substitutions. In certain embodiments, modified variants include 6 or fewer amino acid insertions, deletions or substitutions. In certain embodiments, modified variants include 5 or fewer amino acid insertions, deletions or substitutions. In certain embodiments, modified variants include 4 or fewer amino acid insertions, deletions or substitutions. In certain embodiments, modified variants include 3 or fewer amino acid insertions, deletions or substitutions. In certain embodiments, modified variants include 2 or fewer amino acid insertions, deletions or substitutions. In certain embodiments, modified variants include 1 amino acid insertion, deletion or substitution.

[0152] Also within the scope are post-translationally modified variants. Any of the antibodies provided herein can be post-translationally modified in any manner recognized by those of skill in the art. Typical post-translational modifications for antibodies include interchain disulfide bonding, intrachain disulfide bonding, N-linked glycosylation and proteolysis. Also provided herein are other post-translationally modified antibodies having modifications such as phosphorylation, O-linked glycosylation, methylation, acetylation, lipidation, GPI anchoring, myristoylation, and prenylation. The post-translational modificationAttorney Docket No. : 108843.00484(SU0451WO) can occur during production, in vivo, in vitro or otherwise. In certain embodiments, the post- translational modification can be an intentional modification by a practitioner, for instance, using the methods provided herein.

[0153] Further included within the scope are antibodies fused to further peptides or polypeptides. Exemplary fusions include, but are not limited to, e.g., a methionyl antibody in which a methionine is linked to the N-terminus of the antibody resulting from the recombinant expression, fusions for the purpose of purification (including but not limited to, to polyhistidine or affinity epitopes), fusions for the purpose of linking to other biologically active molecules, fusions with serum albumin binding peptides, and fusions with serum proteins such as serum albumin. The antibodies may comprise protease cleavage sequences, reactive groups, antibody-binding domains (including but not limited to, FLAG or poly-His) or other affinitybased sequences (including but not limited to, FLAG, poly-His, GST, etc.). The antibodies may also comprise linked molecules (including but not limited to, biotin) that improve detection (including but not limited to, GFP), purification or other features of the antibody. In certain embodiments, the antibodies comprise a C-terminal affinity sequence that facilitates purification of full-length antibodies. In certain embodiments, such C-terminal affinity sequence is a poly-His sequence, e.g., a 6-His sequence.

[0154] The antibody can have any antibody form recognized by those of skill in the art. The antibody can comprise a single polypeptide chain - a single heavy chain or a single light chain. The antibody can also form multimers that will be recognized by those of skill in the art including homodimers, heterodimers, homomultimers, and heteromultimers. These multimers can be linked or unlinked. Useful linkages include interchain disulfide bonds typical for antibody molecules. The multimers can also be linked by other amino acids, including the nonnatural amino acids introduced according to the present description. The antibody can be an immunoglobulin such as of any class or subclass including IgA, IgAl, IgA2, IgD, IgE, IgG, IgGl, IgG2, IgG3, IgG4 and IgM. The antibody can be of the form of any antibody fragment including Fv, Fc, Fab, and (Fab')2 and scFv.

[0155] Also provided herein are antibodies that are conjugated to one or more conjugation moieties. The conjugation moiety can be any conjugation moiety deemed useful to one of skill in the art. For instance, the conjugation moiety can be a polymer, such as polyethylene glycol, that can improve the stability of the antibody in vitro or in vivo. The conjugation moiety can have therapeutic activity, thereby yielding an antibody-drug conjugate. The conjugation moiety can be a molecular payload that is harmful to target cells. The conjugation moiety can be aAttorney Docket No. : 108843.00484 (SU0451WO) label useful for detection or diagnosis. In certain embodiments, the conjugation moiety is linked to the antibody via a direct covalent bond. In certain embodiments, the conjugation moiety is linked to the antibody via a linker. In advantageous embodiments, the conjugation moiety or the linker is attached via one of the non-natural amino acids of the antibody. Exemplary conjugation moieties and linkers are discussed in the sections below. Methods of introducing a non-natural amino acid into an antibody are described, for example, in U. S. Patent Publication No. 2015-0017187 Al, which is incorporated herein by reference in its entirety. Exemplary conjugation moieties and linkers are also described in U.S. Patent Publication No. 2015-001787 Al.Non-natural amino acids

[0156] The non-natural amino acids can be any non-natural amino acids known to those of skill in the art. In some embodiments, a non-natural amino acid comprises a functional group. The functional group can be any functional group known to those of skill in the art. In certain embodiments the functional group is a label, a polar group, a non-polar group or a reactive group.

[0157] Reactive groups are particularly advantageous for linking further functional groups to the antibody at the site-specific position of the antibody chain. In certain embodiments, the reactive group is selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, tetrazine and alkynyl.

[0158] In certain embodiments, the non-natural amino acid is according to any of the following formulas:wherein each L is independently a divalent linker; and each R is independently a functional group.

[0159] Those of skill in the art will recognize that antibodies are generally comprised of L- amino acids. However, with non-natural amino acids, the present methods and compositions provide the practitioner with the ability to use L-, D- or racemic non-natural amino acids at the site-specific positions. In certain embodiments, the non-natural amino acids described herein include D- versions of the natural amino acids and racemic versions of the natural amino acids.

[0160] In the above formulas, the wavy lines indicate bonds that connect to the remainder of the polypeptide chains of the antibodies. These non-natural amino acids can be incorporatedAttorney Docket No. : 108843.00484(SU0451WO) into polypeptide chains just as natural amino acids are incorporated into the same polypeptide chains. In certain embodiments, the non-natural amino acids are incorporated into the polypeptide chain via amide bonds as indicated in the formulas.

[0161] In the above formulas R designates any functional group without limitation, so long as the amino acid residue is not identical to a natural amino acid residue. In certain embodiments, R can be a hydrophobic group, a hydrophilic group, a polar group, an acidic group, a basic group, a chelating group, a reactive group, a therapeutic moiety or a labeling moiety. In certain embodiments, R is selected from the group consisting of R1NR2R3, R1C(=O)R2, R1C(=O)OR2, R'N3, RJC(=CH). In these embodiments, R1is selected from the group consisting of a bond, alkylene, heteroalkylene, arylene, heteroarylene. R2andR3are each independently selected from the group consisting of hydrogen, alkyl and heteroalkyl.

[0162] In some embodiments, the non-natural 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, azido, ketone, aldehyde and aminooxy groups) to form stable conjugates. For example, an antigen-binding polypeptide that includes a non- natural amino acid containing an azido functional group can be reacted with a polymer (including but not limited to, polyethylene glycol)) or, alternatively, a second polypeptide containing an alkyne moiety to form a stable conjugate resulting from the selective reaction of the azide and the alkyne functional groups to form a Huisgen [3+2] cycloaddition product. An antigen-binding polypeptide that includes a non-natural amino acid containing a tetrazine functional group can be reacted with a polymer (including but not limited to, polyethylene glycol)) containing a strained alkene moiety to form a stable conjugate resulting from the selective reaction of the tetrazine and strained alkene. Alternatively, a second polypeptide containing a strained alkene moiety may be reacted with the amino acid containing tetrazine functionality to form a stable conjugate resulting from the selective reaction of the tetrazine and strained alkene.

[0163] Exemplary non-natural amino acids that may be suitable for use in the present invention and that are useful for reactions with water soluble polymers include, but are not limited to, those with carbonyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactive groups. In some embodiments, non-natural amino acids comprise a saccharide 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 includeAttorney Docket No. : 108843.00484(SU0451WO) examples where the naturally-occurring N- or O-linkage between the amino acid and the saccharide is replaced by a covalent linkage not commonly found in nature-including but not limited to, an alkene, an oxime, a thioether, an amide and the like. Examples of such amino acids also include saccharides that are not commonly found in naturally-occurring proteins such as 2-deoxy-glucose, 2-deoxygalactose and the like.

[0164] Many of the non-natural amino acids provided herein are commercially available, e.g., from Sigma-Aldrich (St. Louis, Mo., USA), Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), or Peptech (Burlington, Mass., USA). Those that are not commercially available are optionally synthesized as provided herein or using standard methods known to those of skill in the art. For organic synthesis techniques, see, e.g., 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 Application Publications 2003 / 0082575 and 2003 / 0108885, which is incorporated by reference herein. In addition to non-natural amino acids that contain novel side chains, non-natural amino acids that may be suitable for use in the present invention also optionally comprise modified backbone structures, including but not limited to, as illustrated by the structures of Formula II and III:wherein Z typically comprises OH, NH2, SH, NH-R', or S-R'; X and Y, which can be the same or different, typically comprise S or O, and R and R', which are optionally the same or different, are typically selected from the same list of constituents for the R group described above for the non-natural amino acids having Formula I as well as hydrogen. For example, non-natural amino acids optionally comprise substitutions in the amino or carboxyl group as illustrated by Formulas II and III. Unnatural amino acids of this type include, but are not limited to, a- hydroxy acids, a-thioacids, a-aminothiocarboxylates, including but not limited to, with side chains corresponding to the common twenty natural amino acids or unnatural side chains. In addition, substitutions at the a-carbon optionally include, but are not limited to, L, D, or a-a-Attorney Docket No. : 108843.00484(SU0451WO) disubstituted amino acids such as D-glutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Other structural alternatives include cyclic amino acids, such as proline analogues as well as 3, 4, 6, 7, 8, and 9 membered ring proline analogues, P and y amino acids such as substituted P-alanine and y-amino butyric acid.

[0165] Many non-natural amino acids are based on natural amino acids, such as tyrosine, glutamine, phenylalanine, and the like, and are suitable for use in the present invention. Tyrosine analogs include, but are not limited to, para-substituted tyrosines, ortho- substituted tyrosines, and meta substituted tyrosines, where the substituted tyrosine comprises, including but not limited to, a keto group (including but not limited to, an acetyl group), a benzoyl group, an amino group, a hydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropyl group, a methyl group, a C6-C20 straight chain or branched hydrocarbon, a saturated or unsaturated hydrocarbon, an O-methyl group, a polyether group, a nitro group, an alkynyl group or the like. In addition, multiply substituted aryl rings are also contemplated. Glutamine analogs that may be suitable for use in the present invention include, but are not limited to, a- hydroxy derivatives, y-substituted derivatives, cyclic derivatives, and amide substituted glutamine derivatives. Example phenylalanine analogs that may be suitable for use in the present invention include, but are not limited to, para-substituted phenylalanines, orthosubstituted phenylalanines, and meta-substituted phenylalanines, where the substituent comprises, including but not limited to, a hydroxy group, a methoxy group, a methyl group, an allyl group, an aldehyde, an azido, an iodo, a bromo, a keto group (including but not limited to, an acetyl group), a benzoyl, an alkynyl group, or the like. Specific examples of non-natural amino acids that may be suitable for use in the present invention include, but are not limited to, a p -acetyl -L-phenyl alanine, an O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3- methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl- GlcNAcP-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p- azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L- phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, a p- bromophenylalanine, a p-amino-L-phenylalanine, an isopropyl-L-phenylalanine, and a p- propargyloxy-phenylalanine, and the like. Examples of structures of a variety of non-natural amino acids that may be suitable for use in the present invention are provided in, for example, WO 2002 / 085923 entitled "In vivo incorporation of unnatural amino acids." See also Klick et al., (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation, PNAS 99: 19-24, for additional methionine analogs.Attorney Docket No. : 108843.00484(SU0451WO)

[0166] The non-natural amino acids suitable for use in the present invention include those are commercially available, e.g., from Sigma (USA) or Aldrich (Milwaukee, Wis., USA). Examples of non-natural amino acids include those that are not commercially available are optionally synthesized as provided herein or as provided in various publications or using standard methods known to those of skill in the art. For organic synthesis techniques, see, e.g., 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). Additional publications describing the synthesis of non-natural amino acids include, e.g., WO 2002 / 085923 entitled "In vivo incorporation of Unnatural Amino Acids;" Matsoukas et al., (1995) J. Med. Chem., 38, 4660-4669; King, F. E. & Kidd, D. A. A. (1949) A New Synthesis of Glutamine and of y-Dipeptides of Glutamic Acid from Phthylated Intermediates. J. Chem. Soc., 3315-3319; Friedman, O. M. & Chatterrji, R. (1959) Synthesis of Derivatives of Glutamine as Model Substrates for Anti-Tumor Agents. J. Am. Chem. Soc. 81, 3750-3752; Craig, J. C. et al. (1988) Absolute Configuration of the Enantiomers of 7-Chloro-4 [[4-(diethylamino)-l-methylbutyl]amino]quinoline (Chloroquine). J. Org. Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. & Frappier, F. (1991) Glutamine analogues as Potential Antimalarials, Eur. J. Med. Chem. 26, 201-5; Koskinen, A. M. P. & Rapoport, H. (1989) Synthesis of 4-Substituted Prolines as Conformationally Constrained Amino Acid Analogues. J. Org. Chem. 54, 1859-1866; Christie, B. D. & Rapoport, H. (1985) Synthesis of Optically Pure Pipecolates from L-Asparagine. Application to the Total Synthesis of (+)-Apovincamine through Amino Acid Decarbonylation and Iminium Ion Cyclization. J. Org. Chem. 1989: 1859-1866; Barton et al., (1987) Synthesis of Novel a-Amino-Acids and Derivatives Using Radical Chemistry: Synthesis of L- and D-a-Amino-Adipic Acids, L-a- aminopimelic Acid and Appropriate Unsaturated Derivatives. Tetrahedron Lett. 43 :4297-4308; and, Subasinghe et al., (1992) Quisqualic acid analogues: synthesis of beta-heterocyclic 2- aminopropanoic acid derivatives and their activity at a novel quisqualate-sensitized site. J. Med. Chem. 35:4602-7. See also, patent applications entitled "Protein Arrays," filed Dec. 22, 2003, Ser. No. 10 / 744,899 and Ser. No. 60 / 435,821 filed on Dec. 22, 2002.

[0167] In some embodiments, the non-natural amino acids comprise a carbonyl reactive group. Amino acids with a carbonyl reactive group allow for a variety of reactions to link molecules (including but not limited to, PEG or other water-soluble molecules) via nucleophilic addition or aldol condensation reactions among others.Attorney Docket No. : 108843.00484(SU0451WO)Exemplary carbonyl-containing amino acids can be represented as follows:wherein n is 0-10; Ri is an 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, Ri is phenyl and R2 is a simple alkyl (z.e., methyl, ethyl, or propyl) and the ketone moiety is positioned in the para position relative to the alkyl side chain. In some embodiments, n is 1, Ri is phenyl and R2 is a simple alkyl (z.e., methyl, ethyl, or propyl) and the ketone moiety is positioned in the meta position relative to the alkyl side chain.

[0168] In some embodiments, a non-naturally amino acid bearing adjacent hydroxyl and amino groups can be incorporated into the polypeptide as a “masked” aldehyde functionality. For example, 5 -hydroxylysine bears a hydroxyl group adjacent to the epsilon amine. Reaction conditions for generating the aldehyde typically involve addition of 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 the addition of about 1.5 molar excess of sodium meta periodate to a buffered solution of the polypeptide, followed by incubation for about 10 minutes in the dark. See, e.g. U.S. Pat. No. 6,423,685, which is incorporated by reference herein.

[0169] The carbonyl functionality can be reacted selectively with a hydrazine-, hydrazide- , hydroxylamine-, or semicarbazide-containing reagent under mild conditions in aqueous solution to form the corresponding hydrazone, oxime, or semicarbazone linkages, respectively, that are stable under physiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc. 81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc. 117:3893-3899 (1995). Moreover, the unique reactivity of the carbonyl group allows for selective modification in the presence of the other amino acid side chains. See, e.g., Cornish, V. W., et al., J. Am. Chem. Soc. 118:8150- 8151 (1996); Geoghegan, K. F. & Stroh, J. G., Bioconjug. Chem. 3: 138-146 (1992); Mahal, L. K., et al., Science 276: 1125-1128 (1997).

[0170] In some embodiments, the non-natural amino acids comprise nucleophilic group, such as a hydrazine, hydrazide or semicarbazide. Non-natural amino acids comprising a nucleophilic group allow for reaction with a variety of electrophilic groups to form conjugates (including but not limited to, with PEG or other water-soluble polymers).Attorney Docket No. : 108843.00484(SU0451WO)

[0171] Exemplary hydrazine, hydrazide or semicarbazide -containing amino acids can be represented as follows:wherein n is 0-10; Ri is an alkyl, aryl, substituted alkyl, or substituted aryl or not present; X, is O, N, or S 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.

[0172] In some embodiments, n is 4, Ri is not present, and X is N. In some embodiments, n is 2, Ri is not present, and X is not present. In some embodiments, n is 1, Ri is phenyl, X is O, and the oxygen atom is positioned para to the aliphatic group on the aryl ring.

[0173] Hydrazide-, hydrazine-, and semicarbazide-containing amino acids are available from commercial sources. For instance, L-glutamate-y-hydrazide is available from Sigma Chemical (St. Louis, Mo.). Other amino acids not available commercially can be prepared by one skilled in the art. See, e.g., U.S. Pat. No. 6,281,211, which is incorporated by reference herein.

[0174] Polypeptides containing non-natural amino acids that bear hydrazide, hydrazine or semicarbazide functionalities can be reacted efficiently and selectively with a variety of molecules that contain 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 unique reactivity of hydrazide, hydrazine and semicarbazide functional groups makes them significantly more reactive toward aldehydes, ketones and other electrophilic groups as compared to the nucleophilic groups present on the 20 common amino acids (including but not limited to, the hydroxyl group of serine or threonine or the amino groups of lysine and the N-terminus).

[0175] In some embodiments, the non-natural amino acids comprise an aminooxy (also called a hydroxylamine) group. Non-natural amino acids containing an aminooxy group allow for reaction with a variety of electrophilic groups to form conjugates (including but not limited to, with PEG or other water-soluble polymers). Like hydrazines, hydrazides and semicarbazides, the enhanced nucleophilicity of the aminooxy group permits it to react efficiently and selectively with a variety of molecules that contain 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). Whereas the result of reaction with a hydrazine group is the corresponding hydrazone,Attorney Docket No. : 108843.00484(SU0451WO) however, an oxime results generally from the reaction of an aminooxy group with a carbonyl - containing group such as a ketone.

[0176] Exemplary amino acids containing aminooxy groups can be represented as follows:wherein n is 0-10; Ri is an 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, Ri is phenyl, X is O, m is 1, and Y is present. In some embodiments, n is 2, Ri and X are not present, m is 0, and Y is not present.

[0177] Aminooxy-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. et al., Life Sci. 60: 1635-1641 (1997). Other aminooxy-containing amino acids can be prepared by one skilled in the art.

[0178] The unique reactivity of azide and alkyne functional groups makes them extremely useful for the selective modification of polypeptides and other biological molecules. Organic azides, particularly aliphatic azides, and alkynes are generally stable toward common reactive chemical conditions. In particular, both the azide and the alkyne functional groups are inert toward the side chains (z.e., R groups) of the 20 common amino acids found in naturally- occurring polypeptides. When brought into close proximity, however, the "spring-loaded" nature of the azide and alkyne groups is revealed and they react selectively and efficiently via Huisgen [3+2] cycloaddition reaction to generate the corresponding triazole. See, e.g., Chin J., et al., Science 301 :964-7 (2003); Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Chin, J. W., et al., J. Am. Chem. Soc. 124:9026-9027 (2002).

[0179] Because the Huisgen cycloaddition reaction involves a selective cycloaddition reaction (see, e.g., Padwa, A., in COMPREHENSIVE ORGANIC SYNTHESIS, Vol. 4, (ed. Trost, B. M., 1991), p. 1069-1109; Huisgen, R. in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), p. 1-176) rather than a nucleophilic substitution, the incorporation of non-natural amino acids bearing azide and alkyne-containing side chains permits the resultant polypeptides to be modified selectively at the position of the non-naturalAttorney Docket No. : 108843.00484(SU0451WO) amino acid. Cycloaddition reaction involving azide or alkyne-containing antibody can be carried out at room temperature under aqueous conditions by the addition of Cu(II) (including but not limited to, in the form of a catalytic amount of CuSC ) in the presence of a reducing agent for reducing Cu(II) to Cu(I), in situ, in catalytic amount. See, e.g., Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Tornoe, C. W., et al., J. Org. Chem. 67:3057-3064 (2002); Rostovtsev, et al., Angew. Chem. Int. Ed. 41 :2596-2599 (2002). Exemplary reducing agents include, including but not limited to, ascorbate, metallic copper, quinine, hydroquinone, vitamin K, glutathione, cysteine, Fe2+, Co2+, and an applied electric potential.

[0180] In some cases, where a Huisgen [3+2] cycloaddition reaction between an azide and an alkyne is desired, the antigen-binding polypeptide comprises a non-natural amino acid comprising an alkyne moiety and the water-soluble polymer to be attached to the amino acid comprises an azide moiety. Alternatively, the converse reaction (z.e., with the azide moiety on the amino acid and the alkyne moiety present on the water-soluble polymer) can also be performed.

[0181] The azide functional group can also be reacted selectively with a water-soluble polymer containing an aryl ester and appropriately functionalized with an aryl phosphine moiety to generate an amide linkage. The aryl phosphine group reduces the azide in situ and the resulting amine then reacts efficiently with a proximal ester linkage to generate the corresponding amide. See, c.g, 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-l -hexanoic acid) or an aryl azide (p-azido-phenylalanine).

[0182] Exemplary water-soluble polymers containing an aryl ester and a phosphine moiety can be represented as follows:wherein X can be O, N, S or not present, 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 -CH2, -C(CH3)3, -OR', -NR'R", -SR', -halogen, -C(O)R', -CONR'R", - S(O)2R', -S(O)2NR'R", -CN and -NO2. R', R", R'" and R"" each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, including but not limited to, aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound includes more than one R group,Attorney Docket No. : 108843.00484(SU0451WO) for example, each of the R groups is independently selected as are each 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 can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include, but not be limited to, 1-pyrrolidinyl and 4- morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (including but not limited to, -CF3 and -CH2CF3) and acyl (including but not limited to, -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like).

[0183] The azide functional group can also be reacted selectively with a water soluble polymer containing a thioester and appropriately functionalized with an aryl phosphine moiety to generate an amide linkage. The aryl phosphine group reduces the azide in situ and the resulting amine then reacts efficiently with the thioester linkage to generate the corresponding amide. Exemplary water-soluble polymers containing a thioester and a phosphine moiety can be represented as follows:wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and W is a water-soluble polymer.

[0184] Exemplary alkyne-containing amino acids can be represented as follows:wherein n is 0-10; Ri is an 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, Ri 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, Ri is phenyl, X is O, m is 1 and the propargyloxy group is positioned in the para position relative to the alkyl side chain (z.e., O-propargyl -tyrosine). In some embodiments, n is 1, Ri and X are not present and m is 0 (z.e., proparylglycine).

[0185] Alkyne-containing amino acids include those commercially available. For example, propargylglycine is commercially available from Peptech (Burlington, Mass.). Alternatively, alkyne-containing amino acids can be prepared according to standard methods. For instance,Attorney Docket No. : 108843.00484(SU0451WO) p-propargyloxyphenylalanine can be synthesized, for example, as described in Deiters, A., et al., J. Am. Chem. Soc. 125: 11782-11783 (2003), and 4-alkynyl-L-phenylalanine can be synthesized as described in Kayser, B., et al., Tetrahedron 53(7): 2475-2484 (1997). Other alkyne-containing amino acids can be prepared by one skilled in the art.

[0186] Exemplary azide-containing amino acids can be represented as follows:wherein n is 0-10; Ri is an 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, Ri 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 and Ri and X are not present, and m=0. In some embodiments, n is 1, Ri is phenyl, X is O, m is 2 and the P-azidoethoxy moiety is positioned in the para position relative to the alkyl side chain.

[0187] Azide-containing amino acids including those available from commercial sources. For instance, 4-azidophenylalanine can be obtained from Chem-Impex International, Inc. (Wood Dale, Ill.). For those azide-containing amino acids that are not commercially available, the azide group can be prepared relatively readily using standard methods known to those of skill in the art, including but not limited to, via displacement of a suitable leaving group (including but not limited to, halide, mesylate, tosylate) or via opening of a suitably protected lactone. See, e.g., Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York).

[0188] The unique reactivity of beta- substituted aminothiol functional groups makes them extremely useful for the selective modification of polypeptides and other biological molecules that contain aldehyde groups via formation of the thiazolidine. 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 antibodies and then reacted with water soluble polymers comprising an aldehyde functionality. In some embodiments, a water soluble polymer, drug conjugate or other payload can be coupled to an antibody polypeptide comprising a betasubstituted aminothiol amino acid via formation of the thiazolidine.

[0189] Further examples of useful non-natural amino acids 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-GlcNAc b-serine, L-Attorney Docket No. : 108843.00484(SU0451WO)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. Further useful examples 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.

[0190] In particular embodiments, the non-natural amino acids are selected from p-acetyl- phenylalanine, p-ethynyl-phenylalanine, p-propargyloxyphenylalanine, and p-azido- phenylalanine. One particularly useful non-natural amino acid is p-azido phenylalanine. This amino acid residue is known to those of skill in the art to facilitate Huisgen [3+2] cycloaddition reactions (so-called “click” chemistry reactions) with, for example, compounds bearing alkynyl groups. This reaction enables one of skill in the art to readily and rapidly conjugate to the antibody at the site-specific location of the non-natural amino acid.

[0191] In certain embodiments, the first reactive group is an alkynyl moiety (including but not limited to, in the non-natural amino acid p-propargyloxyphenylalanine, where the propargyl group is also sometimes referred to as an acetylene moiety) and the second reactive group is an azido moiety, and [3+2] cycloaddition chemistry can be used. In certain embodiments, the first reactive group is the azido moiety (including but not limited to, in the non-natural amino acid p-azido-L-phenylalanine) and the second reactive group is the alkynyl moiety.

[0192] Another useful amino acid is p-acetyl-L-phenylalanine (pAcF), which is known to those of skill in the art to facilitate oxime ligation. In certain embodiments, the first reactive group is the acetyl moiety (including but not limited to, in the non-natural amino acid p-acetyl- L-phenylalanine) and the second reactive group is the aminooxy moiety.

[0193] In the above formulas, each L represents a divalent linker. The divalent linker can be any divalent linker known to those of skill in the art. Generally, the divalent linker is capable of forming covalent bonds to the functional moiety R and the alpha carbon of the non-natural amino acid. Useful divalent linkers a bond, alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene, substituted arylene, heteroarlyene and substituted heteroarylene. In certain embodiments, L is Ci-io alkylene or Ci-io heteroalkylene.

[0194] In certain embodiments, the non-natural amino acids comprise tetrazine functional groups. Incorporation of tetrazine functional groups in non-natural amino acids enables selective and efficient reaction of the non-natural amino acids with compounds comprisingAttorney Docket No. : 108843.00484(SU0451WO) strained alkenes. Useful strained alkenes include trans-cyclooctenes and norbornenes described herein. These reactions are selective in that the reactive groups - the tetrazines and the strained alkenes - are not reactive with the functional groups of the naturally occurring amino acids or with other well-known reactive groups. Further, the reactions can be carried out in complex environments such as cell extracts, in vitro protein synthesis reaction mixtures and the like.

[0195] The reaction between tetrazine and a strained alkene is known as the “tetrazine ligation.” It is believed that the tetrazine and strained alkene react in an inverse-demand Diels- Alder reaction followed by a retro-Diels- Alder reaction that links the tetrazine to the strained alkene. The reaction is specific, with little to no cross-reactivity with functional groups that occur on biomolecules. The reaction may be carried out under mild conditions, for example at room temperature and without a catalyst.

[0196] The non-natural amino acids used in the methods and compositions described herein have at least one of the following four properties: (1) at least one functional group on the sidechain of the non-natural amino acid has at least one characteristics and / or activity and / or reactivity orthogonal to the chemical reactivity of the 20 common, genetically-encoded amino acids (z.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), or at least orthogonal to the chemical reactivity of the naturally occurring amino acids present in the polypeptide that includes the non-natural amino acid; (2) the introduced non-natural amino acids are substantially chemically inert toward the 20 common, genetically-encoded amino acids; (3) the non-natural amino acid can be stably incorporated into a polypeptide, preferably with the stability commensurate with the naturally-occurring amino acids or under typical physiological conditions, and further preferably such incorporation can occur via an in vivo system; and (4) the non-natural amino acid includes an oxime functional group or a functional group that can be transformed into an oxime group by reacting with a reagent, preferably under conditions that do not destroy the biological properties of the polypeptide that includes the non-natural amino acid (unless of course such a destruction of biological properties is the purpose of the modification / transformation), or where the transformation can occur under aqueous conditions at a pH between about 4 and about 8, or where the reactive site on the non-natural amino acid is an electrophilic site. Any number of non-natural amino acids can be introduced into the polypeptide. Non-natural amino acids may also include protected or masked oximes or protected or masked groups that can be transformed into an oxime group after deprotection ofAttorney Docket No. : 108843.00484(SU0451WO) the protected group or unmasking of the masked group. Non-natural amino acids may also include protected or masked carbonyl or dicarbonyl groups, which can be transformed into a carbonyl or dicarbonyl group after deprotection of the protected group or unmasking of the masked group and thereby are available to react with hydroxylamines or oximes to form oxime groups.

[0197] In further embodiments, non-natural amino acids that may be used in the methods and compositions described herein include, but are not limited to, amino acids comprising a photoactivatable cross-linker, 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 and / or photoisomerizable amino acids, amino acids comprising biotin or a biotin analogue, glycosylated amino acids such as a sugar substituted serine, other carbohydrate non- natural amino acids, keto-containing amino acids, aldehyde-containing amino acids, amino acids comprising polyethylene glycol or other polyethers, heavy atom substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids with an elongated side chains as compared to natural amino acids, including but not limited to, polyethers or long chain hydrocarbons, including but not limited to, greater than about 5 or greater than about 10 carbons, carbon-linked sugar-containing amino acids, redox-active amino acids, amino thioacid containing amino acids, and amino acids comprising one or more toxic moiety.

[0198] In some embodiments, non-natural amino acids comprise a saccharide 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 examples where the naturally-occurring N- or O-linkage between the amino acid and the saccharide is replaced by a covalent linkage not commonly found in nature-including but not limited to, an alkene, an oxime, a thioether, an amide and the like. Examples of such amino acids also include saccharides that are not commonly found in naturally-occurring proteins such as 2-deoxy-glucose, 2-deoxygalactose and the like.

[0199] The chemical moieties incorporated into antibodies via incorporation of non-natural amino acids offer a variety of advantages and manipulations of polypeptides. For example, the unique reactivity of a carbonyl or dicarbonyl functional group (including a keto- or aldehyde- functional group) allows selective modification of antibodies with any of a number of hydrazine- or hydroxylamine-containing reagents in vivo and in vitro. A heavy atom non-Attorney Docket No. : 108843.00484(SU0451WO) natural amino acid, for example, can be useful for phasing x-ray structure data. The site-specific introduction of heavy atoms using non-natural amino acids also provides selectivity and flexibility in choosing positions for heavy atoms. Photoreactive non-natural amino acids (including but not limited to, amino acids with benzophenone and arylazides (including but not limited to, phenylazide) side chains), for example, allow for efficient in vivo and in vitro photocrosslinking of polypeptides. Examples of photoreactive non-natural amino acids include, but are not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine. The antibodies with the photoreactive non-natural amino acids may then be crosslinked at will by excitation of the photoreactive group-providing temporal control. In a non-limiting example, the methyl group of a non-natural amino can be substituted with an isotopically labeled, including but not limited to, with a methyl group, as a probe of local structure and dynamics, including but not limited to, with the use of nuclear magnetic resonance and vibrational spectroscopy.

[0200] In some embodiments, the non-natural amino acids comprise an electrophilic reactive group. Non-natural amino acids with an electrophilic reactive group allow for a variety of reactions to link molecules via various chemical reactions, including, but not limited to, nucleophilic addition reactions. Such electrophilic reactive groups include a carbonyl- or di carbonyl -group (including a keto- or aldehyde group), a carbonyl-like- or dicarbonyl-like- group (which has reactivity similar to a carbonyl- or di carbonyl -group and is structurally similar to a carbonyl- or dicarbonyl-group), a masked carbonyl- or masked dicarbonyl-group (which can be readily converted into a carbonyl- or dicarbonyl-group), or a protected carbonyl - or protected dicarbonyl-group (which has reactivity similar to a carbonyl- or dicarbonyl-group upon deprotection). Such amino acids include amino acids having the structure of Formula (I):wherein: A is optional, 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 when present is a linker selected from the groupAttorney Docket No. : 108843.00484(SU0451WO) 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- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2- -OS(O)2- -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)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2- N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; J isR 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; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or 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 a heterocycloalkyl; or the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising 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 -J-R group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl comprising 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; with a proviso that when A is phenylene and each R3 is H, B is present; and that when A is -(CH2)4- and each R3 is H, B is not -Attorney Docket No. : 108843.00484(SU0451WO)NHC(O)(CH2CH2)-; and that when A and B are absent and each R3 is H, R is not methyl. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0201] In certain embodiments, compounds of Formula (I) are stable in aqueous solution for at least 1 month under mildly acidic conditions. In certain embodiments, compounds of Formula (I) are stable for at least 2 weeks under mildly acidic conditions. In certain embodiments, compound of Formula (I) are stable for at least 5 days under mildly acidic conditions. In certain embodiments, such acidic conditions are pH 2 to 8.

[0202] In certain embodiments of compounds of Formula (I), B is lower alkylene, substituted lower alkylene, -O-(alkylene or substituted alkylene)-, -C(R')=N-N(R')-, - N(R')CO-, -C(O)-, -C(R')=N- -C(O)-(alkylene or substituted alkylene)-, -CON(R')- (alkylene or substituted alkylene)-, -S(alkylene or substituted alkylene)-, -S(O)(alkylene or substituted alkylene)-, or -S(O)2(alkylene or substituted alkylene)-. In certain embodiments of compounds of Formula (I), B is -O(CH2)-, -CH=N-, -CH=N-NH-, -NHCH2-, -NHCO- , -C(O)-, -C(O)-(CH2)-, -CONH-(CH2)-, -SCH2-, -S(=O)CH2-, or -S(O)2CH2-. In certain embodiments of compounds of Formula (I), R is C1-6 alkyl or cycloalkyl. In certain embodiments of compounds of Formula (I) R is -CH3, -CH(CH3)2, or cyclopropyl. In certain embodiments of compounds of Formula (I), Ri is H, tert-butyloxycarbonyl (Boc), 9- Fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In certain embodiments of compounds of Formula (I), Ri is a resin, amino acid, polypeptide, or polynucleotide. In certain embodiments of compounds of Formula (I), R2 is OH, O-methyl, O-ethyl, or O-t-butyl. In certain embodiments of compounds of Formula (I), R2 is a resin, amino acid, polypeptide, or polynucleotide. In certain embodiments of compounds of Formula (I), R2 is a polynucleotide. In certain embodiments of compounds of Formula (I), R2 is ribonucleic acid (RNA). In certain embodiments of compounds of Formula (I), R2 is tRNA. In certain embodiments of compounds of Formula (I), the tRNA specifically recognizes a selector codon. In certain embodiments of compounds of Formula (I) the selector codon is selected from the group consisting of an amber codon, ochre codon, opal codon, a unique codon, a rare codon, an unnatural codon, a five-base codon, and a four-base codon. In certain embodiments of compounds of Formula (I), R2 is a suppressor tRNA.

[0203] In certain embodiments of compounds of Formula (I),Attorney Docket No. : 108843.00484(SU0451WO)is selected from the group consisting of: (i) A is substituted lower alkylene, C4-arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when present is a divalent linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, - S(O)-, -S(O)2-, -NS(O)2-, -OS(O)2-, — C(O)— , -C(O)-(alkylene or substituted alkylene)-, - C(S)-, -N(R')-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, - N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O- -N(R')C(S)-, -S(O)N(R'), - S(O)2N(R'), -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)N(R')-, -N(R')S(O)2N(R')-, - N(R')-N=, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-; (ii) A is optional, and when present is substituted lower alkylene, C4-arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is a divalent linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S(O)-, -S(O)2- -NS(O)2- -OS(O)2- - C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -N(R')-, -C(O)N(R')-, -CON(R')- (alkylene or substituted alkylene)-, -CSN(R')-, -N(R')CO-(alkylene or substituted alkylene)- , -N(R')C(O)O- -N(R')C(S)-, -S(O)N(R'), -S(O)2N(R'), -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)N(R')-, -N(R')S(O)2N(R')-, -N(R')-N=, -C(R')=N-N(R')-, - C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-; (iii) A is lower alkylene; B is optional, and when present is a divalent linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, - O-(alkylene or substituted alkylene)-, -S-, -S(O)-, -S(O)2- -NS(O)2- -OS(O)2- -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -N(R')-, -C(O)N(R')-, -CSN(R')-, - CON(R')-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -N(R')C(S)-, -S(O)N(R'), - S(O)2N(R'), -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)N(R')-, -N(R')S(O)2N(R')-, - N(R')-N=, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-; and (iv) A is phenylene; B is a divalent linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, - O-(alkylene or substituted alkylene)-, -S-, -S(O)-, -S(O)2- -NS(O)2- -OS(O)2- -C(O)-,Attorney Docket No. : 108843.00484(SU0451WO)-C(O)-(alkylene or substituted alkylene)-, -C(S)-, -N(R')-, -C(O)N(R')-, -CON(R')- (alkylene or substituted alkylene)-, -CSN(R')-, -N(R')CO-(alkylene or substituted alkylene)- , -N(R')C(O)O- -N(R')C(S)-, -S(O)N(R'), -S(O)2N(R'), -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)N(R')-, -N(R')S(O)2N(R')-, -N(R')-N=, -C(R')'N-N(R')-, - C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-; J iseach R1is independently H, alkyl, or substituted alkyl; Ri is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; and each R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl; and R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0204] In addition, examples of the non-natural amino acids include amino acids having the structure of Formula (II), including:wherein: A is optional, 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 when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lowerAttorney Docket No. : 108843.00484(SU0451WO) alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2- -OS(O)2- -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)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2- N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide. In certain embodiments, when A is phenylene, B is present; and that when A is -(CH2)4- B is not -NHC(O)(CH2CH2)- ; and that when A and B are absent, R is not methyl. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0205] In addition, examples of the non-natural amino acids include amino acids having the structure of Formula (III), including:wherein: B is a linker 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- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)- , -C(O)-, -NS(O)2-, -OS(O)2-, -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)kN(R')-, -N(R')-N=, -Attorney Docket No. : 108843.00484(SU0451WO)C(R')=N- -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R1is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each Rais independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O)k R' where k is 1, 2, or 3, -C(O)N(R')2, - OR', and -S(O)kR', where each R' is independently H, alkyl, or substituted alkyl. Such nonnatural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0206] In addition, examples of the non-natural amino acids include the following amino

[0207] Such non-natural amino acids may be optionally amino protected group, carboxyl protected and / or in the form of a salt, or may be incorporated into a non-natural amino acidAttorney Docket No. : 108843.00484(SU0451WO) polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0208] In addition, examples of the non-natural amino acids include the following amino acids having the structure of Formula (IV), including:wherein -NS(O)2- -OS(O)2- optional, and when present is a linker 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- where 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)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2- N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each Rais independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O)kR' where k is 1, 2, or 3, -C(O)N(R')2, -OR', and -S(O)kR', where each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8. In certain embodiments, when A is -(CH2)4- B is not -NHC(O)(CH2CH2)-. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0209] In addition, examples of the non-natural amino acids include the following amino acids:Attorney Docket No. : 108843.00484(SU0451WO)wherein such compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0210] In addition, examples of the non-natural amino acids include the following amino acids having the structure of Formula (VIII), including:Attorney Docket No. : 108843.00484(SU0451WO)wherein, A is optional, 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 when present is a linker 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- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2- -OS(O)2- -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)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2- N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0211] In addition, examples of the non-natural amino acids include the following amino acids having the structure of Formula (IX), including:Attorney Docket No. : 108843.00484(SU0451WO)wherein, B is optional, and when present is a linker 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- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2- -OS(O)2- -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)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2- N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; wherein each Rais independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, - N(R')2, -C(O)kR' where k is 1, 2, or 3, -C(0)N(R')2, -OR', and -S(O)k R', where each R' is independently H, alkyl, or substituted alkyl. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0212] In addition, examples of the non-natural amino acids include the following amino acids, including:Attorney Docket No. : 108843.00484(SU0451WO)wherein such compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0213] In addition, examples of the non-natural amino acids include the following amino acids having the structure of Formula (X), including:wherein, B is optional, and when present is a linker 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)-Attorney Docket No. : 108843.00484(SU0451WO), -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2- -OS(O)2- -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)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2- N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each Rais independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O)kR' where k is 1, 2, or 3, -C(0)N(R')2, -OR', and -S(O)kR', where each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0214] In addition, examples of the non-natural amino acids include the following amino acids, including:Attorney Docket No. : 108843.00484(SU0451WO)wherein such compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0215] In addition to monocarbonyl structures, the non-natural amino acids described herein may include groups such as dicarbonyl, dicarbonyl like, masked dicarbonyl and protected dicarbonyl groups. For example, the following amino acids having the structure of Formula (V) are included:Attorney Docket No. : 108843.00484(SU0451WO) wherein, A is optional, 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 when present is a linker 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- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2- -OS(O)2- -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)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2- N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0216] In addition, examples of the non-natural amino acids include the following amino acids having the structure of Formula (VI), including:wherein, B is optional, and when present is a linker 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- where k is 1, 2, or 3, -S(O)k(alkyleneAttorney Docket No. : 108843.00484(SU0451WO) or substituted alkylene)-, -C(O)-, -NS(O)2- -OS(O)2- -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)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; wherein each Rais independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, - N(R')2, -C(O)kR' where k is 1, 2, or 3, -C(O)N(R')2, -OR', and -S(O)kR', where each R' is independently H, alkyl, or substituted alkyl. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0217] In addition, examples of the non-natural amino acids include the following amino acids:wherein such compounds are optionally amino protected and carboxyl protected, or a salt thereof. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.Attorney Docket No. : 108843.00484(SU0451WO)

[0218] In addition, examples of the non-natural amino acids include the following amino acids having the structure of Formula (VII):wherein, B is optional, and when present is a linker 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- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2- -OS(O)2- -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)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2- N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each Rais independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O)kR' where k is 1, 2, or 3, -C(O)N(R')2, -OR', and -S(O)kR', where each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0219] In addition, examples of the non-natural amino acids include the following amino acids:Attorney Docket No. : 108843.00484(SU0451WO)wherein such compounds are optionally amino protected and carboxyl protected, or a salt thereof, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0220] In addition, examples of the non-natural amino acids include the following amino acids having the structure of Formula (XXX):wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lowerAttorney Docket No. : 108843.00484(SU0451WO) 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; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; Xi 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. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0221] In addition, examples of the non-natural amino acids include the following amino acids having the structure of Formula (XXX-A):wherein: A is optional, 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; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or 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. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0222] In addition, examples of the non-natural amino acids include the following amino acids having the structure of Formula (XXX-B):Attorney Docket No. : 108843.00484(SU0451WO)wherein: A is optional, 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; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or 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. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0223] In addition, examples of the non-natural amino acids include the following amino acids having the structure of Formula (XXXI):wherein: A is optional, 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; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; Xi is C, S, or S(O); and n is 0, 1, 2, 3, 4, or 5; and each R8and R9on each CR8R9group is independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R8and R9can together form =0 or a cycloalkyl, or any to adjacent R8groups can together form aAttorney Docket No. : 108843.00484(SU0451WO) cycloalkyl. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0224] In addition, examples of the non-natural amino acids include the following amino acids having the structure of Formula (XXXI- A):wherein: A is optional, 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; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; n is 0, 1, 2, 3, 4, or 5; and each R8and R9on each CR8R9group is independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R8and R9can together form =0 or a cycloalkyl, or any to adjacent R8groups can together form a cycloalkyl. Such non- natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0225] In addition, examples of the non-natural amino acids include the following amino acids having the structure of Formula (XXXI-B):wherein: A is optional, 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,Attorney Docket No. : 108843.00484(SU0451WO) heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; n is 0, 1, 2, 3, 4, or 5; and each R8and R9on each CR8R9group is independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R8and R9can together form =0 or a cycloalkyl, or any to adjacent R8groups can together form a cycloalkyl. Such nonnatural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0226] In addition, examples of the non-natural amino acids include the following amino acids having the structure of Formula (XXXII):wherein: A is optional, 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; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; Xi 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. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0227] The In addition, examples of the non-natural amino acids include the following amino acids having the structure of Formula (XXXII-A):Attorney Docket No. : 108843.00484(SU0451WO)wherein: A is optional, 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; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or 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. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0228] In addition, examples of the non-natural amino acids include the following amino acids having the structure of Formula (XXXII-B):0 0s AA" N — Lr RR-N ARhs wherein: A is optional, 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; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or 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. Such non-natural amino acids may beAttorney Docket No. : 108843.00484(SU0451WO) in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0229] In addition, examples of the non-natural amino acids include amino acids having the structure of Formula (XXXX):wherein: A is optional, 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;where (a) indicates bonding to the A group and (b) indicates bonding to respective carbonyl groups, R3 and R4 are independently chosen 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 a heterocycloalkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; T3 is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; Ri is H, an amino protecting group, resin, aminoAttorney Docket No. : 108843.00484(SU0451WO) acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0230] In addition, examples of the non-natural amino acids include amino acids having the structure of Formula (XXXXI), including:where (a) indicates bonding to the A group and (b) indicates bonding to respective carbonyl groups, R3 and R4 are independently chosen 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 a heterocycloalkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; T3 is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each Rais independently selected from the group consisting ofAttorney Docket No. : 108843.00484(SU0451WO)H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O)kR' where k is 1, 2, or 3, -C(0)N(R')2, - OR', and -S(O)kR', where each R' is independently H, alkyl, or substituted alkyl. Such nonnatural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0231] In addition, examples of the non-natural amino acids include amino acids having the structure of Formula (XXXXII):wherein: R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; and T3 is O, or S. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0232] In addition, examples of the non-natural amino acids include amino acids having the structure of Formula (XXXXIII):wherein: R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0233] In addition, examples of the non-natural amino acids include the following amino acids having structures of Formula (XXXXIII):Attorney Docket No. : 108843.00484(SU0451WO)Such non-natural amino acids may be in the form of a salt, or may be incorporated into a nonnatural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0234] In some embodiments, the non-natural amino acids comprise non-natural amino acids containing a hydroxylamine (also called an aminooxy) group. Non-natural amino acids containing a hydroxylamine group allow for reaction with a variety of electrophilic groups to form conjugates (including but not limited to, with PEG or other water soluble polymers). Like hydrazines, hydrazides and semicarbazides, the enhanced nucleophilicity of the aminooxy group permits it to react efficiently and selectively with a variety of molecules that contain carbonyl- or di carbonyl -groups, including but not limited to, ketones, 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(9): 727-736 (2001). Whereas the result of reaction with a hydrazine group is the corresponding hydrazone, however, an oxime results generally from the reaction of an aminooxy group with a carbonyl- or dicarbonyl-containing group such as, by way of example, a ketones, aldehydes or other functional groups with similar chemical reactivity.

[0235] Thus, in certain embodiments described herein are non-natural amino acids with sidechains comprising a hydroxylamine group, a hydroxylamine-like group (which has reactivity similar to a hydroxylamine group and is structurally similar to a hydroxylamine group), a masked hydroxylamine group (which can be readily converted into a hydroxylamine group), or a protected hydroxylamine group (which has reactivity similar to a hydroxylamine group upon deprotection). Such amino acids include amino acids having the structure of Formula (XIV):Attorney Docket No. : 108843.00484(SU0451WO)wherein: A is optional, 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 when present is a linker 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- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2- -OS(O)2- -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)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2- N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; K is -NReR? or -N=CReR?; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or 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 a heterocycloalkyl; each of Re and R7 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, and substituted aralkyl, -C(O)R", -C(0)2R", -C(0)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; or Re or R7 is L-X, where X is a selected from the group consisting of a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; anAttorney Docket No. : 108843.00484(SU0451WO) antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label; a fluorophore, a metalcontaining moiety; a radioactive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; a photoisomerizable moiety; biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent; an amino thioacid; 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; and any combination thereof; and L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where 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)kN(R')-, -N(R')-N=, -C(R')=N-, - C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0236] In certain embodiments of compounds of Formula (XIV), A is phenylene or substituted phenylene. In certain embodiments of compounds of Formula (XIV), B is - (alkylene or substituted alkylene)-, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, or -C(O)-(alkylene or substituted alkylene)-. In certain embodiments of compounds of Formula (XIV), B is -O(CH2)2-, -S(CH2)2-, -NH(CH2)2-, -CO(CH2)2-, or - (CH2)n- where n is 1 to 4. In certain embodiments of compounds of Formula (XIV), Ri is H, tert-butyloxycarbonyl (Boc), 9-Fluorenylmethoxy carbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In certain embodiments of compounds of Formula (XIV), Ri is a resin, amino acid, polypeptide, or polynucleotide. In certain embodiments of compounds of Formula (XIV), wherein R2is OH, O-methyl, O-ethyl, or O-t-butyl. In certain embodimentsAttorney Docket No. : 108843.00484(SU0451WO) of compounds of Formula (XIV), R2 is a resin, amino acid, polypeptide, or polynucleotide. In certain embodiments of compounds of Formula (XIV), R2 is a polynucleotide. In certain embodiments of compounds of Formula (XIV), R2 is ribonucleic acid (RNA). In certain embodiments of compounds of Formula (XIV), R2 is tRNA. In certain embodiments of compounds of Formula (XIV), the tRNA specifically recognizes a codon selected from the group consisting of an amber codon, ochre codon, opal codon, a unique codon, a rare codon, an unnatural codon, a five-base codon, and a four-base codon. In certain embodiments of compounds of Formula (XIV), R2 is a suppressor tRNA. In certain embodiments of compounds of Formula (XIV), each of Re and R7 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, and substituted aralkyl. In certain embodiments of compounds of Formula (XIV), each of Re and R7 is independently selected from the group consisting of H, methyl, phenyl, and -[(alkylene or substituted alkylene)-O-(hydrogen, alkyl, or substituted alkyl)]x, wherein x is from 1-50. In certain embodiments of compounds of Formula (XIV), K is -NReR?.

[0237] In certain embodiments of compounds of Formula (XIV), X is a biologically active agent selected from the group consisting of a peptide, protein, enzyme, antibody, drug, dye, lipid, nucleosides, oligonucleotide, cell, virus, liposome, microparticle, and micelle. In certain embodiments of compounds of Formula (XIV), X is a drug selected from the group consisting of an antibiotic, fungicide, anti-viral agent, anti-inflammatory agent, anti-tumor agent, cardiovascular agent, anti -anxiety agent, hormone, growth factor, and steroidal agent. In certain embodiments of compounds of Formula (XIV), X is an enzyme selected from the group consisting of horseradish peroxidase, alkaline phosphatase, P-galactosidase, and glucose oxidase. In certain embodiments of compounds of Formula (XIV), X is a detectable label selected from the group consisting of a fluorescent, phosphorescent, chemiluminescent, chelating, electron dense, magnetic, intercalating, radioactive, chromophoric, and energy transfer moiety.

[0238] In certain embodiments, compounds of Formula (XIV) are stable in aqueous solution for at least 1 month under mildly acidic conditions. In certain embodiments, compounds of Formula (XIV) are stable for at least 2 weeks under mildly acidic conditions. In certain embodiments, compounds of Formula (XIV) are stable for at least 5 days under mildly acidic conditions. In certain embodiments, such acidic conditions are pH 2 to 8.

[0239] Such amino acids include amino acids having the structure of Formula (XV):Attorney Docket No. : 108843.00484(SU0451WO)wherein A is optional, 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 when present is a linker 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- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2- -OS(O)2- -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)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2- N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or 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 a heterocycloalkyl. Such non -natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0240] A non-limiting, representative amino acid has the following structure:Attorney Docket No. : 108843.00484(SU0451WO)

[0241] Such a non-natural amino acid may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0242] In some embodiments, the non-natural amino acids comprise non-natural amino acids containing an oxime group. Non-natural amino acids containing an oxime group allow for reaction with a variety of reagents that contain certain reactive carbonyl- or dicarbonylgroups (including but not limited to, ketones, aldehydes, or other groups with similar reactivity) to form new non-natural amino acids comprising a new oxime group. Such an oxime exchange reaction allows for the further functionalization of non-natural amino acid polypeptides. Further, the original non-natural amino acids containing an oxime group may be useful in their own right as long as the oxime linkage is stable under conditions necessary to incorporate the amino acid into a polypeptide (e.g., the in vivo, in vitro and chemical synthetic methods described herein).

[0243] Thus, in certain embodiments described herein are non-natural amino acids with sidechains comprising an oxime group, an oxime-like group (which has reactivity similar to an oxime group and is structurally similar to an oxime group), a masked oxime group (which can be readily converted into an oxime group), or a protected oxime group (which has reactivity similar to an oxime group upon deprotection). Such amino acids include amino acids having the structure of Formula (XI):wherein: A is optional, 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 when present is a linker 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- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2- -OS(O)2- -C(O)-(alkyleneAttorney Docket No. : 108843.00484(SU0451WO) 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)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2- N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 optionally form a cycloalkyl or a heterocycloalkyl; R5 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R")2, -(alkylene or substituted alkylene)-C(O)SR", - (alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -C(O)R", -C(O)2R", or - C(O)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; or R5 is L-X, where X is a selected from the group consisting of a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; 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; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing moiety; a radioactive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; a photoisomerizable moiety; biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent; an amino thioacid; 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; and any combination thereof; and L is optional,Attorney Docket No. : 108843.00484(SU0451WO) and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S- , -S-(alkylene or substituted alkylene)-, -S(O)k- where 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-, - (alkylene or substituted alkylene)-O-N=CR'-, -(alkylene or substituted alkylene)-C(O)NR'- (alkylene or substituted alkylene)-, -(alkylene or substituted alkylene)-S(O)k-(alkylene or substituted alkylene)-S-, -(alkylene or substituted alkylene)-S-S-, -S(O)kN(R')-, - N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)kN(R')-, -N(R')-N=, -C(R')=N-, - C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; with a proviso that when A and B are absent, R is not methyl. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0244] In certain embodiments of compounds of Formula (XI), B is -O-(alkylene or substituted alkylene)-. In certain embodiments of compounds of Formula (XI), B is -O(CH2)- . In certain embodiments of compounds of Formula (XI), R is Ci-4 alkyl. In certain embodiments of compounds of Formula (XI), R is -CH3. In certain embodiments of compounds of Formula (XI), Ri is H, tert-butyloxycarbonyl (Boc), 9- fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In certain embodiments of compounds of Formula (XI), Ri is a resin, amino acid, polypeptide, or polynucleotide. In certain embodiments of compounds of Formula (XI), R2is OH, O-methyl, O-ethyl, or O-t-butyl. In certain embodiments of compounds of Formula (XI), R2is a resin, amino acid, polypeptide, or polynucleotide. In certain embodiments of compounds of Formula (XI), R2is a polynucleotide. In certain embodiments of compounds of Formula (XI), R2is ribonucleic acid (RNA). In certain embodiments of compounds of Formula (XI), R2is tRNA. In certain embodiments of compounds of Formula (XI), the tRNA specifically recognizes a selector codon. In certain embodiments of compounds of Formula (XI), the selector codon is selected from the group consisting of an amber codon, ochre codon, opal codon, a unique codon, a rare codon, an unnatural codon, a five-base codon, and a four-base codon. In certain embodiments of compounds of Formula (XI), R2is a suppressor tRNA. InAttorney Docket No. : 108843.00484 (SU0451WO) certain embodiments of compounds of Formula (XI), R5 is alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, or -C(O)2R". In certain embodiments of compounds of Formula (XI), R5 is -[(alkylene or substituted alkylene)-O- (hydrogen, alkyl, or substituted alkyl)]x, wherein x is from 1-50. In certain embodiments of compounds of Formula (XI), R5 is -(CFhCFy-O-CFh or -COOH.

[0245] In certain embodiments, compounds of Formula (XI) are stable in aqueous solution for at least 1 month under mildly acidic conditions. In certain embodiments, compounds of Formula (XI) are stable for at least 2 weeks under mildly acidic conditions. In certain embodiments, compound of Formula (XI) is stable for at least 5 days under mildly acidic conditions. In certain embodiments, such acidic conditions are pH 2 to 8.

[0246] Amino acids of Formula (XI) include amino acids having the structure of Formula (XII):wherein, B is optional, and when present is a linker 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- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2- -OS(O)2- -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)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2- N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; R5 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl,Attorney Docket No. : 108843.00484(SU0451WO) substituted aralkyl, -(alkylene or substituted alkylene)-ON(R")2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), - C(O)R", -C(0)2R", or -C(0)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; or R5 is L-X, where X is a selected from the group consisting of a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; 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; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing moiety; a radioactive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; a photoisomerizable moiety; biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent; an amino thioacid; 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; and any combination thereof; and L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O-(alkylene or substituted alkylene)-, - S- , -S-(alkylene or substituted alkylene)-, -S(O)k- where 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-, - (alkylene or substituted alkylene)-O-N=CR'-, -(alkylene or substituted alkylene)-C(O)NR'- (alkylene or substituted alkylene)-, -(alkylene or substituted alkylene)-S(O)k-(alkylene or substituted alkylene)-S-, -(alkylene or substituted alkylene)-S-S-, -S(O)kN(R')-, - N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)kN(R')-, -N(R')-N=, -C(R')=N-, - C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl. Such non-natural amino acids may be in theAttorney Docket No. : 108843.00484(SU0451WO) form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0247] Such amino acids include amino acids having the structure of Formula (XIII):wherein, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; R5 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R")2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), - C(O)R", -C(O)2R", or -C(O)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; or R5 is L-X, where X is a selected from the group consisting of a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; 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; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing moiety; a radioactive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; a photoisomerizable moiety; biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent; an amino thioacid; 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; and any combination thereof; and L is optional,Attorney Docket No. : 108843.00484 (SU0451WO) and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O-(alkylene or substituted alkylene)-, - S- , -S-(alkylene or substituted alkylene)-, -S(O)k- where 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-, - (alkylene or substituted alkylene)-O-N=CR'-, -(alkylene or substituted alkylene)-C(O)NR'- (alkylene or substituted alkylene)-, -(alkylene or substituted alkylene)-S(O)k-(alkylene or substituted alkylene)-S-, -(alkylene or substituted alkylene)-S-S-, -S(O)kN(R')-, - N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)kN(R')-, -N(R')-N=, -C(R')=N-, - C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0248] Further non-limiting examples of such amino acids include amino acids having the following structures:

[0249] Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0250] In addition, such amino acids include amino acids having the structure of Formula (XIV):Attorney Docket No. : 108843.00484(SU0451WO)wherein: A is optional, 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 when present is a linker 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- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2- -OS(O)2- -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)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2- N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; K is -NReR? or -N=CReR?; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or 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 a heterocycloalkyl; each of Re and R7 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, and substituted aralkyl, -C(O)R", -C(0)2R", -C(0)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; or Re or R7 is L-X, where X is a selected from the group consisting of a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; anAttorney Docket No. : 108843.00484(SU0451WO) antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label; a fluorophore, a metalcontaining moiety; a radioactive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; a photoisomerizable moiety; biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent; an amino thioacid; 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; and any combination thereof; and L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where 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)kN(R')-, -N(R')-N=, -C(R')=N-, - C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0251] Such amino acids further include amino acids having the structure of Formulawherein: A is optional, 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, orAttorney Docket No. : 108843.00484(SU0451WO) substituted aralkylene; B is optional, and when present is a linker 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- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2- -OS(O)2- -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)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2- N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 optionally form a cycloalkyl or a heterocycloalkyl; each of Re and R7 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, and substituted aralkyl, -C(O)R", -C(O)2R", -C(O)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; or Re or R7 is L-X, where X is a selected from the group consisting of a label; a dye; a polymer; a water- soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; 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; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal -containing moiety; a radioactive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; a photoisomerizable moiety; biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent; an amino thioacid; a toxic moiety; an isotopically labeled moiety; a biophysical probe; a phosphorescent group; aAttorney Docket No. : 108843.00484(SU0451WO) 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; and any combination thereof; and L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O- (alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where 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)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2- N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0252] Further, such amino acids include amino acids having the structure of Formula (XVII):wherein: B is optional, and when present is a linker 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- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -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)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2- N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;Attorney Docket No. : 108843.00484(SU0451WO) and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each of Re and R7 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, and substituted aralkyl, -C(O)R", -C(O)2R", -C(O)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; or Re or R7 is L-X, where X is a selected from the group consisting of a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; 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; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label; a fluorophore, a metalcontaining moiety; a radioactive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; a photoisomerizable moiety; biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent; an amino thioacid; 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; and any combination thereof; and L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where 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)kN(R')-, -N(R')-N=, -C(R')=N-, - C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl.Attorney Docket No. : 108843.00484(SU0451WO)

[0253] Non-limiting examples of such amino acids include amino acids having the following structures:Such non-natural amino acids may be in the form of a salt, or may be incorporated into a nonnatural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0254] Additionally, such amino acids include amino acids having the structure of Formula (XVIII):wherein: B is optional, and when present is a linker 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- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2- -OS(O)2- -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene orAttorney Docket No. : 108843.00484(SU0451WO) 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)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2- N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each of Re and R7 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, and substituted aralkyl, -C(O)R", -C(O)2R", -C(O)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; or Re or R7 is L-X, where X is a selected from the group consisting of a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; 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; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label; a fluorophore, a metalcontaining moiety; a radioactive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; a photoisomerizable moiety; biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent; an amino thioacid; 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; and any combination thereof; and L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where 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')-Attorney Docket No. : 108843.00484(SU0451WO)(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)kN(R')-, -N(R')-N=, -C(R')=N-, - C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; and each Rais independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O)kR' where k is 1, 2, or 3, -C(O)N(R')2, -OR', and -S(O)kR'; where each R' is independently H, alkyl, or substituted alkyl and n is 0 to 8. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0255] Non-limiting examples of such amino acids include amino acids having the following structures:

[0256] Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0257] In certain embodiments, the non-natural amino acid can be according to formula XIX:Formula XIX; or a salt thereof, wherein: D is -Ar-Ws- or -WI-YI-C(O)-Y2-W2-; Ar isAttorney Docket No. : 108843.00484(SU0451WO)each of Wi, W2, and W3 is independently a single bond or lower alkylene; each Xi is independently -NH-, -O-, or -S-; each Yi is independently a single bond, -NH-, or -O-; each Y2 is independently a single bond, -NH-, -O-, or an N-linked or C-linked pyrrolidinylene; and one of Zi, Z2, and Z3 is -N- and the others of Zi, Z2, and Z3 are independently -CH-. In certain embodiments, the non-natural amino acid is according to formula XIXa:Formula XIXa; where D is a defined in the context of formula XIX. In certain embodiments, the non-natural amino acid is according to formula XlXb:Formula XlXb; or a salt thereof, wherein W4 is C1-C10 alkylene. In a further embodiment, W4 is C1-C5 alkylene.In an embodiment, W4 is C1-C3 alkylene. In an embodiment, W4 is Ci alkylene. In particular embodiments, the non-natural amino acid is selected from the group consisting of- I l l -Attorney Docket No. : 108843.00484(SU0451WO)Attorney Docket No. : 108843.00484(SU0451WO)or a salt thereof. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0258] In some embodiments, a non-natural amino acid comprises a reactive group capable of forming a covalent bond to a linker payload. The reactive group can be an amino, carboxy, acetyl, hydrazino, hydrazido, hydroxylamine, semicarbazido, sulfanyl, azido or alkynyl group. Non-limiting examples of non-natural amino acids include p-acetyl-L-phenylalanine, O- methyl-L-tyrosine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L- phenylalanine, p-A benzoyl -L-phenylalanine, p-iodo-phenylalanine, p-bromophenylalanine, p- amino-L-phenylalanine, isopropyl-L-phenylalanine, p-propargyloxy-phenylalanine, and p- azidomethyl-L-phenyl alanine. In one example, a non-natural amino acid is p-azidomethyl-L- phenylalanine. In another example, a non-natural amino acid is p-acetyl-L-phenylalanine.

[0259] Non-natural amino acids comprising azide functional groups, such as the non- natural amino acids provided in any of formulas XIX, XIXa, XlXb, ( 1 )-(30), and (40) may be prepared according to methods provided in PCT / US2013 / 057677, which is incorporated by reference in its entirety.Attorney Docket No. : 108843.00484(SU0451WO)

[0260] In certain embodiments, the non-natural amino acid can be according to formulaAl:Formula Al; or a salt thereof, wherein Ar is:V is a single bond, lower alkylene, or -W1-W2-; one of Wi and W2 is absent or lower alkylene, and the other is -NH-, -O-, or -S-; each Xi is independently -NH-, -O-, or -S-; one of Zi, Z2, and Z3 is -CH- or -N- and the others of Zi, Z2, and Z3 are each independently -CH-; andR is lower alkyl. In certain embodiments, whenthen one of Zi, Z2, and Z3 is -N-. In certain embodiments, V is a single bond, -NH-, or -CH2NH-.

[0261] In certain embodiments, Ar isand Zi, Z2, Z3 and Xi are as defined in the context of formula I. In certain embodiments according to this paragraph, V is -W1-W2-; one of Wi and W2is absent or -CH2-, and the other is -NH-, -O-, or -S-. In certain embodiments according to this paragraph, V is a single bond, -NH-, or -CH2NH-. In certain embodiments according to this paragraph, Zi is N. In certain embodiments according to this paragraph, Z2is N. In certain embodiments accordingAttorney Docket No. : 108843.00484(SU0451WO) to this paragraph, Z3 is N. In certain embodiments according to this paragraph, Zi is CH, Z3 is CH and Xi is S.

[0262] In certain embodiments, Ar isand Zi, Z2, and Z3 are as defined in the context of formula I. In certain embodiments according to this paragraph, V is -W1-W2-; one of Wi and W2 is absent or -CH2-, and the other is - NH-, -O-, or -S-. In certain embodiments according to this paragraph, V is a single bond, - NH-, or -CH2NH-. In certain embodiments according to this paragraph, Zi is N. In certain embodiments according to this paragraph, Z2 is N. In certain embodiments according to this paragraph, Z3 is N.

[0263] In certain embodiments, Ar isand Zi, Z3 and Xi are as defined in the context of formula I. In certain embodiments according to this paragraph, V is -W1-W2-; one of Wi and W2 is absent or -CH2-, and the other is - NH-, -O-, or -S-. In certain embodiments according to this paragraph, V is a single bond, - NH-, or -CH2NH-. In certain embodiments according to this paragraph, Zi is N. In certain embodiments according to this paragraph, Z3 is N. In certain embodiments according to this paragraph, Zi is CH, Z3 is CH and Xi is S.

[0264] In certain embodiments, the non-natural amino acid can be according to formula Ala:Formula Ala; where Ar, V, and R are defined in the context of formula Al.

[0265] In an embodiment, compounds of either of formulas I and la are provided whereinV is a single bond. In another embodiment, compounds of either of formulas I and la areAttorney Docket No. : 108843.00484(SU0451WO) provided wherein V is -NH-. In another embodiment, compounds of either of formulas I and la are provided wherein V is -CH2NH-.

[0266] In certain embodiments, the non-natural amino acid can be according to formula All:Formula All; or a salt thereof, wherein V and R are as defined in Formula Al. In certain embodiments according to this paragraph, V is -W1-W2-; one of Wi and W2 is absent or -CH2-, and the other is -NH-, -O-, or -S-. In certain embodiments, V is a single bond, -NH-, or -CH2NH-. In certain embodiments, V is a single bond or -CH2NH-; and R is methyl.

[0267] In certain embodiments, the non-natural amino acid can be according to formula AIII:Formula AIII; or a salt thereof, wherein V and R are as defined in Formula Al. In certain embodiments according to this paragraph, V is -W1-W2-; one of Wi and W2 is absent or -CH2-, and the other is -NH-, -O-, or -S-. In certain embodiments, V is a single bond, -NH-, or -CH2NH-. In certain embodiments, V is a single bond, -NH-, or -CH2NH-; and R is methyl.

[0268] In certain embodiments, the non-natural amino acid can be according to formula AIV:Attorney Docket No. : 108843.00484(SU0451WO)Formula AIV; or a salt thereof, wherein V and R are as defined in Formula Al. In certain embodiments according to this paragraph, V is -W1-W2-; one of Wi and W2 is absent or -CH2-, and the other is -NH-, -O-, or -S-. In certain embodiments, V is a single bond, -NH-, or -CH2NH-. In certain embodiments, V is a single bond, -NH-, or -CH2NH-; and R is methyl.

[0269] In certain embodiments, the non-natural amino acid can be according to formula AV:Formula AV; or a salt thereof, wherein V and R are as defined in Formula Al. In certain embodiments according to this paragraph, V is -W1-W2-; one of Wi and W2 is absent or -CH2-, and the other is -NH-, -O-, or -S-. In certain embodiments, V is a single bond, -NH-, or -CH2NH-. In certain embodiments, V is a single bond, -NH-, or -CH2NH-; and R is methyl.

[0270] In certain embodiments, the non-natural amino acid can be according to formula AVI:Attorney Docket No. : 108843.00484(SU0451WO)Formula AVI; or a salt thereof, wherein V and R are as defined in Formula Al. In certain embodiments according to this paragraph, V is -W1-W2-; one of Wi and W2 is absent or -CH2-, and the other is -NH-, -O-, or -S-. In certain embodiments, V is a single bond, -NH-, or -CH2NH-. In certain embodiments, V is a single bond, -NH-, or -CH2NH-; and R is methyl.

[0271] In certain embodiments, the non-natural amino acid can be according to formula AVII:Formula AVII; or a salt thereof, wherein V and R are as defined in Formula Al. In certain embodiments according to this paragraph, V is -W1-W2-; one of Wi and W2 is absent or -CH2-, and the other is -NH-, -O-, or -S-. In certain embodiments, V is a single bond, -NH-, or -CH2NH-. In certain embodiments, V is a single bond, -NH-, or -CH2NH-; and R is methyl.

[0272] In certain embodiments, the non-natural amino acid can be according to formula AVIII:Formula A VIII;Attorney Docket No. : 108843.00484(SU0451WO) or a salt thereof, wherein V and R are as defined in Formula Al. In certain embodiments according to this paragraph, V is -W1-W2-; one of Wi and W2 is absent or -CH2-, and the other is -NH-, -O-, or -S-. In certain embodiments, V is a single bond, -NH-, or -CH2NH-. In certain embodiments, V is a single bond, -NH-, or -CH2NH-; and R is methyl.

[0273] In certain embodiments, the non-natural amino acid can be according to formula AIX:Formula AIX; or a salt thereof, wherein V and R are as defined in Formula Al. In certain embodiments according to this paragraph, V is -W1-W2-; one of Wi and W2 is absent or -CH2-, and the other is -NH-, -O-, or -S-. In certain embodiments, V is a single bond, -NH-, or -CH2NH-. In certain embodiments, V is a single bond, -NH-, or -CH2NH-; and R is methyl.

[0274] In certain embodiments, the non-natural amino acid is according to any of formulas 51-62:Attorney Docket No. : 108843.00484(SU0451WO)or a salt thereof.

[0275] In certain embodiments, the non-natural amino acid is selected from the group consisting of compounds 30, 53, 56, 59, 60, 61, and 62 above. In certain embodiments, the nonnatural amino acid is compound 30. In certain embodiments, the non-natural amino acid is compound 56. In some embodiments, the non-natural amino acid is compound 61. In some embodiments, the non-natural amino acid is compound 62.

[0276] In some embodiments, non-natural amino acids comprising tetrazine functional groups are used in conjunction with non-natural amino acids comprising other functional groups. This approach may be used, for example, to produce antibodies comprising non-natural amino acid residues with two or more different types of functional groups. In some embodiments, the antibodies comprise non-natural amino acids with three or more different types of functional groups.

[0277] In advantageous embodiments, provided are antibodies comprising one or more of the non-natural amino acids comprising tetrazine moieties, described herein, along with one or more other non-natural amino acids comprising an azide moiety. This combination of reactiveAttorney Docket No. : 108843.00484(SU0451WO) amino acids facilitates two, independent reactions at specific sites on the antibody. A molecule comprising a strained alkene can selectively react with the one or more tetrazine moieties. Another molecule comprising an alkyne group can react with the one or more azide moieties. Advantageously, there can be little or no cross-reaction between the tetrazine ligations and the azide-alkyne condensations.

[0278] Incorporation of both tetrazine and azide functionality into a single antibody enables, for example, controlled conjugation of more than one payload molecule to the polypeptide chain. For example, in some embodiments, a first payload comprises a strained alkene functional group, enabling reaction with amino acid residues comprising tetrazine, while a second payload comprises an alkyne functional group, enabling reaction with amino acid residues comprising azide. Further payloads, comprising additional functional groups may also be used. The functional groups carried by such further payloads may react with any other suitable functional group, such as a functional group on a further non-natural amino acid residue or a natural amino acid residue.

[0279] In some embodiments, a non-natural amino acid comprising an aliphatic group may be incorporated into an antibody. Compositions and methods for the incorporation of non- natural amino acids comprising aliphatic groups into polypeptides is described in WO 2010 / 139948, which is incorporated by reference in its entirety. Incorporation of non-natural amino acids comprising aliphatic groups can be advantageous, for example, in instances where incorporation of non-natural amino acids comprising aromatic groups would cause misfolding or a loss of protein function. The non-natural amino acids comprising aliphatic groups may comprise any suitable bio-orthogonal functional groups for use in chemical reactions, including any of the bio-orthogonal groups described herein. In certain embodiments, the non-natural amino acids comprising aliphatic groups comprise an azide functional group. In some embodiments, the non-natural amino acids comprising aliphatic groups comprise a tetrazine functional group.

[0280] Any suitable non-natural aliphatic amino acid may be used. Examples of suitable non-natural aliphatic amino acids include N6-[(2-propynyloxy)carbonyl]-L-lysine, N6-[(2- azidoethoxy)carbonyl]-L-lysine, and (S)-2-amino-6((pent-4-enyloxy)carbonylamino)hexanoic acid.

[0281] The non-natural aliphatic amino acids may be incorporated into a polypeptide chain by utilizing a suitable aminoacyl tRNA synthetase, such as any of the suitable aminoacyl tRNA synthetases disclosed in WO 2010 / 139948, which is incorporated by reference in its entirety.Attorney Docket No. : 108843.00484(SU0451WO)In certain embodiments, an orthogonal Methanosarcina barkeri MS pyrrolysyl-tRNA synthetase / tRNAcuA pair may be used to direct efficient, site-specific incorporation of nonnatural aliphatic amino acids into polypeptides. The Methanosarcina barkeri PylS gene encodes the MbPyIRS tRNA synthetase protein, and the PylT gene encodes the MbtRNAcuA tRNA.

[0282] A non-natural aliphatic amino acid may be used to replace any naturally occurring amino acid other than tryptophan, phenylalanine, or tyrosine. In some embodiments, a nonnatural aliphatic amino acid may be used to replace any non-aromatic amino acid. In some embodiments, a non-natural aliphatic amino acid may be used to replace any aliphatic amino acid. In some embodiments, a non-natural aliphatic amino acid may be used to replace an amino acid selected from lysine, aspartic acid, serine, cysteine, threonine, valine or isoleucine. In some embodiments, a non-natural aliphatic amino acid may be used to replace any of serine, cysteine, threonine, valine or isoleucine. In some embodiments, a non-natural aliphatic amino acid may be used to replace a charged amino acid such as lysine or aspartic acid. In some embodiments, a non-natural aliphatic amino acid may be used to replace a hydroxyl-type amino acid such as serine, cysteine, or threonine. In particularly advantageous embodiments, a non- natural aliphatic amino acid may be used to replace valine or isoleucine.Antibody conjugates

[0283] In some aspect, the present disclosure provides antibody conjugates comprising antibodies herein linked to one or more payloads via the one or more non-natural amino acid residues. In certain embodiments, the antibody is linked to one payload. In further embodiments, the antibody is linked to more than one payload. In certain embodiments, the antibody is linked to two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more payloads.Payloads

[0284] The payload can be any molecular entity that one of skill in the art might desire to conjugate to the antibody. In certain embodiments, the payload is a therapeutic moiety. In such embodiment, the antibody conjugate can be used to target the therapeutic moiety to its molecular target. In certain embodiments, the payload comprises a labeling moiety. In such embodiments, the antibody conjugate can be used to detect binding of the antibody to its target. In certain embodiments, the payload is a cytotoxic moiety. In such embodiments, the conjugate can be used target the cytotoxic moiety to a diseased cell, for example a cancer cell, to initiateAttorney Docket No. : 108843.00484(SU0451WO) destruction or elimination of the cell. Conjugates comprising other molecular payloads apparent to those of skill in the art are within the scope of the conjugates described herein.

[0285] In certain embodiments, a conjugate can have a payload selected from the group consisting of a label, a dye, a polymer, a water-soluble polymer, polyethylene glycol, a derivative of polyethylene glycol, a photocrosslinker, a cytotoxic compound, a radionuclide, 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, a DNA, a RNA, an antisense polynucleotide, a peptide, a water-soluble dendrimer, 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 covalently or noncovalently interacts with other molecules, a photocaged moiety, a photoisomerizable moiety, biotin, a derivative of biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, an elongated side chain, a carbon-linked sugar, a redox-active agent, an amino thioacid, 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, or any combination thereof.

[0286] In some embodiments, the payload is an alkylating agent, a DNA-crosslinking agent, an anti-tumor antibiotic, an anti-metabolite, an anti-mitotic agent, a histone-deacetylase (HD AC) inhibitor, a telomerase inhibitor, an immunogenic cell death agent, a tubulin inhibitor, or a topoisomerase inhibitor. In some embodiments, the payload is a topoisomerase I inhibitor, e.g., camptothecin, irinotecan, SN-38, topotecan, or exatecan. In one example, the payload is exatecan.

[0287] In some embodiments, the payload comprises a P-glucuronide linker-payload, e.g., a P-glucuronide exatecan linker-payload. In one example, the P-glucuronide exatecan linkerpayload is or comprises the linker-payload of the following structureAttorney Docket No. : 108843.00484(SU0451WO)salt thereof. Additional examples of 0- glucuronide linker-payloads include those described in W02024006272, which is incorporated by reference herein in its entirety.

[0288] Useful drug payloads include any cytotoxic, cytostatic or immunomodulatory drug. Useful classes of cytotoxic or immunomodulatory agents include, for example, STING agonists, TLR agonists, anti tubulin agents, auri statins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, calmodulin inhibitors, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, maytansinoids, nitrosoureas, platinols, pore-forming compounds, purine antimetabolites, puromycins, radiation sensitizers, rapamycins, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like.

[0289] Individual cytotoxic or immunomodulatory agents include, for example, an androgen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, calicheamicin, calicheamicin derivatives, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin), daunorubicin, decarbazine, DM1, DM4, docetaxel, doxorubicin, etoposide, an estrogen, 5 -fluordeoxyuridine, 5-fluorouracil, gemcitabine, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), maytansine, mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel, palytoxin, plicamycin, procarbizine,Attorney Docket No. : 108843.00484(SU0451WO) rhizoxin, streptozotocin, tenoposide, 6-thioguanine, thioTEPA, topotecan, vinblastine, vincristine, vinorelbine, VP- 16 and VM-26.

[0290] In some embodiments, suitable cytotoxic agents include, for example, DNA minor groove binders (e.g., enediynes and lexitropsins, a CBI compound; see also U.S. Pat. No. 6,130,237), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholinodoxorubicin, echinomycin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophycins, cemadotin, maytansinoids, discodermolide, eleutherobin, and mitoxantrone.

[0291] In some embodiments, the payload is an anti-tubulin agent. Examples of antitubulin agents include, but are not limited to, taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik) and vinca alkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine). Other antitubulin agents include, for example, baccatin derivatives, taxane analogs, epothilones (e.g., epothilone A and B), nocodazole, colchicine and colcimid, estramustine, cryptophycins, cemadotin, maytansinoids, combretastatins, discodermolide, and eleutherobin.

[0292] In certain embodiments, the cytotoxic agent is a maytansinoid, another group of anti-tubulin agents. For example, in specific embodiments, the maytansinoid can be maytansine or DM-1 (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res. 52: 127-131).

[0293] In some embodiments, the payload is an auristatin, such as auristatin E or a derivative thereof. For example, the auristatin E derivative can be an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatin derivatives include AFP, MMAF, and MMAE. The synthesis and structure of auristatin derivatives are described in U.S. Patent Application Publication Nos. 2003-0083263, 2005- 0238649 and 2005-0009751; International Patent Publication No. WO 04 / 010957, International Patent Publication No. WO 02 / 088172, and U.S. Pat. Nos. 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414.

[0294] In some embodiments, the payload is not a radioisotope. In some embodiments, the payload is not radioactive.

[0295] In some embodiments, the payload is an antimetabolite. The antimetabolite can be, for example, a purine antagonist (e.g., azothioprine or mycophenolate mofetil), a dihydrofolateAttorney Docket No. : 108843.00484(SU0451WO) reductase inhibitor (e.g., methotrexate), acyclovir, ganciclovir, zidovudine, vidarabine, ribavarin, azidothymidine, cytidine arabinoside, amantadine, dideoxyuridine, iododeoxyuridine, poscarnet, or trifluridine.

[0296] In other embodiments, the payload is tacrolimus, cyclosporine, FU506 or rapamycin. In further embodiments, the Drug is aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, bexarotene, bexarotene, calusterone, capecitabine, celecoxib, cladribine, Darbepoetin alfa, Denileukin diftitox, dexrazoxane, dromostanolone propionate, epirubicin, Epoetin alfa, estramustine, exemestane, Filgrastim, floxuridine, fludarabine, fulvestrant, gemcitabine, gemtuzumab ozogamicin (MYLOTARG), goserelin, idarubicin, ifosfamide, imatinib mesylate, Interferon alfa-2a, irinotecan, letrozole, leucovorin, levamisole, meclorethamine or nitrogen mustard, megestrol, mesna, methotrexate, methoxsalen, mitomycin C, mitotane, nandrolone phenpropionate, oprelvekin, oxaliplatin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pentostatin, pipobroman, plicamycin, porfimer sodium, procarbazine, quinacrine, rasburicase, Rituximab, Sargramostim, streptozocin, tamoxifen, temozolomide, teniposide, testolactone, thioguanine, toremifene, Tositumomab, Trastuzumab (HERCEPTIN), tretinoin, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine or zoledronate.

[0297] In some embodiments, the payload is an immunomodulatory agent. The immunomodulatory agent can be, for example, ganciclovir, etanercept, tacrolimus, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil or methotrexate. Alternatively, the immunomodulatory agent can be, for example, a glucocorticoid (e.g., cortisol or aldosterone) or a glucocorticoid analogue (e.g., prednisone or dexamethasone).

[0298] In some embodiments, the immunomodulatory agent is an anti-inflammatory agent, such as arylcarboxylic derivatives, pyrazole-containing derivatives, oxicam derivatives and nicotinic acid derivatives. Classes of anti-inflammatory agents include, for example, cyclooxygenase inhibitors, 5 -lipoxygenase inhibitors, and leukotriene receptor antagonists.

[0299] Suitable cyclooxygenase inhibitors include meclofenamic acid, mefenamic acid, carprofen, diclofenac, diflunisal, fenbufen, fenoprofen, indomethacin, ketoprofen, nabumetone, sulindac, tenoxicam and tolmetin.

[0300] Suitable lipoxygenase inhibitors include redox inhibitors (e.g., catechol butane derivatives, nordihydroguaiaretic acid (NDGA), masoprocol, phenidone, lanopalen, indazolinones, naphazatrom, benzofuranol, alkylhydroxylamine), and non-redox inhibitorsAttorney Docket No. : 108843.00484(SU0451WO)(e.g., hydroxythiazoles, methoxyalkylthiazoles, benzopyrans and derivatives thereof, methoxytetrahydropyran, boswellic acids and acetylated derivatives of boswellic acids, and quinolinemethoxyphenylacetic acids substituted with cycloalkyl radicals), and precursors of redox inhibitors.

[0301] Other suitable lipoxygenase inhibitors include antioxidants (e.g., phenols, propyl gallate, flavonoids and / or naturally occurring substrates containing flavonoids, hydroxylated derivatives of the flavones, flavonol, dihydroquercetin, luteolin, galangin, orobol, derivatives of chaicone, 4,2',4'-trihydroxychalcone, ortho-aminophenols, N-hydroxyureas, benzofuranols, ebselen and species that increase the activity of the reducing selenoenzymes), iron chelating agents (e.g., hydroxamic acids and derivatives thereof, N-hydroxyureas, 2 -benzyl- 1 -naphthol, catechols, hydroxylamines, carnosol trolox C, catechol, naphthol, sulfasalazine, zyleuton, 5- hydroxyanthranilic acid and 4-(omega-arylalkyl)phenylalkanoic acids), imidazole-containing compounds (e.g., ketoconazole and itraconazole), phenothiazines, and benzopyran derivatives.

[0302] Yet other suitable lipoxygenase inhibitors include inhibitors of eicosanoids (e.g., octadecatetraenoic, eicosatetraenoic, docosapentaenoic, eicosahexaenoic and docosahexaenoic acids and esters thereof, PGE1 (prostaglandin El), PGA2 (prostaglandin A2), viprostol, 15- monohydroxy eicosatetraenoic, 15-monohydroxy-eicosatrienoic and 15- monohydroxy eicosapentaenoic acids, and leukotrienes B5, C5 and D5), compounds interfering with calcium flows, phenothiazines, diphenylbutylamines, verapamil, fuscoside, curcumin, chlorogenic acid, caffeic acid, 5,8,11,14-eicosatetrayenoic acid (ETYA), hydroxyphenylretinamide, lonapalen, esculin, diethylcarbamazine, phenantroline, baicalein, proxi cromil, thioethers, diallyl sulfide and di-(l -propenyl) sulfide.

[0303] Leukotriene receptor antagonists include calcitriol, ontazolast, Bayer Bay-x-1005, Ciba-Geigy CGS-25019C, ebselen, Leo Denmark ETH-615, Lilly LY-293111, Ono ONO- 4057, Terumo TMK-688, Boehringer Ingleheim BI-RM-270, Lilly LY 213024, Lilly LY 264086, Lilly LY 292728, Ono ONO LB457, Pfizer 105696, Perdue Frederick PF 10042, Rhone-Poulenc Rorer RP 66153, SmithKline Beecham SB-201146, SmithKline Beecham SB- 201993, SmithKline Beecham SB-209247, Searle SC-53228, Sumitamo SM 15178, American Home Products WAY 121006, Bayer Bay-o-8276, Warner-Lambert CI-987, Warner-Lambert CI-987BPC-15LY 223982, Lilly LY 233569, Lilly LY-255283, MacroNex MNX-160, Merck and Co. MK-591, Merck and Co. MK-886, Ono ONO-LB-448, Purdue Frederick PF-5901, Rhone-Poulenc Rorer RG14893, Rhone-Poulenc Rorer RP 66364, Rhone-Poulenc Rorer RP 69698, Shionoogi S-2474, Searle SC-41930, Searle SC-50505, Searle SC-51146, Searle SC-Attorney Docket No. : 108843.00484(SU0451WO)52798, SmithKline Beecham SK&F-104493, Leo Denmark SR-2566, Tanabe T-757 and Teijin TEI-1338.

[0304] Other useful drug payloads include chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include Erlotinib (TARCEVA®, Genentech / OSI Pharm.), Bortezomib (VELCADE®, Millennium Pharm.), Fulvestrant (FASLODEX®, AstraZeneca), Sutent (SU11248, Pfizer), Letrozole (FEMARA®, Novartis), Imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK 222584 (Novartis), Oxaliplatin (Eloxatin®, Sanofi), 5- FU (5 -fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), Lonafamib (SCH 66336), Sorafenib (BAY43- 9006, Bayer Labs), and Gefitinib (IRESSA®, AstraZeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin yll and calicheamicin omegall (Angew Chem. Inti. Ed. Engl. (1994) 33: 183-186); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin,Attorney Docket No. : 108843.00484(SU0451WO) streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamniprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"-tri chlorotri ethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® (doxetaxel; Rhone-Poulenc Rorer, Antony, France); chloranmbucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

[0305] Other useful payloads include: (i) anti -hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example,Attorney Docket No. : 108843.00484(SU0451WO)4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); (iii) anti -androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3- dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-a, Ralf and H-Ras; (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; a topoisomerase 1 inhibitor such as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such as bevacizumab (AVASTIN®, Genentech); and (x) pharmaceutically acceptable salts, acids and derivatives of any of the above. Other anti -angiogenic agents include MMP-2 (matrixmetalloproteinase 2) inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, COX-II (cyclooxygenase II) inhibitors, and VEGF receptor tyrosine kinase inhibitors. Examples of such useful matrix metalloproteinase inhibitors that can be used in combination with the present compounds / compositions are described in WO 96 / 33172, WO 96 / 27583, EP 818442, EP 1004578, WO 98 / 07697, WO 98 / 03516, WO 98 / 34918, WO 98 / 34915, WO 98 / 33768, WO 98 / 30566, EP 606,046, EP 931,788, WO 90 / 05719, WO 99 / 52910, WO 99 / 52889, WO 99 / 29667, WO 99 / 07675, EP 945864, U.S. Pat. No. 5,863,949, U.S. Pat. No. 5,861,510, and EP 780,386, all of which are incorporated herein in their entireties by reference. Examples of VEGF receptor tyrosine kinase inhibitors include 4-(4-bromo-2-fluoroanilino)-6-methoxy-7- (l-methylpiperidin-4-ylmethoxy)qu- -inazoline (ZD6474; Example 2 within WO 01 / 32651), 4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-l-ylpropoxy)- -quinazoline (AZD2171; Example 240 within WO 00 / 47212), vatalanib (PTK787; WO 98 / 35985) and SU11248 (sunitinib; WO 01 / 60814), and compounds such as those disclosed in PCT Publication Nos. WO 97 / 22596, WO 97 / 30035, WO 97 / 32856, and WO 98 / 13354).

[0306] In certain embodiments, the payload is an antibody or an antibody fragment. In certain embodiments, the payload antibody or fragment can be encoded by any of the immunoglobulin genes recognized by those of skill in the art. The immunoglobulin genes include, but are not limited to, the K, , a, y (IgGl, IgG2, IgG3, and IgG4), 5, a and p constant region genes, as well as the immunoglobulin variable region genes. The term includes full- length antibodies and antibody fragments recognized by those of skill in the art, and variantsAttorney Docket No. : 108843.00484(SU0451WO) thereof. Exemplary 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 CDR's, variable regions, framework regions, constant regions, and the like.

[0307] In certain embodiments, the payload is one or more water-soluble polymers. A wide variety of macromolecular polymers and other molecules can be linked to antigen-binding polypeptides to modulate biological properties of the antibody, and / or provide new biological properties to the antibody. These macromolecular polymers can be linked to the antibody via a naturally encoded amino acid, via a non-natural amino acid, or any functional substituent of a natural or non-natural amino acid, or any substituent or functional group added to a natural or non-natural amino acid. The molecular weight of the polymer may be of a wide range, including but not limited to, between about 100 Da and about 100,000 Da or more.

[0308] The polymer selected may be water soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. The polymer may be branched or unbranched. Preferably, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable.

[0309] The proportion of polyethylene glycol molecules to antibody molecules will vary, as will their concentrations in the reaction mixture. In general, the optimum ratio (in terms of efficiency of reaction in that there is minimal excess unreacted protein or polymer) may be determined by the molecular weight of the polyethylene glycol selected and on the number of available reactive groups available. As relates to molecular weight, typically the higher the molecular weight of the polymer, the fewer number of polymer molecules which may be attached to the protein. Similarly, branching of the polymer should be taken into account when optimizing these parameters. Generally, the higher the molecular weight (or the more branches) the higher the polymerprotein ratio.

[0310] The water-soluble polymer may be any structural form including but not limited to linear, forked or branched. Typically, the water soluble polymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG), but other water soluble polymers can also be employed. By way of example, PEG is used to describe certain embodiments of this invention.

[0311] PEG is a well-known, water-soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). The term “PEG” is used broadly to encompass any polyethylene glycolAttorney Docket No. : 108843.00484(SU0451WO) molecule, without regard to size or to modification at an end of the PEG, and can be represented as linked to the antibody by the formula: XO^CEECEEO) ^-CEECEE-Y where n is 2 to 10,000 and X is H or a terminal modification, including but not limited to, a Ci-4 alkyl.

[0312] In some cases, a PEG used in the invention terminates on one end with hydroxy or methoxy, i.e., X is H or CH3 (“methoxy PEG”). Alternatively, the PEG can terminate with a reactive group, thereby forming a bifunctional polymer. Typical reactive groups can include those reactive groups that are commonly used to react with...

Claims

Attorney Docket No. : 108843.00484(SU0451WO)WHAT IS CLAIMED IS:

1. An antibody comprising one or more non-natural amino acid residues at one or more positions selected from the group consisting of HC-S113 according to Kabat numbering, HC- A118, HC-T120, HC-K121, HC-G138, HC-A140, HC-T155, HC-S160, HC-A162, HC-L163, HC-Q196, HC-I199, HC-N203, HC-T209, HC-K210, HC-K246, HC-E258, HC-K290, HC- E294, HC-Q295, HC-Y296, HC-N297, HC-Y300, HC-V303, HC-V305, HC- E318, HC- A330, HC-T335, HC-I336, HC-S337, HC-R344, HC-E345, HC-R355, HC-M358, HC-Q362, HC-E382, HC-E388, HC-N390, HC-Y391, HC-K392, HC-T393, HC-T411, HC- N421, HC- S424, HC-Q438, HC-L443 according to EU index of Kabat, LC-T109, LC-V110, LC-A111, LC-A112, LC-D122, LC-Q124, LC-K126, LC-S127, LC-T129, LC-N138, LC-R142, LC- E143, LC-Q147, LC-K149, LC-D151, LC-A153, LC-L154, LC-Q155, LC-S159, LC-E165, LC-Q166, LC-S168, LC-K169, LC-S171, LC-T172, LC-T180, LC-A184, LC-E187, LC- H189, LC-K190, LC-V191, LC-E195, LC-L201, LC-S202, LC-S203, LC-V205, and LC- T206 according to Kabat numbering, or a post-translationally modified variant thereof.

2. The antibody of claim 1, comprising one or more non-natural amino acid residues at one or more positions selected from the group consisting of HC-S113 according to Kabat numbering, HC-A118, HC-T120, HC-K121, HC-G138, HC-A140, HC-T155, HC-S160, HC- A162, HC-L163, HC-Q196, HC-H99, HC-N203, HC-T209, HC-K210, HC-E258, HC-K290, HC-Y296, HC-T335, HC-I336, HC-S337, HC-R344, HC-E345, HC-R355, HC-M358, HC- Q362, HC-E382, HC-E388, HC-N390, HC-Y391, HC-K392, HC-T393, HC-T411, HC- N421, HC-S424, HC-Q438, HC-L443 according to EU index of Kabat, LC-T109, LC-V110, LC-A111, LC-A112, LC-D122, LC-Q124, LC-K126, LC-S127, LC-T129, LC-N138, LC- R142, LC-E143, LC-Q147, LC-K149, LC-D151, LC-A153, LC-L154, LC-Q155, LC-S159, LC-E165, LC-Q166, LC-S168, LC-K169, LC-S171, LC-T172, LC-T180, LC-A184, LC- E187, LC-H189, LC-K190, LC-V191, LC-E195, LC-L201, LC-S202, LC-S203, LC-V205, and LC-T206 according to Kabat numbering, or a post-translationally modified variant thereof.

3. The antibody of claim 1, which comprises one or more non-natural amino acid residues at one or more positions selected from the group consisting of HC-Q196, HC-Y391, and HC-S337 according to EU index of Kabat, or a post-translationally modified variant thereof.Attorney Docket No. : 108843.00484(SU0451WO)4. The antibody of claim 1, which comprises a non-natural amino acid residue at HC- Y391 according to EU index of Kabat, or a post-translationally modified variant thereof.

5. The antibody of any one of claims 1-4, further comprising one or more additional non-natural amino acid residues at one or more additional positions selected from the group consisting of HC-F404, HC-K121, HC-Y180, HC-F241, HC-221, LC-T22, LC-S7, LC-N152, LC-K42, LC-E161, LC-D170, HC-S136, HC-S25, HC-A40, HC-S119, HC-S190, HC-K222, HC-R19, HC-Y52, and HC-S70 according to EU index of Kabat, or a post translationally modified variant thereof.

6. The antibody of claim 5, wherein the one or more additional non-natural amino acid residues is at one or more positions selected from the group consisting of HC-Y180, HC- F241, HC-F404 HC, and LC-K42 according to EU index of Kabat, or a post-translationally modified variant thereof.

7. The antibody of any of the preceding claims, wherein the antibody comprises a heavy chain of a type selected from the group consisting of a, y, 5, a, and p.

8. The antibody of any of the preceding claims, wherein the antibody comprises a light chain of a type selected from the group consisting of and K.

9. The antibody of claim 5, wherein the light chain is of type K.

10. The antibody of any of the preceding claims, wherein the antibody is of a class or subclass selected from the group consisting of IgA, IgA, IgA2, IgD, IgE, IgG, and IgM.

11. The antibody of claim 10, wherein the antibody is an IgG antibody.

12. The antibody of claim 11, wherein the IgG antibody is an IgGl, IgG2, or IgG3 antibody.

13. The antibody of any of the preceding claims, wherein the antibody is in a form selected from the group consisting of Fv, Fc, Fab, (Fab')2, single chain Fv (scFv) and full- length antibody.Attorney Docket No. : 108843.00484(SU0451WO)14. The antibody of any of the preceding claims, wherein at least one of the one or more non-natural amino acid residues comprises a moiety selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, alkynyl, and tetrazine.

15. The antibody of any of the preceding claims, wherein at least one of the one or more non-natural amino acid residues comprises an azide moiety.

16. The antibody of any of the preceding claims, wherein at least one of the one or more non-natural amino acid residues comprises an acetyl moiety.

17. The antibody of any of the preceding claims, wherein at least one of the one or more non-natural amino acid residues comprises a tetrazine moiety.

18. The antibody of any of the preceding claims, wherein one or more non-natural amino acid residue is according to the formulawherein each L is independently a divalent linker; each R is independently a functional group; and each wavy line indicates a bond that connects to remainder of a polypeptide chain of the antibody.

19. The antibody of claim 18, wherein R is a reactive group, a therapeutic moiety, or a labeling moiety.

20. The antibody of claim 19, wherein R is a reactive group selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, alkynyl, and tetrazine.Attorney Docket No. : 108843.00484 (SU0451WO)21. The antibody of claim 18, wherein L is a divalent linker selected from the group consisting of a bond, alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene, substituted arylene, heteroarlyene and substituted heteroarylene.

22. The antibody of any one of the preceding claims, wherein at least one of the one or more non-natural amino acid residue is selected from the group consisting of orthosubstituted tyrosine, meta substituted tyrosine, para-substituted phenylalanine, orthosubstituted phenylalanine, and meta-substituted phenylalanine.

23. The antibody of claim 22, wherein at least one of the one or more non-natural amino acid residue is selected from the group consisting of / ?-acetyl-L-phenylalanine, O-methyl-L- tyrosine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, fluorinated phenylalanine, isopropyl-L-phenylalanine, / ?-azido-L-phenylalanine, / ?-acyl-L-phenylalanine, / ?-A benzoyl-L-phenylalanine, / ?-iodo-phenyl alanine, / ?-bromophenylalanine, / ?-amino-L- phenylalanine, isopropyl-L-phenylalanine, -propargyloxy -phenylalanine, and p- azi dom ethyl -L-pheny 1 al anine .

24. The antibody of claim 23, wherein at least one of the one or more non-natural amino acid residue is / ?-azidomethyl-L-phenylalanine.

25. The antibody of claim 23, wherein at least one of the one or more non-natural amino acid residue is / ?-acetyl-L-phenylalanine.

26. The antibody of any of the preceding claims, wherein at least one of the one or more additional non-natural amino acid residues comprises a moiety selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, alkynyl, and tetrazine.

27. The antibody of claim 26, wherein at least one of the one or more additional non- natural amino acid residues comprises an azide moiety.

28. The antibody of claim 26, wherein at least one of the one or more additional non- natural amino acid residues comprises an acetyl moiety.Attorney Docket No. : 108843.00484(SU0451WO)29. The antibody of claim 26, wherein at least one of the one or more additional nonnatural amino acid residues comprises a tetrazine moiety.

30. The antibody of any one of the preceding claims, wherein the antibody is aglycosylated.

31. The antibody of any one of the preceding claims, wherein the antibody is glycosylated.

32. An antibody conjugate comprising the antibody of any of the preceding claims linked to one or more payloads via the one or more non-natural amino acid residues.

33. The antibody conjugate of claim 32, wherein the one or more payloads comprises one or more therapeutic moieties.

34. The antibody conjugate of claim 33, wherein one or more therapeutic moieties comprises an immunomodulatory agent, an alkylating agent, a DNA-crosslinking agent, an anti-tumor antibiotic, an anti-metabolite, an anti-mitotic agent, a histone-deacetylase (HD AC) inhibitor, a telomerase inhibitor, an immunogenic cell death agent, a tubulin inhibitor, or a topoisomerase inhibitor.

35. The antibody conjugate of claim 33, wherein one or more therapeutic moieties comprises a topoisomerase I inhibitor and the topoisomerase I inhibitor is selected from the group consisting of camptothecin, irinotecan, SN-38, topotecan, and exatecan.

36. The antibody conjugate of claim 35, wherein the topoisomerase I inhibitor is exatecan.

37. The antibody conjugate of claim 33, wherein the one or more therapeutic moieties comprises a hemiasterlin.

38. The antibody conjugate of claim 33, wherein the one or more therapeutic moieties comprises an immunomodulatory agent.Attorney Docket No. : 108843.00484 (SU0451WO)39. The antibody conjugate of claim 38, wherein the immunomodulatory agent is a STING agonist.

40. The antibody conjugate of claim 38, wherein the immunomodulatory agent is a Tolllike receptor (TLR) agonist.

41. The antibody conjugate of claim 40, wherein the TLR agonist is a TLR 7 / 8 agonist.

42. The antibody conjugate of any one of claims 32-41, wherein the one or more payloads comprises one or more labeling moieties.

43. The antibody conjugate of any of claims 32-42, wherein the antibody is linked to the one or more payloads via one or more linkers.

44. The antibody conjugate of claim 43, wherein the linker is hydrolytically stable.

45. The antibody conjugate of claim 43, wherein the linker is cleavable.

46. The antibody conjugate of claim 43, wherein the linker is a protease cleavable linker, an enzyme cleavable linker, a pH-sensitive linker, or a non-cleavable linker.

47. An antibody conjugate represented by the structure of Formula (I):Formula (I) or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, regioisomer, or mixture of regioisomers thereof; wherein Ab is an anti-HER2 antibody or antigen-binding fragment thereof;Lais a first linker;Lbis a second linker;CY is independently a residue of a cytotoxic agent;STING is independently a residue of a STING agonist; subscript n is an integer selected from 1 to 10; and subscript m is an integer selected from I to KkAttorney Docket No. : 108843.00484(SU0451WO)48. The antibody conjugate of claim 47, wherein STING is independently a residue of a compound of Formula (S-I) or (S-II):or a tautomer or stereoisomer thereof; wherein:X is selected from N and CR3;R20is selected from hydrogen and -CON(R3a)(R3b);Rla, Rlb, R3aand R3bare independently selected from hydrogen and optionally substituted Ci-6 alkyl, wherein the Ci-6 alkyl is optionally substituted with one or more R50;R2aand R2bare independently selected from: (a) optionally substituted Ci- 6 alkyl, wherein the Ci-6 alkyl is optionally substituted with one or more R51and (b) optionally substituted C3-12 carbocycle or optionally substituted 3- to 12-membered heterocycle, each of which is optionally substituted with one or more R53; or Rlaand R2aare joined together with the atoms to which they are attached to form an optionally substituted 3- to 12-membered heterocycle, wherein the heterocycle is optionally substituted with one or more R53; or Rlband R2bare joined together with the atoms to which they are attached to form an optionally substituted 3- to 12-membered heterocycle, wherein the heterocycle is optionally substituted with one or more R53;R3is hydrogen, -OR30, -SR30, -C(O)N(R30)2, -N(R30)C(O)R30, -N(R30)C(O)N(R30)2, -N(R30)2, -C(O)R30, -C(O)OR30, -OC(O)R30, -NO2, or -CN;L1is selected from a bond, -Ci-ioalkylene-, -C2-ioalkenylene-, -C2. walkynylene-, -Ci-ealkylene-O-Ci-ealkylene-, -Ci-ealkylene-NH-Ci-ealkylene-, C3- ecarbocyclene, and -Ci-6alkylene-(C3-6carbocyclene)-Ci-6alkylene-, wherein -Ci-Attorney Docket No. : 108843.00484(SU0451WO)10 alkylene-, -C2-10 alkenylene-, -C2-10 alkynylene-, C3-6 carbocyclene, and each C1-6 alkylene of -Ci-ealkylene-O-Ci-ealkylene-, -Ci-ealkylene-NH-Ci-ealkylene-, and -Ci- 6alkylene-(C3-6carbocyclene)-Ci-6alkylene- are optionally substituted with one or more R50;L2is optionally substituted -C1-6 alkylene- or -Ci- ealkylene-O-, wherein the -C1-6 alkylene- or the C1-6 alkylene of -C1-6 alkylene-O- is optionally substituted with one or more R50and wherein the -C1-6 alkylene-O- is attached to the A ring, or Lbif the A ring is absent, via the oxygen atom;Ring B is an optionally substituted C3-12 carbocycle or an optionally substituted 3- to 12-membered heterocycle, wherein the C3-12 carbocycle and 3- to 12- membered heterocycle are optionally substituted with one or more R52;Ring A is absent; an optionally substituted bridged, fused, or spirocyclic bicyclic heterocycle comprising at least one N atom and at least one O atom, wherein the heterocycle is optionally substituted with one or more R53; or, a 3- to 12- membered heterocycle substituted with R4;R4is an optionally substituted 3- to 12-membered heterocycle optionally comprising at least one NR5and, wherein the heterocycle is optionally substituted with one or more R53;R5is independently selected from hydrogen, R6, -C(O)-Ci-6alkyl, -C(O)- heteroCi-ealkyl, C1-6 alkyl, and heteroCi-ealkyl wherein the C1-6 alkyl, either alone or part of another group, is optionally substituted with one or more R50;R6is independently an amino acid residue;R30is independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-12 carbocycle, and 3- to 12-membered heterocycle, each of which, except hydrogen, is optionally substituted with one or more R55;R50is independently selected from halogen, -OR60, -SR60, -C(O)NR60R61, -N(R60)C(O)R60, -N(R60)C(O)NR60R61, -NR60R61, -C(O)R60, -C(O)OR60, -OC(O)R60, -NO2, =0, =S, =N(R60), -CN, C3-12 carbocycle, and 3- to 12-membered heterocycle;R51is independently selected from halogen, -OR60, -SR60, -C(O)NR60R61, -N(R60)C(O)R60, -N(R60)C(O)NR60R61, -NR60R61, -C(O)R60, -C(O)OR60, -OC(O)R60, -NO2, =0, =S, =N(R60), -CN, optionally substituted C3-12 carbocycle, and optionally substituted 3- to 12-membered heterocycle, wherein the C3-12 carbocycle and 3- to 12- membered heterocycle are optionally substituted with one or more R52;Attorney Docket No. : 108843.00484 (SU0451WO)R52is independently selected from halogen, -OR61, -SR61, -C(O)N(R61)2, -N(R61)C(O)R61, -N(R61)C(O)N(R61)2, -N(R61)2, -C(O)R61, -C(O)OR61, -OC(O)R61, -NO2, =0, =S, =N(R61), -CN, Ci-6alkyl, C2.6alkenyl, and C2-6 alkynyl;R53is independently selected from halogen, -OR60, -SR60, -C(O)NR60R61, -N(R60)C(O)R60, -N(R60)C(O)NR60R61, -NR60R61, -C(O)R60, -C(O)OR60, -OC(O)R60, -NO2, =0, =S, =N(R60), -CN, optionally substituted Ci-6 alkyl, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, optionally substituted C3- 12 carbocycle, and optionally substituted 3- to 12-membered heterocycle, wherein the C1-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl are optionally substituted with one or more R54and the C3-12 carbocycle and 3- to 12-membered heterocycle are optionally substituted with one or more R52;R54is independently selected from halogen, -OR61, -SR61, -C(O)N(R61)2, -N(R61)C(O)R61, -N(R61)C(O)N(R61)2, -N(R61)2, -C(O)R61, -C(O)OR61, -OC(O)R61, - NO2, =0, =S, =N(R61), and -CN;R55is independently selected from halogen, -CN, -NO2, -OH, -NR60R61, -C(O)NR60R61, =0, =S, Ci-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-12 carbocycle, 3- to 12- membered heterocycle, and Ci-io haloalkyl; andR60and R61are independently selected from hydrogen, C1-10 alkyl optionally substituted with NH2or NHC1-6 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-12 carbocycle, and 3- to 12-membered heterocycle; wherein when Ring A is absent, L2is -C1-6 alkylene-O-; wherein the residue of Formula (S-I) is attached to Lbvia an amino group of Ring A, the oxygen of -C1-6 alkylene-O- of L2, an amino group of R54, or an amino group of R4, and the residue of Formula (S-II) is attached to Lbvia an amino group of Ring B.

49. The antibody conjugate of claim 47 or 48, wherein CY is a residue of a compound selected from the group consisting of an alkylating agent, a DNA-crosslinking agent, an antitumor antibiotic, an anti-metabolite, an anti-mitotic agent, a histone-deacetylase (HD AC) inhibitor, a telomerase inhibitor, an immunogenic cell death agent, a tubulin inhibitor, and a topoisomerase inhibitor.Attorney Docket No. : 108843.00484 (SU0451WO)50. The antibody conjugate of claim 49, wherein CY is a topoisomerase I inhibitor selected from the group consisting of camptothecin, irinotecan, SN-38, topotecan, and exatecan.

51. The antibody conjugate of any one of claims 47-50, wherein Laand Lbindependently comprise a protease cleavable linker, an enzyme cleavable linker, a pH-sensitive linker, or a non-cleavable linker.

52. The antibody conjugate of any one of claims 47-51, whereinLais -W1-W2-SG1-HP1-RL1-;Lbis -RL01-HP01-SG01-W02-W01-; and the antibody conjugate of Formula (I) is represented by Formula (la):[CY-W1-W2-SG1-HP1-RL1-]m-Ab-[-RL01-HP01-SG01-W02-W01-STING]n(la) or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, regioisomer, or mixture of regioisomers thereof; wherein:Ab is the anti-TROP2 antibody or antigen-binding fragment thereof;CY is the residue of the cytotoxic agent;STING is the residue of the STING agonist;SG1and SG01are independently, at each occurrence, absent or a divalent spacer group;W1and W01are independently, at each occurrence, absent,wherein the -NH- is bound to W2or W02and eachindicates a point of attachment to the rest of the formula;L3is independently a bond or an optionally substituted Ci-6 alkylene wherein the Ci-ealkylene is optionally substituted with one, two, or three substituents selectedAttorney Docket No. : 108843.00484(SU0451WO) from halogen, Ci-6 alkyl, haloCi-6 alkyl, hydroxyl, amino, Ci-6 alkylamino, and Ci-6 alkoxy;R16is independently a residue of an amino acid sidechain;R11is independently hydrogen or Ci-6 alkyl; w is an integer selected from 0, 1, and 2;W2and W02are independently, at each occurrence, absent, an amino acid residue, or a peptide residue wherein the amino acid residue or peptide residue is optionally substituted with a HP2group;HP1and HP01are independently, at each occurrence, absent or a divalent hydrophilic group;HP2, when present, is a monovalent hydrophilic group;RAis independently, at each occurrence, optionally substituted Ci-6 alkyl wherein the Ci-6 alkyl is optionally substituted with one, two, or three substituents selected from halogen, haloCi-6 alkyl, hydroxyl, amino, alkylCi-6 amino, and Ci-6 alkoxy;O-Su is independently, at each occurrence, a release trigger group;RL1andRL01are independently, at each occurrence, a reactive linker; y is an integer independently selected from 0, 1, and 2; and z is an integer independently selected from 0 and 1.

53. The antibody conjugate of any one of claims 47-52, wherein Rlaand Rlbare both hydrogen.

54. The antibody conjugate of any one of claims 47-53, wherein R2aand R2bare independently selected fromwherein R2cand R2dare independently selected from hydrogen, Ci-6 alkyl optionally substituted with one or more R54, halo, and 3-12 membered heterocycle;R2eis hydrogen or Ci-6 alkyl; andAttorney Docket No. : 108843.00484(SU0451WO)is the point of attachment to the rest of the formula.

55. The antibody conjugate of any one of claims 47-54, wherein the STING is a residue of a compound of Formula (S-I) wherein Ring A is an optionally substituted 3- to 12- membered N-linked spirocyclic bicyclic heterocycle comprising at least one nitrogen atom, including the nitrogen bound to Lb, and at least one oxygen atom; or a 3- to 12- membered N- linked monocyclic heterocycle substituted with R4.

56. The antibody conjugate of any one of claims 47-55, whereinis selected fromAttorney Docket No. : 108843.00484(SU0451WO)57. The antibody conjugate of claim 56, whereinLt— j L2wherein * is the point of attachment to Lband * is the point of attachment to L2.

58. The antibody conjugate of any one of claims 47-57, wherein L2is -Ci-6 alkylene-.

59. The antibody conjugate of any one of claims 47-58, whereinW01is independently absent,L3is bond or -CH2- y is 0 or 1; and z is 0 or 1.

60. The antibody conjugate of claim 59, whereinwhereinrepresents attachment to the remainder of the compound.

61. The antibody conjugate of any one of claims 47-60, whereinW2and W02are independently absent or a group selected from -(C(O)CHR10NRn)o-, -(C(O)CH2CHR10NR11)P-, -(C(O)CHR10CH2NR11)P-, -(C(O)CH2CHR10NR11)P-(C(O)CHR10NR11)O-, and -(C(O)CHR10CH2NR11)P- (C(O)CHR10NRn)o-;Attorney Docket No. : 108843.00484 (SU0451WO) wherein R10is independently an amino acid sidechain residue optionally substituted with HP2;R11is independently hydrogen or Ci-6 alkyl; o and p are independently an integer between 1 and 10, inclusive; and the - C(O)- of W2and W02is bound to W1.

62. The antibody conjugate of claim 61, wherein each W2and W02is independently absent or -(C(O)CH2CHR10NR11)p-(C(O)CHR10NR11)o- whereinR10is an amino acid sidechain residue independently selected from valine, citrulline, alanine, glycine, leucine, asparagine, lysine and 2,3-diaminopropionate and wherein the amino acid sidechain is optionally substituted with HP2;HP2is independentlywherein h is an integer between 1 and 50, inclusivewherein g is an integer between 1 and 50; polypropylene glycoloxy-CH2CH2C(O)-); poly(oxyethylated polyol)oxy-CH2CH2C(O)-; poly(olefinic alcohol)oxy-CH2CH2C(O)-; poly(saccharides)-CH2CH2C(O)-; poly(vinyl alcohol)oxy-CH2CH2C(O)-; or a combination thereof;R11is hydrogen; o is independently an integer between 1 and 4, inclusive; p is 1 ; and eachis the point of attachment to the rest of the formula.

63. The antibody conjugate claim 61 or 62, wherein HP2iswherein g is an integer between 1 and 50, inclusive; and each is the point of attachment to the rest of the formula.Attorney Docket No. : 108843.00484(SU0451WO)64. The antibody conjugate of claim 61, wherein W2and W02is absent or independently65. The antibody conjugate of any one of claims 47-64, wherein SG1is independently -C(O)-Ci-6 alkylene-C(O)-, and SG01is independently absent or -Ci-6 alkylene-C(O)- wherein the -C(O)- is bound to W2and W02.

66. The antibody conjugate of any one of any one of claims 47-65, wherein each HP1andHP01is independently absent orX1wherein R2is hydrogen or methyl and xl is an integer between 1 and 50, inclusive; and eachis the point of attachment to the rest of the formula.

67. The antibody conjugate of any one of claims 47-66, wherein RL1and RL01isAttorney Docket No. : 108843.00484(SU0451WO)each is a point of attachment to the rest of the formula.

68. The antibody conjugate of claim 67, wherein RL1is independently selected fromis a point of attachment to the rest of the formula.

69. The antibody conjugate of claim 67 or 68, wherein RL01isand eachis a point of attachment to the rest of the formula.

70. The antibody conjugate of any one of claims 47-69, wherein CY is independently selected fromAttorney Docket No. : 108843.00484(SU0451WO)each is a point of attachment to the rest of the formula.

71. The antibody conjugate of claim 47-70, wherein CY is independently selected from72. The antibody conjugate of any one of claims 47-71, wherein STING is73. The antibody conjugate of any one of claims 47-71, wherein -[Lb-STING]nis independentlyAttorney Docket No. : 108843.00484(SU0451WO)Attorney Docket No. : 108843.00484(SU0451WO)wherein is the point of attachment to the rest of the formula.

74. The antibody conjugate of any one of claims 47-73, wherein -[Lb-STING]n- is a structure selected from:Attorney Docket No. : 108843.00484(SU0451WO)Attorney Docket No. : 108843.00484(SU0451WO)Attorney Docket No. : 108843.00484(SU0451WO)Attorney Docket No. : 108843.00484(SU0451WO)Attorney Docket No. : 108843.00484(SU0451WO)wherein is the point of attachment to the rest of the formula.

75. The antibody conjugate of any one of claims 44-71, wherein [CY-La-]m- is:Attorney Docket No. : 108843.00484(SU0451WO)Attorney Docket No. : 108843.00484(SU0451WO)whereinis the point of attachment to the rest of the formula76. The antibody conjugate of anyone of claims 44-72, whereinAttorney Docket No. : 108843.00484(SU0451WO)[CY-La-]m- is77. The antibody conjugate of any one of claims 47-76, wherein m and n is an integer independently between 1 and 8, inclusive.

78. The antibody conjugate of any one of claims 47-77, wherein the molar ratio of CY to STING is about 4: 1.

79. The antibody conjugate of any one of claims 47-78, wherein the anti-HER2 antibody or the antigen-binding fragment thereof comprises at least two modified amino acid residues suitable for conjugation that are covalently linked to Laand / or Lb.

80. The antibody conjugate of any one of claims 47-79, wherein (a) the anti-HER2 antibody or antigen-binding fragment thereof is covalently linked to Lavia a modified amino acid and (b) the anti-HER2 antibody or antigen binding fragment thereof is covalently linked to Lbvia a modified amino acid that is different than the modified amino acid in (a).Attorney Docket No. : 108843.00484(SU0451WO)81. The antibody conjugate of claim 79 or 80, wherein the modified amino acid residue is selected from the group consisting of p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, 3- methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L- phenylalanine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, p-propargyloxy-phenylalanine, and p-azidomethyl-L- phenylalanine.

82. The antibody conjugate of claim 81, wherein the modified amino acid residue is independently selected from p-azidomethyl-L-phenylalanine and p-acetyl-L-phenylalanine.

83. The antibody conjugate of any one of claims 47-82, wherein the modified amino acid residues are at positions selected from the group consisting of heavy chain or light chain residues HC-F404, HC-K121, HC-Y180, HC-F241, HC-221, HC-Y391, LC-T22, LC-S7, LC-N152, LC-K42, LC-E161, LC-D170, HC-S136, HC-S25, HC-A40, HC-S119, HC-S190, HC-K222, HC-R19, HC-Y52, or HC-S70, according to the Kabat or Chothia or EU numbering scheme, or a post translationally modified variant thereof.

84. The antibody conjugate of claim 83, wherein the modified amino acid residues are at positions selected from the group consisting of heavy chain or light chain residues HC-F241, HC-F404, HC-Y180, HC-Y391, and LC-K42.

85. The antibody conjugate of claim 84, wherein the modified amino acid residues are p- azidomethyl-L-phenylalanine at HC-F404, HC-F241, HC-Y391 and / or HC-Y180, and p- acetyl-L-phenylalanine at LC-K42, according to the Kabat or Chothia or EU numbering scheme, or a post translationally modified variant thereof. The antibody conjugate of any one of claims 44-82, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein:(i) the heavy chain variable region comprises a CDR1 consisting of an amino acid sequence of SEQ ID NO: 1, a CDR2 consisting of an amino acid sequence of SEQ ID NO: 2, and a CDR3 consisting of an amino acid sequence of SEQ ID NO: 3, and the light chain variable region comprises a CDR1 consisting of an amino acid sequence of SEQ ID NO: 8, aAttorney Docket No. : 108843.00484(SU0451WO)CDR2 consisting of an amino acid sequence of SEQ ID NO: 9, and a CDR3 consisting of an amino acid sequence of SEQ ID NO: 10; or(ii) the heavy chain variable region comprises a CDR1 consisting of an amino acid sequence of SEQ ID NO: 4, a CDR2 consisting of an amino acid sequence of SEQ ID NO: 5, and a CDR3 consisting of an amino acid sequence of SEQ ID NO: 3, and the light chain variable region comprises a CDR1 consisting of an amino acid sequence of SEQ ID NO: 8, a CDR2 consisting of an amino acid sequence of SEQ ID NO: 9, and a CDR3 consisting of an amino acid sequence of SEQ ID NO: 1086. The antibody conjugate of any one of claims 47-85, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 6 and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 11.

87. The antibody conjugate of any one of claims 47-86, wherein the antibody comprises a heavy chain consisting of an amino acid sequence of SEQ ID NO: 7 and a light chain consisting of an amino acid sequence of SEQ ID NO: 12.

88. The antibody conjugate of any one of claims 47-87, wherein the antibody further comprises the amino acid substitution V264S.

89. The antibody conjugate of any one of claims 47-88, wherein the antibody conjugate comprises:(a) either one of:(i) an antibody comprising a heavy chain variable region comprises a CDR1 consisting of an amino acid sequence of SEQ ID NO: 1, a CDR2 consisting of an amino acid sequence of SEQ ID NO: 2, and a CDR3 consisting of an amino acid sequence of SEQ ID NO: 3, and the light chain variable region comprises a CDR1 consisting of an amino acid sequence of SEQ ID NO: 8, a CDR2 consisting of an amino acid sequence of SEQ ID NO: 9, and a CDR3 consisting of an amino acid sequence of SEQ ID NO: 10;(ii) an antibody comprising a heavy chain variable region comprises a CDR1 consisting of an amino acid sequence of SEQ ID NO: 4, a CDR2 consisting of an amino acid sequence of SEQ ID NO: 5, and a CDR3Attorney Docket No. : 108843.00484(SU0451WO) consisting of an amino acid sequence of SEQ ID NO: 3, and the light chain variable region comprises a CDR1 consisting of an amino acid sequence of SEQ ID NO: 8, a CDR2 consisting of an amino acid sequence of SEQ ID NO: 9, and a CDR3 consisting of an amino acid sequence of SEQ ID NO: 10;(b) p-azidom ethyl -L-phenylalanine residues at antibody sites HC-F404, HC-F241, HC- Y391 and HC-Y180; and(c) p-acetyl-L-phenylalanine at antibody site LC-K42,(d) wherein [CY-La-]m- is90. A pharmaceutical composition comprising the antibody of any one of 1-31, or the antibody conjugate of any one of claims 32-89, and a pharmaceutically acceptable carrier.

91. A method of treating or preventing a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of an antibody conjugate of any one of claims 32-89, or a pharmaceutical composition of claim 90.Attorney Docket No. : 108843.00484 (SU0451WO)92. The method of claim 91, wherein the disease is lung cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, uterine cervix cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulval cancer, thyroid cancer, penis cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

93. The method of claim 91 or 92, further comprising administering to the subject one or more additional therapeutic agents.

94. The method of claim 93, wherein the one or more additional therapeutic agents comprises an anti-PD-1 antibody.

95. A method of detecting an antigen, comprising: contacting a sample comprising the antigen with an antibody conjugate of any one of claims 32-89, or a pharmaceutical composition comprising the antibody conjugate, wherein at least one of the non-natural amino acids comprises a labeling moiety or is linked to a payload comprising a labeling moiety; and detecting a complex formed between the antibody and the antigen.

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