Compositions Containing Antibody-TLR Agonist Conjugates, Methods Thereof, and Uses

Abstract

Disclosed herein are trastuzumab-linked TLR agonist derivative analogs containing at least one unnatural amino acid, as well as methods for producing such unnatural amino acids and polypeptides. Trastuzumab-linked TLR agonist derivative analogs can contain a wide range of possible functional groups, but typically have at least one oxime, carbonyl, dicarbonyl, and / or hydroxylamine group. Disclosed herein are unnatural amino acid trastuzumab-linked TLR agonist derivative analogs that are further modified post-translationally, methods for producing such modifications, and methods for purifying such trastuzumab-linked TLR agonist derivative analogs. Typically, modified trastuzumab-linked TLR agonist derivative analogs contain at least one oxime, carbonyl, dicarbonyl, and / or hydroxylamine group. Further disclosed are methods for using such unnatural amino acid trastuzumab-linked TLR agonist derivative analogs and modified unnatural amino acid trastuzumab-linked TLR agonist derivative analogs, including therapeutic, diagnostic, and other biotechnology uses.

Description

Detailed Description of the Invention

[0001] 〔Technical Field〕 Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 804,742, filed on February 12, 2019, entitled "Compositions Containing, Methods And Uses Of Antibody-TLR Agonist Conjugates", the content of which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application is filed in ASCII format via EFS-Web and includes a sequence listing that is hereby incorporated by reference in its entirety. The ASCII copy created on February 7, 2020, is named AMBX_0230_PCT_SL.txt and is 30,527 bytes in size.

[0003] The disclosure of the present invention relates to TLR agonist compounds and TLR agonist conjugates (TC), and their uses. The present invention further relates to pharmaceutical compositions containing (TC) for therapeutic or prophylactic use.

[0004] 〔Background Art〕 Targeted molecules or polypeptides such as antibodies and their fragments, and TLR agonist compounds can be conjugated together using non-naturally encoded amino acids by site-specific conjugation to produce novel TLR-agonist conjugates (TC). The novel TC can be constructed in such a way that during systemic treatment, the circulating TC targets the TLR agonist to the tumor site, stimulating a locally beneficial immune response, thereby minimizing the systemic cytokine release syndrome.

[0005] 〔Summary of the Invention〕 The present invention relates to targeting a polypeptide having one or more non-naturally encoded amino acids conjugated to an agonist compound of TLR, including but not limited to TLR7 and / or TLR8. Such conjugates are herein referred to as TLR-agonist conjugates (TCs). The TCs of the present invention involve targeting a biological molecule or polypeptide and a TLR agonist compound conjugated together using non-naturally encoded amino acids by site-specific conjugation to produce a novel biological TLR-agonist conjugate (BTC). The targeted biological molecule or polypeptide can be a tumor-targeted biological molecule or polypeptide.

[0006] In a further embodiment, the present invention further relates to a TC further conjugated to a water-soluble polymer that forms a stable dimer or multimer.

[0007] The present invention provides a method for inhibiting or reducing the growth of a tumor or cancer, which includes contacting the tumor with an effective amount of the TC of the present invention to stimulate the patient's immune system in proximity to the tumor. The present invention provides a method for inhibiting or reducing the growth of a tumor or cancer, which includes contacting the tumor with an effective amount of the PEGylated TC of the present invention, or a stable dimer or multimer of TC. In one embodiment, the TC is non-PEGylated or mono-PEGylated. In one embodiment, the TC is di-PEGylated. In one embodiment, the TC has two or more and / or different TLR agonist molecules bound thereto. In one embodiment, the TC has two or more and / or the same TLR agonist molecules bound thereto. Another embodiment of the present invention provides a method of using the TC of the present invention to modulate an immune response against tumor cells. In certain embodiments, the TC is co-administered with at least one chemotherapeutic agent and / or at least one immunotherapeutic agent. The chemotherapeutic agent can be selected from the group consisting of temozolomide, gemcitabine, doxorubicin, cyclophosphamide, paclitaxel, cisplatin, fluoropyrimidine, taxane, anthracycline, lapatinib, capecitabine, letrozole, pertuzumab, docetaxel, IFN-α. In another embodiment of the present invention, the TC is co-administered with at least one chemotherapeutic agent and / or at least one immunotherapeutic agent.

[0008] In some embodiments, the TC comprises a targeting polypeptide, including but not limited to an antigen-binding polypeptide (ABP) comprising one or more non-naturally encoded amino acids. In some embodiments, the ABP comprises a complete antibody heavy chain. In some embodiments, the ABP comprises a complete antibody light chain. In some embodiments, the ABP comprises a variable region of an antibody light chain. In some embodiments, the ABP comprises a variable region of an antibody heavy chain. In some embodiments, the ABP comprises at least one CDR of an antibody light chain. In some embodiments, the ABP comprises at least one CDR of an antibody heavy chain. In some embodiments, the ABP comprises at least one CDR of the light chain and at least one CDR of the heavy chain. In some embodiments, the ABP comprises a Fab. In some embodiments, the ABP comprises two or more Fabs. In some embodiments, the ABP comprises a (Fab’)2. In some embodiments, the ABP comprises two or more (Fab’)2s. In some embodiments, the ABP comprises an scFv. In some embodiments, the ABP comprises two or more scFvs. In some embodiments, the ABP comprises a minibody. In some embodiments, the ABP comprises two or more minibodies. In some embodiments, the ABP comprises a diabody. In some embodiments, the ABP comprises two or more diabodies. In some embodiments, the ABP comprises a variable region of the light chain and a variable region of the heavy chain. In some embodiments, the ABP comprises a complete light chain and a complete heavy chain. In some embodiments, the ABP comprises one or more Fc domains or a portion thereof. In some embodiments, the ABP comprises any combination of the above embodiments. In some embodiments, the ABP comprises any homodimer, heterodimer, homotrimer, or heterotrimer of the above embodiments. In some embodiments, the ABP comprises a polypeptide that binds to a binding partner, and the binding partner comprises an antigen, polypeptide, nucleic acid molecule, polymer, or other molecule or substance. In some embodiments, the ABP associates with a non-antibody scaffold molecule or substance.In some embodiments, the antigen is a tumor antigen.

[0009] Toll-like receptors (TLRs) detect a wide range of conserved pathogen-associated molecular patterns (PAMPs). These play an important role in sensing invading pathogens and subsequently initiating the innate immune response. Ten members of the TLR family are known in humans, which are type I transmembrane proteins characterized by an extracellular leucine-rich domain and a cytoplasmic tail containing a conserved Toll / interleukin (IL)-1 receptor (TIR) domain. Within this family, TLR3, TLR7, TLR8, and TLR9 are located within endosomes. TLR7 and TLR8 can be activated by binding to specific small molecule ligands (i.e., TLR7 agonists or TLR8 agonists) or their natural ligands (i.e., single-stranded RNA, ssRNA). After the agonist binds to TLR7 or TLR8, the receptor in its dimeric form is thought to undergo a structural change that leads to the subsequent recruitment of adapter proteins in its cytoplasmic domain, including myeloid differentiation primary response gene 88 (MyD88). After the initiation of the receptor signaling cascade via the MyD88 pathway, cytoplasmic transcription factors such as interferon regulatory factor 7 (IRF-7) and nuclear factor kappa B (NF-κB) are activated. These transcription factors then translocate to the nucleus and initiate the transcription of various genes, such as IFN-alpha and other antiviral cytokine genes. TLR7 is mainly expressed on plasmacytoid cells and B cells. Alterations in the responsiveness of immune cells can contribute to the reduction of the innate immune response in cancer patients. Therefore, agonist-induced activation of TLR7 and / or TLR8 conjugated to a targeting moiety such as an antibody or its fragment may represent a novel approach for cancer treatment. Treatment with TC containing TLR7 or TLR8 agonists represents a promising solution that provides higher efficacy with better tolerance.Suitable TLR7 and / or TLR8 agonists used in the present invention to produce TC are found in the following U.S. patents, each of which is incorporated herein by reference: U.S. Patent No. 6,825,350, U.S. Patent No. 6,656,389, U.S. Patent No. 6,656,398, U.S. Patent No. 6,683,088, U.S. Patent No. 6,756,382, U.S. Patent No. 6,825,350, U.S. Patent No. 6,667,312, U.S. Patent No. 6,677,347, U.S. Patent No. 7,598,382, U.S. Patent No. 8,673,932.

[0010] In some embodiments, the TC further comprises a targeted polypeptide having an amino acid substitution, addition, or deletion that increases the compatibility of the TC polypeptide with a pharmaceutical preservative (e.g., m-cresol, phenol, benzyl alcohol) as compared to the compatibility of the corresponding wild-type TC without substitution, addition, or deletion. This increased compatibility will enable the preparation of a preserved pharmaceutical formulation that maintains the physiochemical properties and biological activity of the protein during storage.

[0011] In some embodiments, one or more engineered linkages are made with one or more non-natural amino acids. Intramolecular linkages can be made in many ways including, but not limited to, reactions between two amino acids in a protein under suitable conditions (where one or both amino acids can be non-natural amino acids), reactions with two amino acids, each of which can be naturally encoded or non-naturally encoded by a linker, polymer, or other molecule under suitable conditions.

[0012] In some embodiments, one or more amino acid substitutions in the TC polypeptide can be by one or more naturally occurring or non-naturally occurring amino acids. In some embodiments, the amino acid substitutions in the TC can be by naturally occurring or non-naturally occurring amino acids, provided that at least one substitution is by a non-naturally encoded amino acid. In some embodiments, one or more amino acid substitutions in the TC polypeptide can be by one or more naturally occurring amino acids, and in addition, at least one substitution is by a non-naturally encoded amino acid. In some embodiments, the TC polypeptide can be an antibody or an antibody fragment. In some embodiments, the TC polypeptide can be a tumor-targeting polypeptide.

[0013] In some embodiments, the non-naturally encoded amino acid contains a carbonyl group, an acetyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group.

[0014] In some embodiments, the non-naturally encoded amino acid contains a carbonyl group. In some embodiments, the non-naturally encoded amino acid has the structure:

[0015]

Chemical formula

[0016] wherein n is from 0 to 10, R1 is alkyl, aryl, substituted alkyl, or substituted aryl, R2 is H, alkyl, aryl, substituted alkyl, or substituted aryl, R3 is H, an amino acid, a polypeptide, or an amino-terminal modifying group, and R4 is H, an amino acid, a polypeptide, or a carboxy-terminal modifying group.

[0017] In some embodiments, the non-naturally encoded amino acid contains an aminooxy group. In some embodiments, the non-naturally encoded amino acid contains a hydrazide group. In some embodiments, the non-naturally encoded amino acid contains a hydrazine group. In some embodiments, the non-naturally encoded amino acid residue contains a semicarbazide group.

[0018] In some embodiments, the non-naturally encoded amino acid residue contains an azide group. In some embodiments, the non-naturally encoded amino acid has the structure:

[0019]

Chemical formula

[0020] wherein n is from 0 to 10, R1 is alkyl, aryl, substituted alkyl, substituted aryl, or absent, X is O, N, S, or absent, m is from 0 to 10, R2 is H, an amino acid, a polypeptide, or an amino-terminal modifying group, and R3 is H, an amino acid, a polypeptide, or a carboxy-terminal modifying group.

[0021] In some embodiments, the non-naturally encoded amino acid contains an alkyne group. In some embodiments, the non-naturally encoded amino acid has the structure:

[0022]

Chemical formula

[0023] wherein n is from 0 to 10, R1 is alkyl, aryl, substituted alkyl, or substituted aryl, X is O, N, S, or absent, m is from 0 to 10, R2 is H, an amino acid, a polypeptide, or an amino-terminal modifying group, and R3 is H, an amino acid, a polypeptide, or a carboxy-terminal modifying group.

[0024] In some embodiments, the polypeptide is a TC comprising a non-naturally encoded amino acid linked to a water-soluble polymer. In some embodiments, the water-soluble polymer comprises a poly(ethylene glycol) moiety. In some embodiments, the TC comprises a non-naturally encoded amino acid and one or more post-translational modifications, linkers, polymers, or bioactive molecules.

[0025] The invention also provides an isolated nucleic acid comprising a polynucleotide encoding a targeted polypeptide of the TC, and the invention provides an isolated nucleic acid comprising a polynucleotide that hybridizes to the polynucleotide under stringent conditions. The invention also provides an isolated nucleic acid comprising a polynucleotide encoding a targeted polypeptide, wherein the polynucleotide comprises at least one selector codon. It will be readily apparent to those skilled in the art that several different polynucleotides can encode any polypeptide of the invention.

[0026] In some embodiments, the selector codon is selected from the group consisting of an amber codon, an ochre codon, an opal codon, a unique codon, a rare codon, a five-base codon, and a four-base codon.

[0027] The present invention also provides a method of making a TC polypeptide that is linked to a water-soluble polymer or to one or more TC polypeptides to form a homodimer or homotrimer. In some embodiments, the method comprises contacting an isolated TC polypeptide comprising a non-naturally encoded amino acid with a water-soluble polymer or linker comprising a moiety that reacts with the non-naturally encoded amino acid. In some embodiments, the non-naturally encoded amino acid incorporated into the TC polypeptide is reactive with a water-soluble polymer or linker that is otherwise non-reactive with any of the 20 common amino acids. In some embodiments, the non-naturally encoded amino acid incorporated into the TC polypeptide is reactive with a linker, polymer, or biologically active molecule that is otherwise non-reactive with any of the 20 common amino acids.

[0028] In some embodiments, the TC polypeptide linked to the water-soluble polymer or linker is made by reacting a TC polypeptide comprising a carbonyl-containing amino acid with a poly(ethylene glycol) molecule or a linker comprising an aminooxy, hydrazine, hydrazide, or semicarbazide group. In some embodiments, the aminooxy, hydrazine, hydrazide, or semicarbazide group is linked to the poly(ethylene glycol) molecule or linker via an amide linkage. In some embodiments, the aminooxy, hydrazine, hydrazide, or semicarbazide group is linked to the poly(ethylene glycol) molecule or linker via a carbamate linkage.

[0029] In some embodiments, the TC polypeptide linked to the water-soluble polymer is made by reacting a poly(ethylene glycol) molecule or linker comprising a carbonyl group with a polypeptide comprising a non-naturally encoded amino acid comprising an aminooxy, hydrazine, hydrazide, or semicarbazide group.

[0030] In some embodiments, the TC polypeptide linked to the water-soluble polymer or linker is prepared by reacting a TC comprising an alkyne-containing amino acid with a poly(ethylene glycol) molecule comprising an azide moiety. In some embodiments, the azide or alkyne group is linked to the poly(ethylene glycol) molecule or linker via an amide linkage.

[0031] In some embodiments, the TC polypeptide linked to the water-soluble polymer or linker is prepared by reacting a TC polypeptide comprising an azide-containing amino acid with a poly(ethylene glycol) molecule comprising an alkyne moiety. In some embodiments, the azide or alkyne group is linked to the poly(ethylene glycol) molecule or linker via an amide linkage.

[0032] In some embodiments, the poly(ethylene glycol) molecule or linker has a molecular weight of from about 0.1 kDa to about 100 kDa. In some embodiments, the poly(ethylene glycol) molecule or linker has a molecular weight of 0.1 kDa to 50 kDa. In some embodiments, the poly(ethylene glycol) molecule or linker is a branched polymer or linker. In some embodiments, each branch of the poly(ethylene glycol) branched polymer or linker has a molecular weight of 1 kDa to 100 kDa, or 1 kDa to 50 kDa.

[0033] In some embodiments, the water-soluble polymer linked to the TC polypeptide comprises a polyalkylene glycol moiety. In some embodiments, the non-naturally encoded amino acid residue incorporated into the TC comprises a carbonyl group, an aminooxy group, a hydrazide group, hydrazine, a semicarbazide group, an azide group, or an alkyne group. In some embodiments, the non-naturally encoded amino acid residue incorporated into the TC polypeptide comprises a carbonyl moiety and the water-soluble polymer comprises an aminooxy, hydrazide, hydrazine, or semicarbazide moiety. In some embodiments, the non-naturally encoded amino acid residue incorporated into the TC polypeptide comprises an alkyne moiety and the water-soluble polymer comprises an azide moiety. In some embodiments, the non-naturally encoded amino acid residue incorporated into the TC polypeptide comprises an azide moiety and the water-soluble polymer comprises an alkyne moiety. The present invention also provides a composition comprising a TC polypeptide comprising a non-naturally encoded amino acid and a pharmaceutically acceptable carrier. In some embodiments, the non-naturally encoded amino acid is linked to the water-soluble polymer.

[0034] The present invention also provides a cell comprising a polynucleotide encoding a targeted polypeptide of TC comprising a selector codon. In some embodiments, the cell comprises an orthogonal RNA synthetase and / or an orthogonal tRNA for substituting a non-naturally encoded amino acid for the targeted polypeptide of TC.

[0035] The present invention also provides a method for producing a targeted polypeptide of TC comprising a non-naturally encoded amino acid. In some embodiments, the method comprises culturing a cell comprising a polynucleotide (s) encoding a targeted polypeptide of TC, an orthogonal RNA synthetase, and / or an orthogonal tRNA under conditions that allow expression of the targeted polypeptide of TC or a variant thereof, and purifying the TC polypeptide from the cell and / or the culture medium.

[0036] The present invention also provides a method of increasing the therapeutic half-life, serum half-life, or circulation time of a TC. The present invention also provides a method of modulating the immunogenicity of a TC. In some embodiments, the method comprises using non-naturally encoded amino acids in place of any one or more amino acids in the naturally occurring targeting polypeptide of the TC, and / or linking the targeting polypeptide to a linker, polymer, water-soluble polymer, or bioactive molecule.

[0037] The present invention also provides a method of treating a patient in need of such treatment with an effective amount of a TC molecule of the present invention. In some embodiments, the method comprises administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of a TC comprising a non-naturally encoded amino acid and a pharmaceutically acceptable carrier. In some embodiments, the non-naturally encoded amino acid is linked to a water-soluble polymer. In some embodiments, the TC is glycosylated. In some embodiments, the TC is not glycosylated.

[0038] The present invention also provides a TC comprising a water-soluble polymer or linker linked by a covalent bond to the TC at a single amino acid. In some embodiments, the water-soluble polymer comprises a poly(ethylene glycol) moiety. In some embodiments, the amino acid covalently bonded to the water-soluble polymer or linker is a non-naturally encoded amino acid present in the targeting polypeptide of the TC.

[0039] The present invention provides a TC polypeptide comprising at least one linker, polymer, or biologically active molecule, wherein the linker, polymer, or biologically active molecule is attached to the polypeptide via a functional group of a non-naturally encoded amino acid incorporated into the targeting polypeptide of TC by ribosomes. In a TC conjugate, PEG or another water-soluble polymer, another TC, polypeptide, or biologically active molecule can be directly conjugated to the TC via a linker. In one embodiment, the linker is of sufficient length to allow mobility and enable dimer formation. In one embodiment, the linker is at least 3 amino acids or 18 atoms in length to enable dimer formation. In some embodiments, the polypeptide is linked to the linker to enable the formation of multimers. In some embodiments, the linker is a bifunctional linker. In some embodiments, the compositions and / or TC of the present invention may include multiple linkers. In other embodiments, each linker may include one or more attached compounds. The linker may also include alkylene, alkenylene, alkynylene, polyether, polyester, polyamide group(s), as well as polyamino acid, polypeptide, cleavable peptide, or aminobenzyl carbamate. In some embodiments, the linkers may be the same or different. Suitable linkers include, for example, cleavable and non-cleavable linkers. Suitable cleavable linkers include, for example, peptide linkers cleavable by intracellular proteases such as lysosomal protease or endosomal protease. Cleavable linkers may include valine-citrulline linkers or valine-alanine peptides. In some embodiments, the linker may be a dipeptide linker such as valine-citrulline or phenylalanine-lysine linker. Valine-citrulline or valine-alanine-containing linkers may contain maleimide or succinimide groups. Valine-citrulline or valine-alanine-containing linkers may contain para-aminobenzyl alcohol (PABA) groups or para-aminobenzyl carbamate (PABC).Other suitable linkers include linkers that are hydrolysable at a pH below 5.5, such as hydrazone linkers. Further suitable cleavable linkers include disulfide linkers. In some embodiments, the cleavable linker can be a linker cleaved in the tumor microenvironment, which can include, for example, tumor-infiltrating T cells. In some embodiments, non-cleavable linkers include, but are not limited to, maleimidocaproyl linkers. Maleimidocaproyl linkers can include, but are not limited to, N-maleimidomethylcyclohexane-1-carboxylate, succinimide groups, pentafluorophenyl groups, and / or one or more PEG molecules. In some embodiments, any one of the compositions, compounds, or salts thereof of the present invention can be linked to a polypeptide by a linker. In some embodiments, any one of the compounds or salts thereof disclosed herein in Tables 3, 4, 5, 6, and 7 can be linked to a polypeptide by a linker. In some embodiments, the polypeptide is a targeted polypeptide or a biologically targeted polypeptide or a tumor-targeted polypeptide. In some embodiments, the targeted polypeptide is an antibody or an antibody fragment.

[0040] In some embodiments, the TC polypeptide is mono-PEGylated. The present invention also provides a TC comprising a linker, polymer, or bioactive molecule attached to one or more non-naturally encoded amino acids, which non-naturally encoded amino acids are incorporated into the polypeptide by ribosome at a preselected site.

[0041] In some embodiments, the present invention provides a composition comprising one or more targeted polypeptides having one or more non-naturally encoded amino acids incorporated therein, wherein at least one of the polypeptides is linked to a TLR agonist molecule via a linker covalently attached to the non-natural amino acid of the polypeptide.

[0042] In another embodiment, the present invention provides a composition in which one or more targeting polypeptides are the same or different targeting polypeptides. In another embodiment, the present invention provides a composition in which one or more targeting polypeptides bind to a cell surface target or a tumor cell target or a cancer cell target. In another embodiment, the one or more targeting polypeptides are monospecific, bispecific, or multispecific targeting polypeptides.

[0043] In other embodiments, the monospecific, bispecific, or multispecific targeting polypeptides comprise a drug conjugate or a checkpoint inhibitor. Any suitable immune checkpoint inhibitor is contemplated for use with the compositions of the present invention or with TC. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins. In another embodiment, the immune checkpoint inhibitor reduces the interaction between one or more immune checkpoint proteins and their ligands. Inhibitory nucleic acids that reduce the expression and / or activity of immune checkpoint molecules can also be used in the present invention. In some embodiments, the immune checkpoint inhibitor is CTLA4, TIGIT, glucocorticoid-induced TNFR-related protein (GITR), inducible T cell co-stimulator (ICOS), CD96, poliovirus receptor-related 2 (PVRL2), PD-1, PD-L1, PD-L2, LAG-3, B7-H4, killer immunoglobulin receptor (KIR), OX40, OX40-L indoleamine 2,3-dioxygenase 1 (IDO-1), indoleamine 2,3-dioxygenase 2 (IDO-2), CEACAM1, CD272, TEVI3, adenosine A2A receptor, and VISTA protein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA4, PD-1, or PD-L1.

[0044] In another embodiment, the targeting polypeptide comprises an antibody or an antibody fragment. In other embodiments, the targeting polypeptide is an antibody or an antibody fragment that binds to an antigen of a cell. In another embodiment, the target polypeptide is an antibody or an antibody fragment that binds to a target selected from the group consisting of HER2, HER3, PD-1, PDL-1, EGFR, TROP2, PSMA, VEGFR, CTLA-4, EpCAM, MUC1, MUC16, c-met, GPC3, ENPP3, TIM-1, FOLR1, STEAP1, mesothelin, 5T4, CEA, CA9, cadherin 6, ROR1, SLC34A2, SLC39A6, SLC44A4, LY6E, DLL3, ePhA2, GPNMB, SLITRK6, CD3, CD19, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD47, CD52, CD56, CD70, CD96, CD97, CD99, CD117, CD123, CD179, CD223, and CD276. In some embodiments, the targeting polypeptide comprises an antibody or an antibody fragment that binds to HER2. In another embodiment, the targeting polypeptide is trastuzumab.

[0045] In another embodiment, the antibody or antibody fragment comprises IgG, Fab, (Fab’)2, Fv, or single-chain Fv (scFv). In some embodiments, the antibody or antibody fragment comprises one or more Fab, (Fab’)2, Fv, or single-chain Fv (scFv) mutations. In some embodiments, the antibody or antibody fragment comprises one or more Fc mutations. In other embodiments, the antibody or antibody fragment comprises from 1 to 6 Fc mutations. In some embodiments, the antibody or antibody fragment comprises two or more Fc mutations. In other embodiments, the antibody or antibody fragment comprises three or more Fc mutations. In some embodiments, the antibody or antibody fragment comprises four or more Fc mutations. In other embodiments, the antibody or antibody fragment comprises five or more Fc mutations. In other embodiments, the antibody or antibody fragment comprises six Fc mutations.

[0046] In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy and light chains. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains, and further comprises one or more Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into each of the heavy and light chains, and the antibody or antibody fragment further comprises one or more Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains, and further comprises at least two Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into each of the heavy and light chains, and the antibody or antibody fragment further comprises at least two Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains, and further comprises at least three Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into each of the heavy and light chains, and the antibody or antibody fragment further comprises at least three Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains, and further comprises at least four Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into each of the heavy and light chains, and the antibody or antibody fragment further comprises at least four Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains, and further comprises at least five Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into each of the heavy and light chains, and the antibody or antibody fragment further comprises at least five Fc mutations.In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, light chain, or both the heavy and light chains, and further comprises at least six Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into each of the heavy and light chains, and the antibody or antibody fragment further comprises at least six Fc mutations.

[0047] In another embodiment, the targeted polypeptide comprises one or more non-naturally encoded amino acids selected from the group consisting of para-acetylphenylalanine, p-nitrophenylalanine, p-sulfotyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-boronylphenylalanine, o-boronylphenylalanine, m-boronylphenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-OMe phenylalanine, o-OMe phenylalanine, m-OMe phenylalanine, p-sulfophenylalanine, o-sulfophenylalanine, m-sulfophenylalanine, 5-nitroHis, 3-nitroTyr, 2-nitroTyr, nitro-substituted Leu, nitro-substituted His, nitro-substituted De, nitro-substituted Trp, 2-nitroTrp, 4-nitroTrp, 5-nitroTrp, 6-nitroTrp, 7-nitroTrp, 3-aminotyrosine, 2-aminotyrosine, O-sulfotyrosine, 2-sulfooxyphenylalanine, 3-sulfooxyphenylalanine, o-carboxyphenylalanine, m-carboxyphenylalanine, p-acetyl-L-phenylalanine, p-propynyl-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propynyloxy-L-phenylalanine, 4-azido-L-phenylalanine, para-azidoethoxyphenylalanine, and para-azidomethyl-phenylalanine.In another embodiment, the unnatural amino acid is selected from the group consisting of para-acetyl-phenylalanine, 4-azido-L-phenylalanine, para-azidoethoxyphenylalanine, or para-azidomethyl-phenylalanine. In other embodiments, the unnatural encoded amino acid is site-specifically incorporated into one or more targeted polypeptides.

[0048] In another embodiment, the TLR agonist is a TLR7 agonist, a TLR8 agonist, or a TLR7 / TLR8 dual agonist. In other embodiments, the TLR agonist is a TLR agonist comprising the molecular structure described in any one of Structures 1, 2, 3, 4, or 5 of FIG. 1. In another embodiment, the TLR agonist is any one of the TLR agonists selected from the group of structures described in Tables 3, 4, 5, 6, 7 of the present invention.

[0049] In other embodiments, the targeted polypeptide is conjugated to one or more linkers, polymers, or bioactive molecules. In some embodiments, the targeted polypeptide is directly or indirectly conjugated to one or more linkers, polymers, or bioactive molecules. In some embodiments, one or more of the linkers are cleavable or non-cleavable linkers.

[0050] In some embodiments, one or more of the linkers are from 0.1 kDa to 50 kDa. In other embodiments, one or more of the linkers are from 0.1 kDa to 10 kDa. In other embodiments, one or more of the linkers or polymers are linear, branched, multimeric, or dendrimeric. In another embodiment, one or more of the linkers or polymers are bifunctional or polyfunctional linkers or bifunctional or polyfunctional polymers.

[0051] In other embodiments, the one or more polymers are water-soluble polymers. In other embodiments, the water-soluble polymer is polyethylene glycol (PEG). In some embodiments, the PEG has a molecular weight of 0.1 kDa to 100 kDa. In other embodiments, the PEG has a molecular weight of 0.1 kDa to 50 kDa. In other embodiments, the PEG has a molecular weight of 0.1 kDa to 40 kDa. In other embodiments, the PEG has a molecular weight of 0.1 kDa to 30 kDa. In other embodiments, the PEG has a molecular weight of 0.1 kDa to 20 kDa. In other embodiments, the PEG has a molecular weight of 0.1 kDa to 10 kDa. In some embodiments, the poly(ethylene glycol) molecules have a molecular weight of about 0.1 kDa to about 100 kDa. In some embodiments, the poly(ethylene glycol) molecules have a molecular weight of 0.1 kDa to 50 kDa. In some embodiments, the poly(ethylene glycol) has a molecular weight of 1 kDa to 25 kDa, or 2 to 22 kDa, or 5 kDa to 20 kDa. For example, the molecular weight of the poly(ethylene glycol) polymer can be about 5 kDa, or about 10 kDa, or about 20 kDa, or about 30 kDa. For example, the molecular weight of the poly(ethylene glycol) polymer can be 5 kDa, or 10 kDa, or 20 kDa, or 30 kDa. In some embodiments, the poly(ethylene glycol) molecules are branched PEG. In some embodiments, the poly(ethylene glycol) molecules are branched 5K PEG. In some embodiments, the poly(ethylene glycol) molecules are branched 10K PEG. In some embodiments, the poly(ethylene glycol) molecules are branched 20K PEG. In some embodiments, the poly(ethylene glycol) molecules are linear PEG. In some embodiments, the poly(ethylene glycol) molecules are linear 5K PEG. In some embodiments, the poly(ethylene glycol) molecules are linear 10K PEG. In some embodiments, the poly(ethylene glycol) molecules are linear 20K PEG.In some embodiments, the poly(ethylene glycol) molecule is a linear 30K PEG. In some embodiments, the molecular weight of the poly(ethylene glycol) polymer is the average molecular weight. In certain embodiments, the average molecular weight is the number average molecular weight (Mn). The average molecular weight can be determined or measured using GPC or SEC, SDS / PAGE analysis, RP-HPLC, mass spectrometry, or capillary electrophoresis.

[0052] In another embodiment, at least one linker, polymer, or biologically active molecule is linked to at least one non-naturally encoded amino acid. In some embodiments, the linker is PEG. In other embodiments, the linker is a PEG having a molecular weight of 0.1 kDa to 50 kDa. In other embodiments, the linker is a PEG having a molecular weight of 0.1 kDa to 40 kDa. In other embodiments, the linker is a PEG having a molecular weight of 0.1 kDa to 30 kDa. In other embodiments, the linker is a PEG having a molecular weight of 0.1 kDa to 20 kDa. In other embodiments, the linker is a PEG having a molecular weight of 0.1 kDa to 10 kDa. In other embodiments, the linker is a PEG having a molecular weight of 0.1 kDa to 5 kDa.

[0053] In another embodiment, the targeted polypeptide comprises one or more amino acid substitutions, additions, or deletions that increase the stability or solubility of the composition. In another embodiment, the targeted polypeptide comprises one or more amino acid substitutions, additions, or deletions that enhance / reduce ADCP or ADCC activity. In another embodiment, the targeted polypeptide comprises one or more amino acid substitutions, additions, or deletions that increase the pharmacokinetics of the composition. In other embodiments, the composition comprises one or more amino acid substitutions, additions, or deletions that increase the expression of the targeted polypeptide in recombinant host cells or synthesized in vitro.

[0054] In another embodiment, the non-naturally encoded amino acid is reactive towards a linker, polymer, or bioactive molecule that is otherwise non-reactive towards any of the 20 common amino acids in a polypeptide. In another embodiment, the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group. In other embodiments, the non-naturally encoded amino acid comprises a carbonyl group.

[0055] In another embodiment, the targeted polypeptide is linked to a cytotoxic agent or an immunostimulatory agent. In another embodiment, the TC or BTC of the present invention is linked to a cytotoxic agent or an immunostimulatory agent. In another embodiment, the targeted polypeptide comprises a cytotoxic agent or an immunostimulatory agent. In another embodiment, the TC or BTC of the present invention comprises a cytotoxic agent or an immunostimulatory agent.

[0056] In another embodiment, the present invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist comprising the structure described in any of the structures of FIG. 1, wherein the TLR agonist is conjugated to the antibody or antibody fragment via a linker covalently attached to one or more non-naturally encoded amino acids incorporated into the antibody or antibody fragment. In another embodiment, the TLR agonist is a TLR7 agonist, a TLR8 agonist, or a TLR7 / TLR8 dual agonist. In another embodiment, the TLR agonist comprises the structure described in Structure 1 of FIG. 1.In another embodiment, the TLR agonist comprising the structure described in Structure 1 is selected from the group of AXC-621, AXC-622, AXC-625, AXC-626, AXC-627, AXC-638, AXC-639, AXC-640, AXC-642, AXC-662, AXC-665, AXC-666, AXC-667, AXC-668, AXC-669, AXC-670, AXC-671, AXC-672, AXC-675, AXC-678, AXC-679, AXC-681, AXC-687, AXC-688, AXC-689, AXC-690, AXC-691, AXC-696, AXC-697, AXC-698, AXC-699, AXC-700, AXC-701, AXC-702, AXC-709, AXC-710, AXC-711, AXC-712, AXC-713, AXC-714, AXC-715, AXC-716, AXC-717, AXC-718, AXC-719, AXC-722, AXC-723, AXC-724, AXC-725, AXC-726, AXC-727, AXC-729, AXC-731, AXC-732, AXC-733, AXC-734, AXC-735, AXC-736, AXC-737, AXC-738, AXC-739, AXC-740, AXC-741, AXC-743, AXC-742, AXC-747, AXC-748, AXC-749, AXC-750, AXC-751, AXC-752, AXC-754, AXC-755, AXC-756, AXC-757, AXC-758, AXC-759, AXC-760, AXC-761, AXC-762, AXC-764, AXC-771, AXC-772, AXC-773, AXC-777, AXC-778, AXC-779, AXC-789, AXC-793, AXC-799, AXC-800, AXC-801, AXC-802, AXC-803, AXC-804, AXC-805, AXC-806, AXC-807, AXC-808, AXC-809, AXC-810, AXC-831, and AXC-910 compounds.In another embodiment, the present invention provides a TLR agonist of any one of the compounds AXC-621, AXC-622, AXC-625, AXC-626, AXC-627, AXC-638, AXC-639, AXC-640, AXC-642, AXC-662, AXC-665, AXC-666, AXC-667, AXC-668, AXC-669, AXC-670, AXC-671, AXC-672, AXC-675, AXC-678, AXC-679, AXC-681, AXC-687, AXC-688, AXC-689, AXC-690, AXC-691, AXC-696, AXC-697, AXC-698, AXC-699, AXC-700, AXC-701, AXC-702, AXC-709, AXC-710, AXC-711, AXC-712, AXC-713, AXC-714, AXC-715, AXC-716, AXC-717, AXC-718, AXC-719, AXC-722, AXC-723, AXC-724, AXC-725, AXC-726, AXC-727, AXC-729, AXC-731, AXC-732, AXC-733, AXC-734, AXC-735, AXC-736, AXC-737, AXC-738, AXC-739, AXC-740, AXC-741, AXC-743, AXC-742, AXC-747, AXC-748, AXC-749, AXC-750, AXC-751, AXC-752, AXC-754, AXC-755, AXC-756, AXC-757, AXC-758, AXC-759, AXC-760, AXC-761, AXC-762, AXC-764, AXC-771, AXC-772, AXC-773, AXC-777, AXC-778, AXC-779, AXC-789, AXC-793, AXC-799, AXC-800, AXC-801, AXC-802, AXC-803, AXC-804, AXC-805, AXC-806, AXC-807, AXC-808, AXC-809, AXC-810, AXC-831, or AXC-910, further comprising a linker. In another embodiment, the TLR agonist further comprises a linker and comprises the structure set forth in Structure 1.

[0057] In other embodiments, the TLR agonist comprising the structure described in Structure 1 is selected from the group of compounds AXC-625, AXC-626, AXC-638, AXC-639, AXC-640, AXC-642, AXC-662, AXC-667, AXC-668, AXC-669, AXC-670, AXC-671, AXC-672, AXC-675, AXC-681, AXC-687, AXC-688, AXC-689, AXC-690, AXC-691, AXC-697, AXC-699, AXC-700, AXC-701, AXC-702, AXC-709, AXC-710, AXC-711, AXC-713, AXC-714, AXC-717, AXC-719, AXC-722, AXC-723, AXC-724, AXC-725, AXC-726, AXC-727, AXC-731, AXC-732, AXC-733, AXC-734, AXC-735, AXC-736, AXC-737, AXC-738, AXC-739, AXC-740, AXC-741, AXC-743, AXC-742, AXC-747, AXC-748, AXC-750, AXC-751, AXC-752, AXC-754, AXC-755, AXC-756, AXC-757, AXC-758, AXC-759, AXC-760, AXC-761, AXC-762, AXC-764, AXC-771, AXC-772, AXC-773, AXC-777, AXC-778, AXC-779, AXC-789, AXC-793, AXC-800, AXC-801, AXC-802, AXC-803, AXC-804, AXC-805, AXC-806, AXC-807, AXC-808, AXC-809, AXC-810, AXC-831, and AXC-910. In other embodiments, the TLR agonist comprising the structure described in Structure 1 is selected from the group of compounds AXC-801, AXC-802, AXC-831, and AXC-910. In other embodiments, the TLR agonist comprising the structure described in Structure 1 and selected from the group of compounds AXC-801, AXC-802, AXC-831, and AXC-910 further comprises a linker.

[0058] In another embodiment, the anti-HER2 antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, light chain, or both the heavy and light chains. In another embodiment, the one or more non-naturally encoded amino acids are selected from the group consisting of para-acetylphenylalanine, p-nitrophenylalanine, p-sulfotyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-boronylphenylalanine, o-boronylphenylalanine, m-boronylphenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-OMe phenylalanine, o-OMe phenylalanine, m-OMe phenylalanine, p-sulfophenylalanine, o-sulfophenylalanine, m-sulfophenylalanine, 5-nitroHis, 3-nitroTyr, 2-nitroTyr, nitro-substituted Leu, nitro-substituted His, nitro-substituted De, nitro-substituted Trp, 2-nitroTrp, 4-nitroTrp, 5-nitroTrp, 6-nitroTrp, 7-nitroTrp, 3-aminotyrosine, 2-aminotyrosine, O-sulfotyrosine, 2-sulfooxyphenylalanine, 3-sulfooxyphenylalanine, o-carboxyphenylalanine, m-carboxyphenylalanine, p-acetyl-L-phenylalanine, p-propargyl-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propargyloxy-L-phenylalanine, 4-azido-L-phenylalanine, para-azidoethoxyphenylalanine, and para-azidomethyl-phenylalanine.In other embodiments, the unnatural amino acid is para-acetyl-phenylalanine, 4-azido-L-phenylalanine, para-azidomethyl-phenylalanine, or para-azidoethoxyphenylalanine.

[0059] In another embodiment, the anti-HER2 antibody or antibody fragment further comprises one or more mutations in the Fc region. In another embodiment, the anti-HER2 antibody or antibody fragment further comprises two or more mutations in the Fc region. In another embodiment, the anti-HER2 antibody or antibody fragment further comprises three or more mutations in the Fc region. In another embodiment, the anti-HER2 antibody or antibody fragment further comprises four or more mutations in the Fc region. In another embodiment, the anti-HER2 antibody or antibody fragment further comprises five or more mutations in the Fc region. In another embodiment, the anti-HER2 antibody or antibody fragment further comprises six or more mutations in the Fc region. In another embodiment, the anti-HER2 antibody or antibody fragment further comprises six mutations in the Fc region.

[0060] In another embodiment, one or more linkers are cleavable or non-cleavable linkers. In other embodiments, one or more linkers are bifunctional or polyfunctional linkers.

[0061] In another embodiment, the TLR agonist comprises the structure described in Structure 2 of FIG. 1. In another embodiment, the TLR agonist comprising the structure described in Structure 2 is selected from the group consisting of the AXC-745, AXC-746, and AXC-753 compounds. In another embodiment, the TLR agonist comprising the structure described in any one of the AXC-745, AXC-746, and AXC-753 compounds further comprises a linker. In another embodiment, the TLR agonist further comprises a linker and comprises the structure described in Structure 2.

[0062] In another embodiment, the TLR agonist comprises the structure described in Structure 3 of FIG. 1. In another embodiment, the TLR agonist comprises the structure described in Structure 3 which is the AXC-837 or AXC-847 compound. In another embodiment, the TLR agonist comprises the structure described in the AXC-837 or AXC-847 compound which further comprises a linker. In another embodiment, the TLR agonist comprises the structure described in the AXC-847 compound which further comprises a linker. In another embodiment, the TLR agonist comprises the structure described in Structure 3 which further comprises a linker.

[0063] In another embodiment, the TLR agonist comprises the structure described in Structure 4 of FIG. 1. In another embodiment, the TLR agonist comprising the structure described in Structure 4 is selected from the group of AXC-844, AXC-842, AXC-843, AXC-845, AXC-846, AXC-836, or AXC-841 compounds. In another embodiment, the TLR agonist comprising the structure described in Structure 4 of any one of the AXC-844, AXC-842, AXC-843, AXC-845, AXC-846, AXC-836, or AXC-841 compounds further comprises a linker. In another embodiment, the TLR agonist comprises the structure described in Structure 4 which further comprises a linker.

[0064] In another embodiment, the TLR agonist comprises the structure described in structure 5 of FIG. 1. In another embodiment, the TLR agonist comprising the structure described in structure 5 is selected from the group of AXC-862, AXC-863, AXC-867, AXC-868, AXC-869, AXC-872, AXC-873, AXC-876, AXC-877, AXC-878, AXC-879, AXC-880, AXC-881, AXC-882, AXC-883, AXC-884, AXC-885, AXC-886, AXC-887, AXC-888, AXC-889, AXC-890, AXC-891, AXC-892, AXC-893, AXC-895, AXC-896, AXC-897, AXC-898, AXC-901, AXC-903, AXC-904, AXC-905, AXC-906, AXC-907, AXC-908, AXC-909, AXC-911, AXC-912, AXC-913, AXC-914, AXC-915, or AXC-916 compounds. In other embodiments, the TLR agonist comprising the structure described in structure 5 is selected from the group of AXC-862, AXC-863, AXC-867, AXC-868, AXC-869, AXC-873, AXC-876, AXC-879, AXC-880, AXC-882, AXC-889, AXC-893, AXC-896, AXC-897, AXC-901, AXC-907, AXC-909, AXC-913, and AXC-914 compounds.In another embodiment, a TLR agonist comprising the structure recited in any one of the AXC-862, AXC-863, AXC-867, AXC-868, AXC-869, AXC-872, AXC-873, AXC-876, AXC-877, AXC-878, AXC-879, AXC-880, AXC-881, AXC-882, AXC-883, AXC-884, AXC-885, AXC-886, AXC-887, AXC-888, AXC-889, AXC-890, AXC-891, AXC-892, AXC-893, AXC-895, AXC-896, AXC-897, AXC-898, AXC-901, AXC-903, AXC-904, AXC-905, AXC-906, AXC-907, AXC-908, AXC-909, AXC-911, AXC-912, AXC-913, AXC-914, AXC-915, or AXC-916 compounds further comprises a linker. In another embodiment, the TLR agonist further comprises a linker and comprises the structure recited in Structure 5.

[0065] In another embodiment, the anti-HER2 antibody or antibody fragment comprises at least one amino acid sequence among SEQ ID NOs: 1 to 13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises at least two amino acid sequences among SEQ ID NOs: 1 to 13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises a) SEQ ID NO: 1 or 2, and b) any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises a) the heavy chain of SEQ ID NO: 1 or 2, and b) the light chain of any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises a) SEQ ID NO: 1, and b) any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises a) SEQ ID NO: 2, and b) any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 3. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 4. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 5. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 6. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 7. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 8. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 9. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 10. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 11. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 12. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 13.In another embodiment, the present invention provides an anti-HER2 antibody or antibody fragment, wherein the non-naturally encoded amino acid is specifically incorporated at position 114 according to Kabat numbering.

[0066] In another embodiment, the present invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist having a structure described in any of the structures of FIG. 1, wherein the TLR agonist is conjugated to the antibody or antibody fragment via a linker covalently attached to one or more non-naturally encoded amino acids incorporated into the antibody or antibody fragment, and the TC further comprises a chemotherapeutic agent or an immunotherapeutic agent. In another embodiment, the present invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist selected from any one of the compounds of Tables 3-7, wherein the TLR agonist is conjugated to the antibody or antibody fragment via a linker covalently attached to one or more non-naturally encoded amino acids incorporated into the antibody or antibody fragment. In another embodiment, the present invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist selected from any one of the compounds of Tables 3-7, wherein the TLR agonist is conjugated to the antibody or antibody fragment via a linker covalently attached to one or more non-naturally encoded amino acids incorporated into the antibody or antibody fragment, and the TC further comprises a chemotherapeutic agent or an immunotherapeutic agent.

[0067] In another embodiment, the present invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist comprising the structure described in any of the structures of FIG. 1, wherein the TLR agonist is conjugated to the antibody or antibody fragment via a linker covalently attached to one or more non-naturally encoded amino acids incorporated into the antibody or antibody fragment, and the TC further comprises a drug conjugate. In other embodiments, the drug conjugate is an antibody-drug conjugate. In another embodiment, the present invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist selected from any one of the compounds of Tables 3-7, wherein the TLR agonist is conjugated to the antibody or antibody fragment via a linker covalently attached to one or more non-naturally encoded amino acids incorporated into the antibody or antibody fragment. In another embodiment, the present invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist selected from any one of the compounds of Tables 3-7, wherein the TLR agonist is conjugated to the antibody or antibody fragment via a linker covalently attached to one or more non-naturally encoded amino acids incorporated into the antibody or antibody fragment, and the TC further comprises a drug conjugate. In other embodiments, the drug conjugate is an antibody-drug conjugate. In other embodiments, the TC further comprises a cytokine or a cytotoxin.

[0068] In another embodiment, the present invention provides a method of treating a subject or patient having cancer or a disease or condition or indication or disorder, comprising administering to the subject or patient a therapeutically effective amount of the composition or TC of the present invention. In certain embodiments, the tumor or cancer is a HER2-positive tumor or cancer. In certain embodiments, the tumor, cancer, indication, disease, disorder, or condition is a HER2-positive tumor, cancer, indication, disease, disorder, or condition. In certain embodiments, the tumor or cancer is selected from the group consisting of colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, glioblastoma, prostate cancer, bladder cancer, cervical cancer, pancreatic cancer, kidney cancer, esophageal cancer, vaginal cancer, gastric cancer, and leukemia.

[0069] In another embodiment, the present invention provides a method of treating a subject or patient having cancer or a disease or condition, comprising administering to the subject or patient a therapeutically effective amount of the composition or TC of the present invention, further comprising a chemotherapeutic agent or an immunotherapeutic agent. In certain embodiments, the TC is co-administered with at least one chemotherapeutic agent. The chemotherapeutic agent can be selected from the group consisting of temozolomide, gemcitabine, doxorubicin, cyclophosphamide, paclitaxel, cisplatin, fluoropyrimidine, taxane, anthracycline, lapatinib, capecitabine, letrozole, pertuzumab, docetaxel, IFN-α. In another embodiment of the present invention, the TC is co-administered with at least one chemotherapeutic agent.

[0070] In another embodiment, the present invention provides a method of treating a subject or patient having cancer or a disease or condition, comprising administering to the subject or patient a therapeutically effective amount of the composition or TC of the present invention, further comprising an antibody-drug conjugate, a cytotoxic agent, or a checkpoint inhibitor.

[0071] In another embodiment, the present invention provides a method of killing cells, comprising contacting the cells with the TC of the present invention. In other embodiments, the cells are tumor or cancer cells. In certain embodiments, the tumor or cancer cells are colon, ovarian, breast, melanoma, lung, glioblastoma, prostate, bladder, cervical, pancreatic, kidney, esophageal, vaginal, gastric, or leukemia cancer cells. In certain embodiments, the tumor or cancer is a HER2-positive tumor or cancer. In certain embodiments, the tumor, cancer, indication, disease, disorder, or condition being treated is a HER2-positive tumor, cancer, indication, disease, disorder, or condition.

[0072] The present invention provides a method of inhibiting or reducing the growth of a tumor or cancer, comprising contacting the tumor with an effective amount of the TC of the present invention to stimulate the patient's immune system in proximity to the tumor. The present invention provides a method of inhibiting or reducing the growth of a tumor or cancer, comprising contacting the tumor with an effective amount of the PEGylated TC of the present invention, or a stable dimer or multimer of TC. In one embodiment, the TC is non-PEGylated or mono-PEGylated. In one embodiment, the TC is di-PEGylated. In one embodiment, the TC has two or more and / or different TLR agonist molecules attached thereto. Another embodiment of the present invention provides a method of using the TC of the present invention to modulate an immune response against tumor cells.

[0073] In some embodiments, the present invention provides a method of using TC to treat cancer. In some embodiments, the TC of the present invention can be used for the treatment or prevention of cancer-related diseases, disorders, and conditions, including cancer and conditions directly or indirectly related to pre-cancerous states such as angiogenesis and dysplasia. In some embodiments, the tumor is a liquid or solid tumor. In some embodiments, the condition being treated is cancer. The cancer can be breast cancer, brain cancer, pancreatic cancer, skin cancer, lung cancer, liver cancer, gallbladder cancer, colon cancer, ovarian cancer, prostate cancer, uterine cancer, bone cancer, and blood cancer (leukemic cancer), or a cancer or disease or condition associated with any of these cancers, but is not limited thereto. Carcinomas are cancers that start from epithelial cells, which are cells that cover the body's surface, produce hormones, and form glands. As non-limiting examples, carcinomas include breast cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, rectal cancer, kidney cancer, bladder cancer, stomach cancer, prostate cancer, liver cancer, ovarian cancer, brain cancer, vaginal cancer, vulvar cancer, uterine cancer, oral cancer, penile cancer, testicular cancer, esophageal cancer, skin cancer, fallopian tube cancer, head and neck cancer, gastrointestinal stromal cancer, adenocarcinoma, skin or intraocular melanoma, cancer of the anal region, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid, cancer of the parathyroid, cancer of the adrenal gland, cancer of the urethra, renal pelvic cancer, cancer of the ureter, endometrial cancer, cervical cancer, pituitary cancer, tumors of the central nervous system (CNS), primary CNS lymphoma, brainstem glioma, and spinal cord axis tumors. In some cases, the cancer is a skin cancer such as basal cell carcinoma, squamous cell carcinoma, melanoma, non-melanoma, or actinic (solar) keratosis. In some embodiments, the present invention also relates to a method for treating acute leukemia in a mammal, which includes administering a therapeutically effective amount of the TC of the present invention to the mammal. The present invention also provides a method for inhibiting the proliferation of acute leukemia blast cells, which includes administering a therapeutically effective amount of the TC of the present invention to a mammal suffering from acute leukemia.

[0074] In another embodiment, the TC disclosed herein can be used to modulate the immune response. Modulation of the immune response can include stimulation, activation, increase, enhancement, or upregulation of the immune response. Modulation of the immune response can include suppressing, inhibiting, preventing, reducing, or downregulating the immune response.

[0075] In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the composition or TC of the present invention and a pharmaceutically acceptable carrier or excipient.

[0076] In another embodiment, the present invention provides the use of the composition of the present invention in the manufacture of a formulation.

[0077] In another embodiment, the present invention provides an immunostimulatory antibody conjugate (ISAC) comprising a TLR agonist as described in any one of the structures of FIG. 1. In another embodiment, the present invention provides an immunostimulatory antibody conjugate (ISAC) comprising a TLR agonist as described in any one of the compounds of Tables 3, 4, 5, 6, 7. In another embodiment, the present invention provides an ISAC in which the TLR agonist comprises a compound selected from the group of compounds AXC-862, AXC-863, AXC-867, AXC-868, AXC-869, AXC-874, AXC-875, AXC-876, AXC-879, AXC-880, AXC-882, AXC-893, AXC-896, AXC-897, AXC-901, AXC-907, and AXC-910.

[0078] In another embodiment, the present invention provides a salt of any one of the compounds having the structure described in FIG. 1. In another embodiment, the present invention provides a salt of any one of the compounds of Tables 3, 4, 5, 6, 7. In another embodiment, the present invention provides a pharmaceutical composition or a salt thereof with the composition, compound, and TC of the present disclosure. In other embodiments, the pharmaceutical composition or salt further comprises a pharmaceutically acceptable excipient.

[0079] [Brief Description of the Drawings] [FIG. 1] shows the general structure of a TLR agonist suitable for use in the present invention.

[0080] [FIG. 2] shows the structures of various TC conjugates.

[0081] [Figure 3] shows the structure of a further TC conjugate.

[0082] [Figure 4] shows the biological activity of selected TC conjugates in a cell proliferation assay.

[0083] [Figure 5] A and B show the TLR7 activity of various TLR7 agonists.

[0084] [Figure 6] shows the TLR7 activity of various TLR7 agonists linked to a linker.

[0085] [Figure 7] shows the TLR7 activity of further TLR7 agonists and TLR7 agonists linked to a linker.

[0086] [Figure 8] shows the TLR7 activity of further TLR7 agonists and TLR7 agonists linked to a linker.

[0087] [Figure 9] shows the TLR7 activity of different TLR7 agonists (drug linker or DL) linked to a linker compared to the unnatural amino acid pAF (DL-pAF).

[0088] [Figure 10] shows the HPLC chromatograms of an unconjugated anti-HER2 antibody having an unnatural amino acid at amino acid position HA114 (A), and anti-HER2 antibodies conjugated at amino acid position HA114 having the TLR agonists AXC-875 (B) and AXC-880 (C).

[0089] [Figure 11] compares the tumor-dependent ISAC activities of various payload linkers conjugated to an anti-HER2 antibody in SKOV3 HER2 high-expressing tumor cell line (A), JIMT-1 HER2 medium / low-expressing tumor cell line (B), and A431 HER2 low-expressing tumor cell line (C).

[0090] Compare the tumor-dependent ISAC activity of additional payload linkers conjugated to anti-HER2 antibodies in SKBR3 HER2-high-expressing tumor cell line (A) and HCC1806 HER2-low-expressing tumor cell line (B).

[0091] 〔Figure 13〕Compare the tumor-dependent ISAC activity of additional payload linkers conjugated to anti-HER2 antibodies in SKBR3 HER2-high-expressing tumor cell line (A) and HCC1806 HER2-low-expressing tumor cell line (B).

[0092] 〔Figure 14〕Compare the tumor-dependent ISAC activity of additional payload linkers conjugated to anti-HER2 antibodies in SKBR3 HER2-high-expressing tumor cell line (A) and HCC1806 HER2-low-expressing tumor cell line (B).

[0093] 〔Figure 15〕Compare the tumor-dependent ISAC activity of three (3) payload linkers conjugated to anti-HER2 antibodies in SKBR3 HER2-high-expressing tumor cell line (A) and HCC1806 HER2-low-expressing tumor cell line (B), showing that HER2-AXC-879 has the best ISAC activity.

[0094] 〔Mode for Carrying Out the Invention〕 Disclosed herein are TCs comprising a targeting moiety such as an antibody and one or more TLR agonists. The TLR agonist may further comprise one or more linker(s). The TCs of the present invention may comprise a TLR agonist linked to a non-natural amino acid of the targeting moiety. Also included is a method for making such TCs comprising non-natural amino acids incorporated into the targeting moiety polypeptide.

[0095] In certain embodiments, provided are pharmaceutical compositions comprising any of the described compounds and a pharmaceutically acceptable carrier, excipient, or binder.

[0096] A further or alternative embodiment is a method for detecting the presence of a polypeptide in a patient, the method comprising administering a polypeptide comprising at least one heterocyclic-containing unnatural amino acid, wherein the resulting heterocyclic-containing unnatural amino acid polypeptide modulates the immunogenicity of the polypeptide relative to the homologous naturally occurring amino acid polypeptide.

[0097] It is to be understood that the methods and compositions described herein are not limited to the specific methodologies, protocols, cell lines, constructs, and reagents described herein and may, therefore, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the methods and compositions described herein, which is limited only by the appended claims.

[0098] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0099] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention described herein belongs. Any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the invention described herein, but the preferred methods, devices, and materials are described herein.

[0100] All publications and patents mentioned herein are hereby incorporated by reference in their entirety for the purpose of, for example, describing and disclosing the constructs and methodologies described in the publications. This may be used in connection with the presently described invention. The publications discussed herein are provided only for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors described herein are not entitled to antedate such disclosure by virtue of prior invention or for any other reason.

[0101] The term "aldol-based linkage" or "mixed aldol-based linkage" refers to the acid or base-catalyzed condensation of a carbonyl compound with the enolate / enol of another carbonyl compound, which may or may not be the same, to produce a β-hydroxycarbonyl compound-aldol.

[0102] As used herein, the term "affinity label" refers to a label that binds reversibly or irreversibly to another molecule for the purpose of modifying it, destroying it, or forming a compound with it. Examples include enzymes and their substrates, or antibodies and their antigens.

[0103] The terms "alkoxy", "alkylamino", and "alkylthio" (or thioalkoxy) are used in their conventional meanings and refer to their alkyl groups linked to the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.

[0104] The term "alkyl", by itself or as part of another molecule, unless otherwise specified, means a straight-chain, branched-chain, or cyclic hydrocarbon radical, or combinations thereof, which may be fully saturated, monounsaturated or polyunsaturated, and has the specified number of carbon atoms (i.e., C1-C 10(which means from 1 to 10 carbons) may include divalent and polyvalent radicals. Examples of saturated hydrocarbon radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers such as, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl groups have one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers. The term "alkyl" also means, unless otherwise stated, to include those derivatives of alkyl as more particularly defined herein such as "heteroalkyl", "haloalkyl", and "homoalkyl".

[0105] The term "alkylene" means a divalent radical derived from an alkane, as exemplified by (-CH2-), n wherein n can be from 1 to about 24. By way of mere example, such groups include, but are not limited to, groups having up to 10 carbon atoms such as the structures -CH2CH2- and -CH2CH2CH2CH2-. "Lower alkyl" or "lower alkylene" are generally shorter chain alkyl or alkylene groups having up to 8 carbon atoms. The term "alkylene" also means, unless otherwise stated, to include those groups described herein as "heteroalkylene".

[0106] The term "amino acid" refers to naturally occurring and non-natural amino acids, as well as amino acid analogs and mimetics that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrrolidine, and selenocysteine. Amino acid analogs refer to compounds having the same basic chemical structure as the α-carbon bonded to hydrogen, a carboxyl group, an amino group, and an R group, which is, for example, a naturally occurring amino acid. Such analogs may have a modified R group (e.g., norleucine), or may have a modified peptide backbone while still retaining the same basic chemical structure as a naturally occurring amino acid. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium.

[0107] Amino acids can be referred to herein by either their names, their commonly known three-letter symbols, or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. In addition, nucleotides may be referred to by their generally accepted one-letter codes.

[0108] The "amino-terminal modifying group" refers to any molecule that can bind to the terminal amine group. By way of example, such a terminal amine group may be at the end of a polymer molecule, and such polymer molecules include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. The terminal modifying groups include, but are not limited to, various water-soluble polymers, peptides, or proteins. By way of one example only, the terminal modifying groups include polyethylene glycol or serum albumin. The terminal modifying groups can be used to modify the therapeutic characteristics of polymer molecules, including, but not limited to, increasing the serum half-life of a peptide.

[0109] As used herein, "antibody" means a protein consisting of one or more polypeptides substantially encoded by all or a portion of an antibody gene. Immunoglobulin genes include, but are not limited to, kappa, lambda, alpha, gamma (IgG1, IgG2, IgG3, and IgG4), delta, epsilon, and mu constant region genes, as well as myriad immunoglobulin variable region genes. Antibodies herein include full-length antibodies and antibody fragments, and mean antibodies that occur naturally within any organism or are engineered (e.g., variants).

[0110] "Antibody fragment" means any form of an antibody other than the full-length form. Antibody fragments as used herein include antibodies that are smaller components present within a full-length antibody, and antibodies that have been engineered. Antibody fragments include, but are not limited to, Fv, Fc, Fab, and (Fab’)2, single-chain Fv (scFv), diabody, triabody, tetrabody, bifunctional hybrid antibody, combinations of CDR1, CDR2, CDR3, CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, and variable regions, as well as alternative scaffold non-antibody molecules, bispecific antibodies, etc. (Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9:395-402). Another functional substructure is the single-chain Fv (scFv) consisting of the variable regions of the immunoglobulin heavy and light chains covalently linked by a peptide linker (S-z Hu et al., 1996, Cancer Research, 56, 3055-3061). These small (Mr 25,000) proteins generally retain the specificity and affinity for an antigen in a single polypeptide and can provide convenient building blocks for larger antigen-specific molecules. Unless otherwise specified, the terms "antibody(ies)" as used in the specification and claims specifically include "antibody fragment(s)".

[0111] As used herein, "antibody-drug conjugate" or "ADC" refers to an antibody molecule or fragment thereof that is covalently linked to one or more biologically active molecule(s). The biologically active molecule can be conjugated to the antibody via a linker, polymer, or other covalent bond.

[0112] As used herein, the terms "aromatic" or "aryl" refer to a closed ring structure having at least one ring with a conjugated π - electron system, including both carbocyclic aryl and heterocyclic aryl (or "heteroaryl" or "heteroaromatic") groups. The carbocyclic or heterocyclic aromatic group may contain 5 to 20 ring atoms. The term includes monocyclic rings or fused - ring polycyclic (i.e., rings sharing a pair of adjacent carbon atoms) groups that are covalently linked. The aromatic group may be unsubstituted or substituted. Non - limiting examples of "aromatic" or "aryl" groups include phenyl, 1 - naphthyl, 2 - naphthyl, 4 - biphenyl, anthracenyl, and phenanthracenyl. The substituents for each of the above - mentioned aryl and heteroaryl ring systems are selected from the group of acceptable substituents described herein.

[0113] Briefly stated, the terms "aromatic" or "aryl", when used in combination with other terms (including but not limited to aryloxy, arylthioxy, aralkyl), include both the aryl ring and the heteroaryl ring as defined above. Thus, the term "aralkyl" or "alkaryl" means those radicals in which an aryl group is bonded to an alkyl group (including but not limited to benzyl, phenethyl, pyridylmethyl, etc.), including those alkyl groups in which a carbon atom (including but not limited to a methylene group) is replaced by a heteroatom, for example, an oxygen atom. Examples of such aryl groups include, but are not limited to, phenoxymethyl, 2 - pyridyloxymethyl, 3-(1 - naphthyloxy)propyl, etc.

[0114] As used herein, the term "arylene" refers to a divalent aryl radical. Non - limiting examples of "arylene" include phenylene, pyridinylene, pyrimidinylene, and thiophenylene. The substituents of the arylene group are selected from the group of acceptable substituents described herein.

[0115] "Bifunctional polymer", also referred to as "bifunctional linker", refers to a polymer containing two functional groups capable of specifically reacting with other moieties to form covalent or non-covalent bonds. Such moieties can include, but are not limited to, natural or unnatural amino acids, or side groups on peptides containing such natural or unnatural amino acids. The other moieties that can be linked to the bifunctional linker or bifunctional polymer may be the same or different. By way of example only, a bifunctional linker can have a functional group reactive with a group on a first peptide and another functional group reactive with a group on a second peptide, thereby forming a conjugate comprising the first peptide, the bifunctional linker, and the second peptide. Many procedures and linker molecules are known for attaching various compounds to peptides. See, for example, European Patent Application No. 188,256, U.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784, 4,680,338, and 4,569,789 (which are hereby incorporated by reference in their entirety). "Multifunctional polymer", also referred to as "multifunctional linker", refers to a polymer containing two or more functional groups capable of reacting with other moieties. Such moieties can include, but are not limited to, natural or unnatural amino acids, or side groups on peptides containing such natural or unnatural amino acids. (Including but not limited to amino acid side groups) for forming covalent or non-covalent bonds. The bifunctional polymer or multifunctional polymer can be of any desired length or molecular weight and can be selected to provide a specific desired spacing or conformation between one or more molecules linked to a compound and the molecule or compound to which it is attached.

[0116] As used herein, the term "bioavailability" refers to the rate and extent to which a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. An increase in bioavailability refers to an increase in the rate and extent to which a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. By way of example, an increase in bioavailability can be shown as an increase in the concentration of the substance or its active moiety in the blood when compared to other substances or active moieties. Methods for assessing an increase in bioavailability are known in the art and can be used to assess the bioavailability of any polypeptide.

[0117] As used herein, the terms "biologically active molecule", "biologically active moiety", or "biologically active agent" mean any substance that can affect any physical or biochemical property of a biological system, pathway, molecule, or interaction related to a living organism, including but not limited to viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans. In particular, as used herein, biologically active molecules include, but are not limited to, any substance intended for the diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals or, alternatively, for enhancing the physical or mental health of humans or animals. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, prodrugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles, and micelles. Classes of biologically active agents suitable for use with the methods and compositions described herein include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, bactericides, antiviral agents, anti-inflammatory agents, antitumor agents, cardiovascular therapeutics, anxiolytics, hormones, growth factors, steroid agents, microbial-derived toxins, and the like.

[0118] "Regulation of biological activity" means increasing or decreasing the reactivity of a polypeptide, changing the selectivity of a polypeptide, or enhancing or decreasing the substrate selectivity of a polypeptide. Analysis of modified biological activity can be carried out by comparing the biological activity of a non-natural polypeptide with that of a natural polypeptide.

[0119] As used herein, the term "biomaterial" refers to materials of biological origin, including but not limited to materials obtained from bioreactors and / or recombinant methods and techniques.

[0120] As used herein, the term "biophysical probe" refers to a probe that can detect or monitor structural changes in a molecule. Such molecules include, but are not limited to, proteins, and "biophysical probes" can be used to detect or monitor protein interactions with other macromolecules. Examples of biophysical probes include, but are not limited to, spin labels, fluorophores, and photoactivatable groups.

[0121] As used herein, the term "biosynthetically" refers to any method that utilizes a translation system (cellular or cell-free) that involves the use of at least one of the following components: polynucleotide, codon, tRNA, and ribosome. For example, unnatural amino acids can be "biosynthetically incorporated" into unnatural amino acid polypeptides using the methods and techniques described in WO2002 / 085923 (incorporated herein by reference in its entirety). In addition, a useful method for selecting unnatural amino acids that can be "biosynthetically incorporated" into unnatural amino acid polypeptides is described in WO2002 / 085923, which is incorporated herein by reference in its entirety.

[0122] As used herein, the term "biotin analog" or also referred to as "biotin mimetic" is any molecule other than biotin that binds to avidin and / or streptavidin with high affinity.

[0123] As used herein, the term "carbonyl" refers to a group containing a moiety selected from the group consisting of -C(O)-, -S(O)-, -S(O)2-, and -C(S)-, including but not limited to groups containing at least one ketone group, and / or at least one aldehyde group, and / or at least one ester group, and / or at least one carboxylic acid group, and / or at least one thioester group. Such carbonyl groups include ketones, aldehydes, carboxylic acids, esters, and thioesters. In addition, such groups can be part of linear, branched, or cyclic molecules.

[0124] The term "carboxy-terminal modifying group" refers to any molecule that can be attached to a terminal carboxy group. By way of example, such a terminal carboxy group may be at the end of a polymer molecule, such polymer molecules including but not limited to polypeptides, polynucleotides, and polysaccharides. Carboxy-terminal modifying groups include, but are not limited to, various water-soluble polymers, peptides, or proteins. By way of a single example, carboxy-terminal modifying groups include polyethylene glycol or serum albumin. Carboxy-terminal modifying groups can be used to modify the therapeutic characteristics of polymer molecules, including but not limited to increasing the serum half-life of a peptide.

[0125] As used herein, the term "chemically cleavable group" is also referred to as "chemically labile" and refers to a group that decomposes or cleaves upon exposure to an acid, base, oxidizing agent, reducing agent, chemical reaction initiator, or radical reaction initiator.

[0126] As used herein, "co-folding" refers to a refolding process, reaction, or method that uses at least two molecules that interact with each other to effect the conversion of non-folded or improperly folded molecules into properly folded molecules. By way of example only, "co-folding" uses at least two polypeptides that interact with each other to effect the conversion of non-folded or improperly folded polypeptides into a naturally occurring properly folded polypeptide. Such polypeptides can contain natural amino acids and / or at least one non-natural amino acid.

[0127] As used herein, "conjugate" refers to a polypeptide that is linked, e.g., covalently linked, either directly or via a linker, to a compound or compound-linker described herein, such as any one of the compounds or salts of the structures described in FIG. 1, or any one of the structures in Tables 3-7. "Targeting moiety" refers to a structure that has a selective affinity for a target molecule as compared to other non-target molecules. The targeting moiety of the present invention binds to the target molecule. The targeting moiety can include, for example, an antibody, a peptide, a ligand, a receptor, or a binding portion thereof. The target biological molecule can be another structure of a cell, such as a biological receptor or a tumor antigen. As used herein, the terms "conjugate of the present invention", "targeting moiety conjugate", "targeted conjugate", "targeting moiety active molecule conjugate", or "TC" refer to a targeting polypeptide that binds to a target present on a cell or a subunit thereof that is conjugated to a biologically active molecule, a portion thereof, or an analog thereof, including but not limited to TLR7 and / or TLR8 agonists. As used herein, the terms "tumor targeting moiety conjugate", "tumor targeting moiety - biologically active molecule conjugate" or "BTC" refer to a tumor targeting polypeptide that binds to a target present on a tumor cell or a subunit thereof that is conjugated to a biologically active molecule, a portion thereof, or an analog thereof, including but not limited to TLR7 and / or TLR8 agonists. Unless otherwise indicated, the terms "compound of the present invention" and "composition of the present invention" are used as alternatives to the term "conjugate of the present invention".

[0128] The term "conservatively modified variant" applies to both natural and unnatural amino acids, as well as to both natural and unnatural nucleic acid sequences, and combinations thereof. With respect to a particular nucleic acid sequence, a "conservatively modified variant" refers to those natural and unnatural nucleic acids that encode the same or essentially the same natural and unnatural amino acid sequences, or that do not encode natural and unnatural amino acid sequences for an essentially identical sequence. By way of example, due to the degeneracy of the genetic code, a number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be modified to any of the corresponding codons described without modifying the encoded polypeptide. Such nucleic acid changes are "silent changes", which are one species of conservatively modified changes. Thus, by way of example, all natural or unnatural nucleic acid sequences herein that encode a natural or unnatural polypeptide also describe all possible silent changes of the natural or unnatural nucleic acids. One of ordinary skill in the art will recognize that each codon in a natural or unnatural nucleic acid (except for the AUG, which is typically the only codon for methionine, and the TGG, which is typically the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Thus, each silent change of a natural and unnatural nucleic acid encoding a natural and unnatural polypeptide is implicit in each described sequence.

[0129] With respect to amino acid sequences, individual substitutions, deletions, or additions to the nucleic acid, peptide, polypeptide, or protein sequences which modify, add, or delete a single natural and unnatural amino acid, or a small number of natural and unnatural amino acids in the coding sequence are “conservatively modified variants” where the modification results in an amino acid deletion, an amino acid addition, or substitution by an amino acid chemically similar to a natural and unnatural amino acid. Tables of conserved substitutions providing functionally similar amino acids are well known in the art. Such conservatively modified variants are added to, and do not exclude, the polymorphic variants, interspecies homologs, and alleles of the methods and compositions described herein.

[0130] Tables of conserved substitutions providing functionally similar amino acids are known to those of skill in the art. The following eight groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M). (See, e.g., Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2nd edition (December 1993)). The terms "cycloalkyl" and "heterocycloalkyl", alone or in combination with other terms, unless otherwise specified, each represent a cyclic form of "alkyl" and "heteroalkyl", respectively. Thus, cycloalkyl or heterocycloalkyl includes saturated, partially unsaturated, and fully unsaturated ring linkages. In addition, in the case of heterocycloalkyl, the heteroatom can occupy the position where the heterocycle is attached to the rest of the molecule. The heteroatom can include, but is not limited to, oxygen, nitrogen, or sulfur. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, etc. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, etc. In addition, the term encompasses polycyclic structures including, but not limited to, bicyclic and tricyclic ring structures. Similarly, the term "heterocycloalkylene" means a divalent radical derived from heterocycloalkyl, either alone or as part of another molecule, and the term "cycloalkylene" means a divalent radical derived from cycloalkyl, either alone or as part of another molecule.

[0131] As used herein, the term "cyclodextrin" refers to a cyclic carbohydrate consisting of at least 6 - 8 glucose molecules in the ring formation. The outer portion of the ring contains water-soluble groups, and in the center of the ring is a relatively nonpolar cavity capable of accommodating small molecules.

[0132] As used herein, the term "cytotoxic" refers to a compound that harms cells.

[0133] As used herein, "Denaturing agent" or "denaturant" refers to any compound or material that causes reversible unfolding of a polymer. By way of example only, a "denaturing agent" or "denaturant" can cause reversible unfolding of a protein. The strength of a denaturing agent or denaturant is determined by both the properties and concentration of a particular denaturing agent or denaturant. By way of example, denaturing agents or denaturants include, but are not limited to, chaotropes, detergents, organic water-miscible solvents, phospholipids, or combinations thereof. Non-limiting examples of chaotropes include, but are not limited to, urea, guanidine, and sodium thiocyanate. Non-limiting examples of detergents can include strong detergents such as sodium dodecyl sulfate, or polyoxyethylene ethers (such as Tween or Triton detergents), sarcosyl, mild non-ionic detergents (such as digitonin), mild cationic detergents such as N->2,3-(dioleyloxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents (such as sodium cholate or sodium deoxycholate), or zwitterionic detergents (Zwittergent, 3-(3-chloroamidopropyl)dimethylammonio-1-propanesulfate (CHAPS), and 3-(3-chloroamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO) may be included, but are not limited thereto). Non-limiting examples of organic water-miscible solvents that can be used as denaturing agents include acetonitrile, lower alkanols (especially C2-C4 alkanols such as ethanol or isopropanol), or lower alkanediols (C2-C4 alkanediols such as ethylene-glycol), but are not limited thereto.Non-limiting examples of phospholipids include naturally occurring phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol, or synthetic phospholipid derivatives or variants such as dihexanoyl phosphatidylcholine or diheptanoyl phosphatidylcholine, but are not limited thereto.

[0134] As used herein, the term "diamine" refers to a group / molecule containing at least two amine functional groups, including but not limited to hydrazine groups, amidine groups, imine groups, 1,1-diamine groups, 1,2-diamine groups, 1,3-diamine groups, and 1,4-diamine groups. Additionally, such groups can be part of a linear, branched, or cyclic molecule.

[0135] As used herein, the term "detectable label" refers to a label that can be observed using analytical techniques including but not limited to fluorescence, chemiluminescence, electron-spin resonance, ultraviolet / visible absorption spectroscopy, mass spectrometry, nuclear magnetic resonance, magnetic resonance, and electrochemical methods.

[0136] As used herein, the term "dicarbonyl" refers to a group containing at least two moieties selected from the group consisting of -C(O)-, -S(O)-, -S(O)2-, and -C(S)-, including, but not limited to, 1,2-dicarbonyl groups, 1,3-dicarbonyl groups, and 1,4-dicarbonyl groups, as well as at least one ketone group, and / or at least one aldehyde group, and / or at least one ester group, and / or at least one carboxylic acid group, and / or at least one thioester group. Such dicarbonyl groups include diketones, ketoaldehydes, ketoacids, ketoesters, and ketothioesters. In addition, such groups can be part of a linear, branched, or cyclic molecule. The two moieties in the dicarbonyl group may be the same or different, and either of the two moieties may contain substituents that produce, by way of example only, an ester, ketone, aldehyde, thioester, or amide.

[0137] As used herein, the term "drug" refers to any substance used in the prevention, diagnosis, alleviation, treatment, or cure of a disease or condition.

[0138] As used herein, the term "effective amount" refers to a sufficient amount of an administered agent or compound to effect some alleviation of one or more of the symptoms of the disease or condition being treated. The result can be a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. By way of example, the administered agent or compound can be, but is not limited to, a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-amino acid polypeptide. Compositions containing such natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides can be administered for prophylactic, enhancing, and / or therapeutic treatment. The appropriate "effective" amount in any individual case can be determined using techniques such as dose escalation studies.

[0139] The term "enhance" or "enhancement" means that either the potency or the duration of the desired effect is increased or extended. As an example, "enhancing" the effect of a therapeutic agent refers to the ability to increase or extend the effect of the therapeutic agent in either potency or duration during the treatment of a disease, disorder, or condition. As used herein, an "enhancing effective amount" refers to an amount sufficient to enhance the effect of a therapeutic agent in the treatment of a disease, disorder, or condition. When used in a patient, the effective amount for such use depends on the severity and course of the disease, disorder, or condition, previous therapy, the patient's health status and response to the drug, and the judgment of the physician performing the treatment.

[0140] As used herein, the term "eukaryote" refers to organisms belonging to the eukaryotic taxonomic domain, including but not limited to animals (including but not limited to mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to monocots, dicots, and algae), fungi, yeasts, flagellates, microsporidia, and protists.

[0141] As used herein, the term "fatty acid" refers to carboxylic acids having hydrocarbon side chains of about C6 or longer.

[0142] As used herein, the term "fluorophore" refers to a molecule that emits photons when excited and is thereby fluorescent.

[0143] As used herein, the terms "functional group", "active moiety", "active group", "leaving group", "reactive site", "chemically reactive group", and "chemically reactive moiety" refer to a part or unit of a molecule at which a chemical reaction occurs. These terms are to some extent synonymous in the chemical art and are used herein to denote a part of a molecule that performs some function or activity and is reactive with other molecules.

[0144] The term "halogen" includes fluorine, chlorine, iodine, and bromine.

[0145] As used herein, the term "haloacyl" refers to an acyl group containing a halogen moiety including, but not limited to, -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.

[0146] As used herein, the term "haloalkyl" refers to an alkyl group containing a halogen moiety including, but not limited to, -CF3 and -CH2CF3, etc.

[0147] As used herein, the term "heteroalkyl" refers to an alkyl group, and a straight-chain, branched-chain, or cyclic hydrocarbon radical consisting of at least one heteroatom selected from the group consisting of O, N, Si, and S, or combinations thereof, wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The O, N, S, and Si heteroatom(s) may be located at any internal position of the heteroalkyl group or at the position where the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. In addition, up to two heteroatoms may be consecutive, for example, by way of example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

[0148] The term "heterocyclic ring-based linkage" or "heterocyclic linkage" refers to a moiety formed from the reaction of a dicarbonyl group and a diamine group. The resulting reaction product is a heterocyclic ring containing a heteroaryl group or a heterocycloalkyl group. The resulting heterocyclic group functions as a chemical linkage between a non-natural amino acid or a non-natural amino acid polypeptide and another functional group. In one embodiment, the heterocyclic linkage includes nitrogen-containing heterocyclic linkages including, by way of example only, pyrazole linkages, pyrrole linkages, indole linkages, benzodiazepine linkages, and pyrazolone linkages.

[0149] Similarly, the term "heteroalkylene" refers to a divalent radical derived from heteroalkyl, exemplified by, but not limited to, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. With respect to the heteroalkylene group, the same or different heteroatoms can also occupy one or both of the chain termini (including, but not limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, etc.). Still further, with respect to the alkylene and heteroalkylene linking groups, the orientation of the linking group is not implied by the direction in which the formula of the linking group is written. By way of example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-.

[0150] As used herein, the term "heteroaryl" or "heteroaromatic" refers to an aryl group containing at least one heteroatom selected from N, O, and S, wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen atom(s) may optionally be quaternized. The heteroaryl group may be substituted or unsubstituted. The heteroaryl group may be attached to the remainder of the molecule via a heteroatom. Non-limiting examples of heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.

[0151] As used herein, the term "homoalkyl" refers to an alkyl group that is a hydrocarbon group.

[0152] As used herein, the term "identical" refers to two or more arrays or sub-arrays that are the same. In addition, as used herein, the term "substantially identical" refers to two or more arrays that have a percentage of array units that are the same when compared and aligned for maximum match over a specified region measured using a comparison window, or a comparison algorithm, or by manual alignment and visual inspection. By way of example only, two or more arrays may be "substantially identical" if the array units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. Such percentages describe the "percent identity" of two or more arrays. Array identity can exist over a region of at least about 75 to 100 array units in length, over a region of about 50 array units in length, or, if not specified, over the entire array. This definition also refers to the complementarity of test arrays. By way of example only, two or more polypeptide sequences are identical when the amino acid residues are the same, while two or more polypeptide sequences are "substantially identical" if the amino acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. Identity can exist over a region of at least about 75 to about 100 amino acids in length, over a region of about 50 amino acids in length, or, if not specified, over the entire polypeptide sequence. In addition, by way of example only, two or more polynucleotide sequences are identical when the nucleic acid residues are the same, while two or more polynucleotide sequences are "substantially identical" if the nucleic acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. Identity can exist over a region of at least about 75 to about 100 nucleic acids in length, over a region of about 50 nucleic acids in length, or, if not specified, over the entire polynucleotide sequence.

[0153] For array comparison, typically, one array functions as a reference array against which a test array is compared. When using an array comparison algorithm, the test and reference arrays are input into a computer, and optionally, subsequence coordinates are specified and the parameters of the array algorithm program are specified. Default program parameters may be used or alternative parameters may be specified. The array comparison algorithm then calculates the percent sequence identity of the test array relative to the reference array based on the program parameters.

[0154] As used herein, the term "immunogenicity" refers to the antibody response to the administration of a therapeutic agent. The immunogenicity of a therapeutic unnatural amino acid polypeptide can be obtained using quantitative and qualitative assays for the detection of anti-unnatural amino acid polypeptide antibodies in biological fluids. Such assays include, but are not limited to, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), luminescent immunoassay (LIA), and fluorescence immunoassay (FIA). Analysis of the immunogenicity of a therapeutic unnatural amino acid polypeptide involves comparing the antibody response upon administration of the therapeutic unnatural amino acid polypeptide to the antibody response upon administration of a therapeutic natural amino acid polypeptide.

[0155] As used herein, the term "isolated" refers to separating and removing a component of interest from components that are not of interest. An isolated substance can be in a dry or semi-dry state, or in a solution, including but not limited to an aqueous solution. An isolated component can be in a homogeneous state, or the isolated component can be part of a pharmaceutical composition that includes additional pharmaceutically acceptable carriers and / or excipients. Purity and homogeneity can be determined using analytical chemistry techniques including, but not limited to, polyacrylamide gel electrophoresis or high performance liquid chromatography. Further, when the component of interest is isolated and is the major species present in the preparation, the component is described herein as being substantially purified. As used herein, the term "purified" can refer to a component of interest that is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or more pure. By way of example only, a nucleic acid or protein is "isolated" when such nucleic acid or protein does not include at least some of the cellular components with which it is associated in its natural state, or when the nucleic acid or protein is concentrated to a level higher than its in vivo or in vitro production concentration. Also, by way of example, a gene is isolated when it is separated from open reading frames adjacent to the gene that encode proteins other than the gene of interest.

[0156] As used herein, the term "label" refers to a substance that is incorporated into a compound and is readily detectable, whereby the physical distribution thereof can be detected and / or monitored.

[0157] As used herein, the terms "linkage" or "linker" refer to a bond or chemical moiety formed from a chemical reaction between a functional group of the linker and another molecule. Such bonds can include, but are not limited to, covalent and non-covalent bonds. On the other hand, such chemical moieties can include, but are not limited to, esters, carbonates, iminophosphates, hydrazones, acetals, orthoesters, peptide linkages, and oligonucleotide linkages. A hydrolytically stable linkage means that the linkage is substantially stable in water and does not react with water at useful pH values, including but not limited to physiological conditions, for a long time, perhaps even permanently. A hydrolytically unstable or degradable linkage means that the linkage is degradable, for example, in water or an aqueous solution containing blood. An enzymatically unstable or degradable linkage means that the linkage can be degraded by one or more enzymes. As just one example, PEG and related polymers may contain degradable linkages within the polymer backbone or within a linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. Such degradable linkages include, but are not limited to, ester linkages formed by the reaction of PEG carboxylic acid or activated PEG carboxylic acid with an alcohol group on a biologically active agent, and such ester groups generally hydrolyze under physiological conditions to release the biologically active agent. Other hydrolytically degradable linkages include carbonate linkages, imine linkages resulting from the reaction of an amine and an aldehyde, phosphate ester linkages formed by the reaction of an alcohol and a phosphate group, hydrazone linkages which are reaction products of a hydrazide and an aldehyde, acetal linkages which are reaction products of an aldehyde and an alcohol, orthoester linkages which are reaction products of a formate and an alcohol, peptide linkages formed by amine groups including but not limited to the termini of polymers such as PEG and carboxyl groups of peptides, and oligonucleotide linkages formed by phosphoramidite groups including but not limited to the termini of polymers and 5'-hydroxyl groups of oligonucleotides, but are not limited to these.Linkers include, but are not limited to, short linear, branched, multi-arm, or dendrimer molecules such as polymers. In some embodiments of the present invention, the linker may be branched. In other embodiments, the linker can be a bifunctional linker. In some embodiments, the linker can be a trifunctional linker. Those skilled in the art are aware of several different cleavable linkers. See U.S. Pat. Nos. 4,618,492, 4,542,225, and 4,625,014. Mechanisms for releasing the agent from these linker groups include, for example, irradiation of a photolabile bond and acid-catalyzed hydrolysis. For example, U.S. Pat. No. 4,671,958 includes a description of an immunoconjugate that includes a linker that is cleaved at the target site in vivo by a proteolytic enzyme of the patient's complement system. The length of the linker can be predetermined or selected according to the desired spatial relationship between the polypeptide and the molecule linked thereto. In view of the numerous methods reported for conjugating various radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and other agents to antibodies, those skilled in the art will be able to determine a suitable method for conjugating a given agent or molecule to a polypeptide.

[0158] As used herein, the term "modified" refers to the presence of a change to a natural amino acid, unnatural amino acid, natural amino acid polypeptide, or unnatural amino acid polypeptide. Such changes or modifications can be obtained by post-synthetic modification of a natural amino acid, unnatural amino acid, natural amino acid polypeptide, or unnatural amino acid polypeptide, or by co-translation, or by post-translational modification of a natural amino acid, unnatural amino acid, natural amino acid polypeptide, or unnatural amino acid polypeptide. The phrase "modified or unmodified" means that the natural amino acid, unnatural amino acid, natural amino acid polypeptide, or unnatural amino acid polypeptide being discussed is optionally modified, i.e., the natural amino acid, unnatural amino acid, natural amino acid polypeptide, or unnatural amino acid polypeptide being discussed can be either modified or unmodified.

[0159] As used herein, the term "modulated serum half-life" refers to a positive or negative change in the circulating half-life of a modified biologically active molecule relative to its unmodified form. By way of example, modified biologically active molecules include, but are not limited to, natural amino acids, unnatural amino acids, natural amino acid polypeptides, or unnatural amino acid polypeptides. By way of example, serum half-life is measured by taking blood samples at various time points after administration of the biologically active molecule or modified biologically active molecule and determining the concentration of that molecule in each sample. Correlation of serum concentration with time enables calculation of the serum half-life. By way of example, a modulated serum half-life can be an increase in serum half-life that enables an improved dosing regimen or avoids toxic effects. Such an increase in serum can be at least about 2-fold, at least about 3-fold, at least about 5-fold, or at least about 10-fold. Methods for assessing an increase in the serum half-life of any polypeptide are well known to those of skill in the art.

[0160] As used herein, the term "modulated therapeutic half-life" refers to a positive or negative change in the half-life of a therapeutically effective amount of a modified biologically active molecule relative to its unmodified form. By way of example, modified biologically active molecules include, but are not limited to, natural amino acids, unnatural amino acids, natural amino acid polypeptides, or unnatural amino acid polypeptides. By way of example, therapeutic half-life is measured by measuring the pharmacokinetic and / or pharmacodynamic properties of the molecule at various time points after administration. An increase in therapeutic half-life can enable a particular beneficial dosing regimen, a particular beneficial total dose, or avoid undesirable effects. By way of example, an increase in therapeutic half-life can result from an increase or decrease in efficacy, an increase or decrease in binding of the modified molecule to its target, an increase or decrease in another parameter or mechanism of action of the unmodified molecule, or, by way of example only, an increase or decrease in degradation of the molecule by an enzyme such as a protease. Methods for assessing an increase in the therapeutic half-life of any polypeptide are well known to those of skill in the art.

[0161] "Unnatural amino acid" refers to an amino acid that is not one of the 20 common amino acids, or pyrrolidine or selenocysteine. Other terms that may be used synonymously with the term "unnatural amino acid" are "non-naturally encoded amino acid", "unnatural amino acid", "amino acid that does not occur naturally", and various hyphenated and non-hyphenated forms thereof. The term "unnatural amino acid" includes, but is not limited to, amino acids that occur naturally by modification of a naturally encoded amino acid (including, but not limited to, the 20 common amino acids or pyrrolidine and selenocysteine), but are not themselves incorporated into a polypeptide chain that grows by a translation complex. Examples of such amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine. In addition, the term "unnatural amino acid" includes, but is not limited to, amino acids that do not occur naturally and can be obtained synthetically or by modification of an unnatural amino acid. In some embodiments, the unnatural amino acid includes lysine analogs, such as N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, or allyloxycarbonyl lysine. In some embodiments, the unnatural amino acid includes a sugar moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine. Examples of such amino acids also include cases where the naturally occurring N- or O-linkage between the amino acid and the sugar is replaced by a covalent bond that is not commonly found in nature, including, but not limited to, alkenes, oximes, thioethers, amides, etc. Examples of such amino acids also include sugars that are not commonly found in naturally occurring proteins, such as 2-deoxy-glucose, 2-deoxygalactose, etc.Specific examples of non-natural amino acids include, but are not limited to, p-acetyl-L-phenylalanine, p-propynyloxyphenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propynyloxy-L-phenylalanine, 4-azido-L-phenylalanine, para-azidoethoxyphenylalanine, and para-azidomethyl-phenylalanine. In some embodiments, the non-natural amino acid is selected from the group consisting of para-acetyl-phenylalanine, 4-azido-L-phenylalanine, para-azidoethoxyphenylalanine, or para-azidomethyl-phenylalanine.

[0162] As used herein, the term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides in either single-stranded or double-stranded form, as well as polymers thereof. By way of example only, such nucleic acids and nucleic acid polymers include: (i) analogs of natural nucleotides that have binding properties similar to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides; (ii) oligonucleotide analogs including, but not limited to, PNA (peptide nucleic acid), analogs of DNA used in antisense technology (such as phosphorothioates, phosphoramidates, etc.); (iii) their conservatively modified variants (including, but not limited to, degenerate codon substitutions), and complementary sequences and sequences explicitly shown, among others. By way of example, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991), Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985), and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0163] As used herein, the term "oxidizing agent" refers to a compound or material capable of removing electrons from a compound that is oxidized. By way of example, oxidizing agents include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythritol, and oxygen. A wide variety of oxidizing agents are suitable for use in the methods and compositions described herein.

[0164] As used herein, the term "pharmaceutically acceptable" refers to materials, including but not limited to salts, carriers, or diluents that do not inhibit the biological activity or properties of a compound and are relatively non-toxic, i.e., the materials do not cause undesirable biological effects or interact in a harmful manner with any of the components of the composition in which it is contained and can be administered to an individual.

[0165] As used herein, the term "polyalkylene glycol" or "poly(alkene glycol)" refers to a linear or branched polymeric polyether polyol. Such polyalkylene glycols include, but are not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof. Other exemplary embodiments are listed in catalogs of commercial suppliers such as, for example, Shearwater Corporation’s catalog “Polyethylene Glycol and Derivatives for Biomedical Applications” (2001). By way of example only, such polymeric polyether polyols have an average molecular weight of from about 0.1 kDa to about 100 kDa. By way of example, such polymeric polyether polyols include, but are not limited to, from about 100 Da to about 100,000 Da or more. The molecular weight of the polymer can be from about 100 Da to about 100,000 Da, including, but not limited to, about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da, about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, about 1,000 Da, about 900 Da, about 800 Da, about 700 Da, about 600 Da, about 500 Da, 400 Da, about 300 Da, about 200 Da, and about 100 Da. In some embodiments, the molecular weight of the polymer is from about 100 Da to about 50,000 Da. In some embodiments, the molecular weight of the polymer is from about 100 Da to about 40,000 Da. In some embodiments, the molecular weight of the polymer is from about 1,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the polymer is from about 2,000 to about 50,000 Da.In some embodiments, the molecular weight of the polymer is from about 5,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the polymer is from about 10,000 Da to about 40,000 Da. In some embodiments, the poly(ethylene glycol) molecule is a branched polymer. The molecular weight of the branched-chain PEG can be from about 1,000 Da to about 100,000 Da, including, but not limited to, about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da, about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, and about 1,000 Da. In some embodiments, the molecular weight of the branched-chain PEG is from about 1,000 Da to about 50,000 Da. In some embodiments, the molecular weight of the branched-chain PEG is from about 1,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the branched-chain PEG is from about 5,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the branched-chain PEG is from about 5,000 Da to about 20,000 Da. In other embodiments, the molecular weight of the branched-chain PEG is from about 2,000 to about 50,000 Da.

[0166] As used herein, the term "polymer" refers to a molecule composed of repeating subunits. Such molecules include, but are not limited to, polypeptides, polynucleotides, or polysaccharides or polyalkylene glycols.

[0167] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to polymers of amino acid residues. That is, descriptions regarding polypeptides apply equally to descriptions of peptides and proteins, and vice versa. The term applies to both naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-natural amino acids. In addition, such "polypeptides", "peptides", and "proteins" include amino acid chains of any length, including full-length proteins, and the amino acid residues are linked by covalent peptide bonds.

[0168] The term "post-translational modification" refers to any modification of a natural or non-natural amino acid that occurs after such amino acid has been incorporated into the polypeptide chain by translation. Such modifications include, but are not limited to, co-translational in vivo modifications, co-translational in vitro modifications (such as in a cell-free translation system), post-translational in vivo modifications, and post-translational in vitro modifications.

[0169] As used herein, the terms "prodrug" or "pharmaceutically acceptable prodrug" refer to an agent that does not inhibit the biological activity or properties of a drug and is relatively non-toxic, and is converted in vivo or in vitro to the parent drug, i.e., the material can be administered to an individual without causing undesirable biological effects or interacting in a harmful manner with any of the components of the composition containing it. A prodrug is generally a drug precursor that is converted to an active species, or a more active species, via some process such as conversion by a metabolic pathway after administration to a subject and subsequent absorption. Some prodrugs have chemical groups present on the prodrug that reduce the activity of the prodrug and / or impart solubility or some other property to the drug. When the chemical group is cleaved and / or modified from the prodrug, the active drug is produced. Prodrugs are converted to active drugs in the body through enzymatic or non-enzymatic reactions. Prodrugs can provide improved physiochemical properties, such as better solubility, enhanced delivery characteristics, such as specifically targeting certain cells, tissues, organs, or ligands, and improved therapeutic value of the drug. Advantages of such prodrugs include, but are not limited to, (i) ease of administration compared to the parent drug, (ii) the prodrug is bioavailable by oral administration while the parent drug is not, and (iii) the prodrug may also have improved solubility in pharmaceutical compositions compared to the parent drug. Prodrugs include derivatives of active drugs that are pharmacologically inactive or have reduced activity. Prodrugs can be designed to modulate the amount of a drug or biologically active molecule that reaches the desired site of action through manipulation of the properties of the drug, such as physiochemical, biopharmaceutical, or pharmacokinetic properties. Examples of prodrugs include, but are not limited to, non-natural amino acid polypeptides (the "prodrug") that are administered as esters to facilitate transmembrane transport and are metabolically hydrolyzed to carboxylic acids that are active substances once they enter cells where water solubility is harmful to mobility but beneficial later.Prodrugs can be designed as reversible drug derivatives for use as modifiers to enhance drug transport to site-specific tissues.

[0170] As used herein, the term "prophylactically effective amount" refers to the amount of a composition containing at least one unnatural amino acid polypeptide or at least one modified unnatural amino acid polypeptide that is prophylactically applied to a patient and that alleviates to some extent one or more of the symptoms of the disease, condition, or disorder being treated. In such prophylactic applications, such amount may depend on the health status, weight, etc. of the patient. It is considered well within the scope of those of ordinary skill in the art to determine such prophylactically effective amount by routine experimentation, including but not limited to dose escalation clinical trials.

[0171] As used herein, the term "protected" refers to the presence of a "protecting group" or moiety that prevents the reaction of a chemically reactive functional group under certain reaction conditions. Protecting groups vary depending on the type of chemically reactive group being protected. By way of example only, (i) when the chemically reactive group is an amine or hydrazide, the protecting group can be selected from tert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc), (ii) when the chemically reactive group is a thiol, the protecting group can be orthopyridyldisulfide, and (iii) when the chemically reactive group is a carboxylic acid such as butanoic acid or propionic acid, or a hydroxyl group, the protecting group can be benzyl or an alkyl group such as methyl, ethyl, or tert-butyl.

[0172] By way of example only, the blocking / protecting group is selected from the following.

[0173]

Chemical formula

[0174] Furthermore, the protecting groups include, but are not limited to, photo-labile groups such as Nvoc and MeNvoc, as well as other protecting groups known in the art. Other protecting groups are described in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999 (incorporated herein by reference in its entirety).

[0175] The term "recombinant host cell", also referred to as "host cell", refers to a cell containing an exogenous polynucleotide, and the methods used to insert the exogenous polynucleotide into the cell include, but are not limited to, direct uptake, transduction, f-mating, or other methods known in the art for producing recombinant host cells. By way of example only, such exogenous polynucleotides can be non-integrating vectors including, but not limited to, plasmids, or can be integrated into the host genome.

[0176] As used herein, the term "redox active agent" refers to a molecule that oxidizes or reduces another molecule, thereby causing the redox active agent to be reduced or oxidized. Examples of redox active agents include, but are not limited to, ferrocene, quinone, Ru 2+ / 3+ complexes, Co 2+ / 3+ complexes, and Os 2+ / 3+ complexes.

[0177] As used herein, the term "reducing agent" refers to a compound or material capable of adding electrons to a compound to be reduced. By way of example, reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2-aminoethanethiol), and reduced glutathione. Such reducing agents can be used, by way of example only, to maintain sulfhydryl groups in a reduced state and reduce intra- or intermolecular disulfide bonds.

[0178] As used herein, "refolding" describes any process, reaction, or method that converts an improperly folded or unfolded state to a native or properly folded conformation. By way of example only, refolding converts a disulfide bond-containing polypeptide from an improperly folded or unfolded state to a native or properly folded conformation with respect to disulfide bonds. Such disulfide bond-containing polypeptides can be natural amino acid polypeptides or non-natural amino acid polypeptides.

[0179] As used herein, the terms "safety" or "safety profile" refer to side effects that may be associated with the administration of a drug relative to the number of times the drug is administered. By way of example, a drug that is administered multiple times and has little or no side effects is said to have an excellent safety profile. Methods for assessing the safety profile of any polypeptide are known in the art.

[0180] As used herein, the phrases "selectively hybridizes to" or "specifically hybridizes to" refer to the binding, duplex formation, or hybridization of a molecule to a particular nucleotide sequence under stringent hybridization conditions when its sequence is present in a complex mixture, including but not limited to whole cell or library DNA or RNA.

[0181] The term "stringent hybridization conditions" refers to the hybridization of the sequences of DNA, RNA, PNA, or other nucleic acid mimetics, or combinations thereof, under conditions of low ionic strength and high temperature. By way of example, under stringent conditions, a probe hybridizes to its target sequence in a complex mixture of nucleic acids (including but not limited to whole cell or library DNA or RNA), but does not hybridize to other sequences in the complex mixture. Stringent conditions are sequence-dependent and will be different under different circumstances. By way of example, longer sequences will hybridize specifically at higher temperatures. Stringent hybridization conditions include (i) being about 5-10 °C lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength and pH, (ii) the salt concentration being about 0.01 M to about 1.0 M at about pH 7.0 to about pH 8.3, and the temperature being at least about 30 °C for short probes (including but not limited to those containing about 10 to about 50 nucleotides) and at least about 60 °C for long probes (including but not limited to those over 50 nucleotides), (iii) the addition of destabilizing agents including but not limited to formamide, (iv) incubation at 42 °C in 50% formamide, 5× SSC, and 1% SDS, or incubation at 65 °C in 5× SSC, about 1% SDS, washing with 0.2× SSC, and incubation at about 0.1% SDS, about 5 minutes to about 120 minutes at 65 °C. By way of one example only, the detection of selective or specific hybridization includes, but is not limited to, a positive signal of at least 2-fold background. A general guide to nucleic acid hybridization can be found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993).

[0182] As used herein, the term "subject" refers to an animal that is the subject of treatment, observation, or experimentation. By way of example only, the subject can be, but is not limited to, a mammal including a human.

[0183] As used herein, the term "substantially purified" refers to a component of interest that does not substantially or essentially contain other components that are normally associated with or interact with the component of interest prior to purification. By way of example only, a preparation of a component of interest may be "substantially purified" if it contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (dry weight) of contaminating components. Thus, a "substantially purified" component of interest can have a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or more. By way of example only, a natural amino acid polypeptide or unnatural amino acid polypeptide can be purified from host cells in the case of natural cells, or recombinantly produced natural amino acid polypeptides or unnatural amino acid polypeptides. By way of example, a preparation of a natural amino acid polypeptide or unnatural amino acid polypeptide may be "substantially purified" if the preparation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (dry weight) of contaminating material. By way of example, if a natural amino acid polypeptide or unnatural amino acid polypeptide is recombinantly produced by a host cell, the natural amino acid polypeptide or unnatural amino acid polypeptide can be present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cells. By way of example, if a natural amino acid polypeptide or unnatural amino acid polypeptide is recombinantly produced by a host cell, the natural amino acid polypeptide or unnatural amino acid polypeptide can be present in the culture medium at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L or less of the dry weight of the cells.By way of example, a “substantially purified” natural amino acid polypeptide or unnatural amino acid polypeptide can have a purity level of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more as determined by suitable methods including, but not limited to, SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0184] The term “substituent,” also referred to as “non-interfering substituent,” refers to a group that can be used to replace another group on a molecule. Such groups include halo, C1-C 10 alkyl, C 2- C 10 alkenyl, C2-C 10 alkynyl, C1-C 10 alkoxy, C5-C 12 aralkyl, C3-C 12 cycloalkyl, C4-C 12 cycloalkenyl, phenyl, substituted phenyl, tolyl, xylyl, biphenyl, C2-C 12 alkoxyalkyl, C5-C 12 alkoxyaryl, C5-C 12 aryloxyalkyl, C7-C 12 oxyaryl, C1-C6 alkylsulfinyl, C1-C 10 alkylsulfonyl, -(CH2) m -O-(C1-C 10 alkyl) (wherein m is from 1 to 8), aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic radical, nitroalkyl, -NO2, -CN, -NRC(O)-(C1-C 10 alkyl), -C(O)-(C1-C 10 alkyl), C2-C 10 alkylthioalkyl, -C(O)O-(C1-C 10 alkyl), -OH, -SO2, =S, -COOH, -NR2, carbonyl, -C(O)-(C1-C 10 alkyl)-CF3, -C(O)-CF3, -C(O)NR2, -(C1-C10 aryl)-S-(C6-C 10 aryl), -C(O)-(C6-C 10 aryl), -(CH2) m -O-(CH2) m -O-(C1-C 10 alkyl) (each m is from 1 to 8), -C(O)NR2, -C(S)NR2, -SO2NR2, -NRC(O)NR2, -NRC(S)NR2, their salts, and the like, but are not limited thereto. Each R group in the aforementioned list includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl, or alkaryl. When the substituents are specified by their conventional chemical formulas written from left to right, they equally include chemically identical substituents that would result from writing the structure from right to left. For example, -CH2O- is equal to -OCH2-.

[0185] By way of example only, substituents of alkyl and heteroalkyl radicals (including those groups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) include, but are not limited to: -OR, =O, =NR, =N-OR, -NR2, -SR, -halogen, -SiR3, -OC(O)R, -C(O)R, -CO2R, -CONR2, -OC(O)NR2, -NRC(O)R, -NRC(O)NR2, -NR(O)2R, -NR-C(NR2)=NR, -S(O)R, -S(O)2R, -S(O)2NR2, -NRSO2R, -CN, and -NO2. Each R group in the aforementioned list includes, but is not limited to, hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (including, but not limited to, aryl substituted with 1 to 3 halogens), substituted or unsubstituted alkyl, alkoxy, or thioalkoxy group, or aralkyl group. When two R groups are attached to the same nitrogen atom, they can combine with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR2 is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl.

[0186] As an example, substituents of aryl and heteroaryl groups are numbers in the range of zero to the total number of open valences on the aromatic system, and are not limited to, but include -OR, =O, =NR, =N-OR, -NR2, -SR, -halogen, -SiR3, -OC(O)R, -C(O)R, -CO2R, -CONR2, -OC(O)NR2, -NRC(O)R, -NRC(O)NR2, -NR(O)2R, -NR-C(NR2)=NR, -S(O)R, -S(O)2R, -S(O)2NR2, -NRSO2R, -CN, -NO2, -R, -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy, and fluoro(C1-C4)alkyl, and each R group in the aforementioned list includes, but is not limited to, hydrogen, alkyl, heteroalkyl, aryl, and heteroaryl.

[0187] As used herein, the term "therapeutically effective amount" refers to the amount of a composition containing at least one unnatural amino acid polypeptide and / or at least one modified unnatural amino acid polypeptide that, when administered to a patient already suffering from a disease, condition, or disorder, is sufficient to cure, or at least partially prevent, or to some extent alleviate, one or more of the symptoms of the disease, disorder, or condition being treated. The effectiveness of such a composition depends on conditions including, but not limited to, the severity and course of the disease, disorder, or condition, previous therapies, the health status and response of the patient to the drug, and the judgment of the physician administering the treatment. By way of example only, a therapeutically effective amount can be determined by routine experimentation, including but not limited to dose escalation clinical trials.

[0188] As used herein, the term "thioalkoxy" refers to a sulfur-containing alkyl group linked to a molecule via an oxygen atom.

[0189] As used herein, the terms "toxic moiety" or "toxic group" refer to compounds that can cause harm, impairment, or death. Toxic moieties include auristatin, DNA minor groove binder, DNA minor groove alkylating agent, enediyne, lexitropsin, duocarmycin, taxane, puromycin, TLR agonist, maytansinoid, vinca alkaloid, AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, lysosomotropic agent, cyanomorpholino-doxorubicin, TLR-agonist-10, echinomycin, combretastatin, calicheamicin, maytansine, DM-1, netropsin, podophyllotoxin (e.g., etoposide, teniposide, etc.), baccatin and its derivatives, anti-tubulin agent, cryptophycin, combretastatin, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, epothilone A, epothilone B, nocodazole, colchicine, colcemid, estramustine, semadotin, discodermolide, maytansine, erythrobicin, mechlorethamine, cyclophosphamide, melphalan, carmustine, lomustine, semustine, streptozocin, chlorozotocin, uracil mustard, chloromethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramide, busulfan, dacarbazine, and temozolomide, cladribine, cytosine arabinoside, fluorouracil, floxuridine, 6-thioguanine, 6-mercaptopurine, pentostatin, 5-fluorouracil, methotrexate, 10-propargyl-5,8-dideazafolate, 5,8-dideazatetrahydrofolate, leucovorin, fludarabine phosphate, pentostatin, gemcitabine, Ara-C, paclitaxel, docetaxel, deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine, brequinar, antibiotics (e.g., anthracycline, gentamicin, cephalothin, vancomycin, telavancin, daptomycin, azithromycin, erythromycin, roxithromycin, furazolidone, amoxicillin,ampicillin, carbenicillin, furcloxacillin, methicillin, penicillin, ciprofloxacin, moxifloxacin, ofloxacin, doxycycline, minocycline, oxytetracycline, tetracycline, streptomycin, rifabutin, ethambutol, rifaximin, etc.), antiviral agents (e.g., abacavir, acyclovir, ampligen, cidofovir, delavirdine, didanosine, efavirenz, entecavir, phosphonet, ganciclovir, ibacitabine, immunovir, idoxuridine, inosine, lopinavir, methisazone, nevirapine, oseltamivir, penciclovir, stubidine, trifluridine, torbada, valacyclovir, zanamivir, etc.), daunorubicin hydrochloride, daunomycin, rubidomycin, celbidine, idarubicin, doxorubicin, epirubicin, and morpholino derivatives, phenoxazone biscyclopeptide (e.g., dactinomycin), basic sugar peptide (e.g., bleomycin), anthraquinone glycoside (e.g., plicamycin, mitomycin), anthracenedione (e.g., mitoxantrone), aziridinopyrroloindole region (e.g., mitomycin), macrocyclic immunosuppressants (e.g., cyclosporine, FK-506, tacrolimus, prograf, rapamycin, etc.), navelbine, CPT-11, anastrozole, letrozole, capecitabine, raloxifene, cyclophosphamide, ifosfamide, droloxifene, allocolchicine, halicondrin B, colchicine, colchicine derivatives, maytansine, lysocine, paclitaxel, paclitaxel derivatives, docetaxel, thiocolchicine, tritylcysteine, vincblastine sulfate, vincristine sulfate, cisplatin, carboplatin, hydroxyurea, N-methylhydrazine, epidophyllotoxin, procarbazine, mitoxantrone, leucovorin, and tegafur are included, but not limited to these. "Taxane" includes paclitaxel and any active taxane derivative or prodrug.,

[0190] As used herein, the terms "treat", "treating", or "treatment" include reducing, decreasing, or ameliorating the symptoms of a disease or condition, preventing additional symptoms, ameliorating or preventing the underlying metabolic cause of the symptoms, inhibiting the disease or condition, e.g., preventing the onset of the disease or condition, alleviating the disease or condition, causing regression of the disease or condition, alleviating the condition caused by the disease or condition, or arresting the symptoms of the disease or condition. The terms "treat", "treating", or "treatment" include, but are not limited to, prophylactic and / or therapeutic measures.

[0191] As used herein, the term "water-soluble polymer" refers to any polymer that is soluble in an aqueous solvent. Such water-soluble polymers include polyethylene glycol, polyethylene glycol propionaldehyde, mono C1-C 10Alkoxy or its aryloxy derivatives (described in U.S. Patent No. 5,252,714 incorporated herein by reference), monomethoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives (including but not limited to methylcellulose and carboxymethylcellulose), serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycols and their derivatives, copolymers of polyalkylene glycols and their derivatives, polyvinyl ethyl ether, and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide], etc., or mixtures thereof, but not limited thereto. By way of example only, the conjugation of such water-soluble polymers to natural amino acid polypeptides or unnatural polypeptides can result in changes including, but not limited to, increased water solubility, increased or modulated serum half-life, increased or modulated therapeutic half-life, increased bioavailability, modulation of biological activity, extended circulation time, modulation of immunogenicity, changes in aggregation and multimer formation, receptor binding, effector regulation, or other targeted polypeptide binding, changes in binding to one or more binding partners, and changes in dimerization or multimerization of targeted polypeptide receptors, including but not limited to modulation of physical association characteristics. In addition, such water-soluble polymers may or may not have their own biological activity and may be utilized as a linker for conjugating a targeted polypeptide, including but not limited to one or more targeted polypeptides or one or more biologically active molecules, to other substances.

[0192] Unless otherwise indicated, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, within the skill of the art, are used.

[0193] The compounds presented in this specification (including, but not limited to, unnatural amino acids, unnatural amino acid polypeptides, modified unnatural amino acid polypeptides, and reagents for producing the aforementioned compounds) are the same as those listed in the various formulas and structures presented herein, except that one or more atoms are replaced with atoms having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. This fact relates to the inclusion of isotope-labeled compounds. Examples of isotopes that can be incorporated into these compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, such as 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 35 S, 18 F, 36 Cl. Certain isotope-labeled compounds described herein, for example, compounds incorporating radioactive isotopes such as 3 H and 14 C, are useful in drug and / or substrate tissue distribution assays. Further, substitution with isotopes such as deuterium, i.e., 2 H, can result in certain therapeutic advantages due to greater metabolic stability, e.g., an increase in the in vivo half-life or a reduction in the required dosage.

[0194] Some of the compounds of this specification (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, as well as reagents for producing the aforementioned compounds) have asymmetric carbon atoms and can therefore exist as enantiomers or diastereomers. A mixture of diastereomers can be separated into their individual diastereomers based on their physicochemical differences by methods known, for example, by chromatography and / or fractional crystallization. Enantiomers can be separated by converting a mixture of enantiomers into a mixture of diastereomers by reaction with a suitable optically active compound (e.g., an alcohol), separating the diastereomers, and converting the individual diastereomers into the corresponding pure enantiomers (e.g., by hydrolysis). All such isomers, including diastereomers, enantiomers, and mixtures thereof, are considered part of the compositions described herein.

[0195] In additional or further embodiments, the compounds described herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, as well as reagents for producing the aforementioned compounds) are used in the form of prodrugs. In additional or further embodiments, the compounds described herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, as well as reagents for producing the aforementioned compounds) are metabolized upon administration to an organism that needs to produce metabolites and are then used to produce the desired effects, including the desired therapeutic effect. In further or additional embodiments, they are active metabolites of non-natural amino acids and "modified or unmodified" non-natural amino acid polypeptides.

[0196] The methods and formulations described herein include the use of N-oxides, crystalline forms (also known as polymorphs), or pharmaceutically acceptable salts of unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides. In certain embodiments, the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides may exist as tautomers. All tautomers are included within the scope of the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides presented herein. Additionally, the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water and ethanol. The solvated forms of the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides presented herein are also considered to be disclosed herein.

[0197] Some of the compounds herein (including, but not limited to, unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides, as well as reagents for producing the aforementioned compounds) may exist in several tautomeric forms. All such tautomeric forms are considered to be part of the compositions described herein. Also, for example, all enol-keto forms of any compound herein (including, but not limited to, unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides, as well as reagents for producing the aforementioned compounds) are considered to be part of the compositions described herein.

[0198] Some of the compounds of the present specification (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, as well as reagents for producing any of the foregoing compounds) are acidic and can form salts with pharmaceutically acceptable cations. Some of the compounds of the present invention (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, as well as reagents for producing the foregoing compounds) can be basic and, therefore, can form salts with pharmaceutically acceptable anions. All such salts containing dibasic salts are within the scope of the compositions described herein and can be prepared by conventional methods. For example, the salts can be prepared by contacting acidic and basic substances in any of aqueous, non-aqueous, or partially aqueous media. The salts are recovered by using at least one of the following techniques: filtration, precipitation with a non-solvent followed by filtration, evaporation of the solvent, or freeze-drying in the case of an aqueous solution.

[0199] Pharmaceutically acceptable salts of the non-natural amino acid polypeptides disclosed herein can be formed when the acidic protons present in the parent non-natural amino acid polypeptide are replaced by metal ions such as, by way of example, alkali metal ions, alkaline earth metal ions, or aluminum ions, or when coordinated with an organic base. In addition, the salt forms of the disclosed non-natural amino acid polypeptides can be prepared using salts of the starting materials or intermediates. The non-natural amino acid polypeptides described herein can be prepared as pharmaceutically acceptable acid addition salts (one type of pharmaceutically acceptable salt) by reacting the free base form of the non-natural amino acid polypeptides described herein with a pharmaceutically acceptable inorganic or organic acid. Alternatively, the non-natural amino acid polypeptides described herein can be prepared as pharmaceutically acceptable base addition salts (one type of pharmaceutically acceptable salt) by reacting the free acid form of the non-natural amino acid polypeptides described herein with a pharmaceutically acceptable inorganic or organic base.

[0200] Pharmaceutically acceptable salts include, but are not limited to, the following: (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., or organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, etc., or (2) salts formed when the acidic proton present in the parent compound is replaced by a metal ion such as an alkali metal ion, alkaline earth ion, or aluminum ion, or coordinates with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, etc.

[0201] The corresponding counterions of pharmaceutically acceptable salts of non-natural amino acid polypeptides can be analyzed and identified using a variety of methods including, but not limited to, ion exchange chromatography, ion chromatography, capillary electrophoresis, inductively coupled plasma, atomic absorption spectroscopy, mass spectrometry, or any combination thereof. In addition, the therapeutic activity of such pharmaceutically acceptable salts of non-natural amino acid polypeptides can be tested using the techniques and methods described in the examples.

[0202] References to salts are to be understood to include solvent addition forms or crystal forms thereof, particularly solvates or polymorphs. Solvates contain either a stoichiometric or non-stoichiometric amount of solvent and are often formed during the crystallization process with pharmaceutically acceptable solvents such as water, ethanol, etc. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Polymorphs include different crystal packing arrangements of the same elemental composition of a compound. Polymorphs typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optical and electrical properties, stability, and solubility. Various factors such as recrystallization solvent, crystallization rate, and storage temperature can govern the single crystal form.

[0203] Screening and characterization of polymorphs and / or solvates of pharmaceutically acceptable salts of non-natural amino acid polypeptides can be achieved using a variety of techniques including, but not limited to, thermal analysis, X-ray diffraction, spectroscopy, vapor sorption, and microscopy. Thermal analysis methods address thermochemical decomposition or thermophysical processes including, but not limited to, polymorph transitions and such methods are used to analyze relationships between polymorphs, determine weight loss, find glass transition temperatures, or for excipient compatibility studies. Such methods include, but are not limited to, differential scanning calorimetry (DSC), modulated differential scanning calorimetry (MDCS), thermogravimetric analysis (TGA), and thermogravimetry and infrared analysis (TG / IR). X-ray diffraction methods include, but are not limited to, single crystal and powder diffractometers and synchrotron sources. The various spectroscopic techniques used include, but are not limited to, Raman, FTIR, UVIS, and NMR (liquid and solid state). The various microscopy techniques include, but are not limited to, polarized microscopy, scanning electron microscopy (SEM) using energy dispersive X-ray analysis (EDX), environmental scanning electron microscopy (in gas or water vapor atmosphere) using EDX, IR microscopy, and Raman microscopy.

[0204] Preferred embodiments of the present invention have been shown and described herein, and it will be apparent to those skilled in the art that such embodiments are provided by way of example. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in practicing the present invention. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims, and their equivalents, be covered thereby.

[0205] TLR-agonist linker derivative Tools (methods, compositions, techniques) are described herein for making and using a targeted polypeptide of a TC or analog thereof that includes at least one unnatural amino acid or modified unnatural amino acid having a carbonyl, dicarbonyl, oxime, or hydroxylamine group at a certain level. Such targeted polypeptides of a TC that include an unnatural amino acid may contain additional functional groups including, but not limited to, a polymer, a water-soluble polymer, a derivative of polyethylene glycol, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, and any combination thereof. It should be noted that the various foregoing functional groups are not meant to imply that a member of one functional group cannot be classified as a member of another functional group. In fact, there is overlap depending on the particular situation. As just one example, a water-soluble polymer overlaps in scope with a derivative of polyethylene glycol, but the overlap is not complete, and thus both functional groups are cited above.

[0206] In one aspect, a method of selecting and designing TLR-agonist linker derivatives and targeted polypeptides modified using the methods, compositions, and techniques described herein. Novel TLR-agonist linker derivatives and targeted polypeptides, including but not limited to, can be designed as part of a high-throughput screening process (wherein a large number of polypeptides can be designed, synthesized, characterized, and / or tested), or newly designed based on the interests of the researcher. Novel TLR agonist linker derivatives and targeted polypeptides can also be designed based on the structure of known or partially characterized polypeptides. As but one example, TLR-agonists have been the subject of intensive research by the scientific community, and novel compounds can be designed based on the structure of TLR-agonists. The principles for selecting which amino acid(s) to substitute and / or modify are described separately herein. The choice of which modifications to use is also described herein and can be used to meet the needs of the experimenter or end-user. Such needs include, but are not limited to, manipulating the therapeutic efficacy of the polypeptide, improving the safety profile of the polypeptide, modulating the pharmacokinetics, pharmacology, and / or pharmacodynamics of the polypeptide, e.g., as but one example, increasing water solubility, increasing bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending circulation time. In addition, such modifications include, but are not limited to, providing additional functional groups to the polypeptide, incorporating antibodies, and any combination of the foregoing modifications.

[0207] TLR-agonist linker derivatives and targeted polypeptides that can be modified to have or contain an oxime, carbonyl, dicarbonyl, or hydroxylamine group are also described herein. This aspect includes methods for producing, purifying, characterizing, and using such TLR-agonist linker derivatives and targeted polypeptides.

[0208] The TLR-agonist linker derivative or the targeted polypeptide may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or 10 or more of a carbonyl or dicarbonyl group, an oxime group, a hydroxylamine group, or a protected form thereof. The TLR-agonist linker derivatives or the targeted polypeptides may be the same or different. For example, in derivatives containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different reactive groups, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different sites may be present.

[0209] As described herein, the present disclosure provides a targeted polypeptide conjugated to another molecule having the formula "targeted polypeptide-L-M", where L is a linking group or a chemical bond, and M is any other molecule including, but not limited to, another targeted polypeptide. In some embodiments, L is stable in vivo. In some embodiments, L is hydrolysable in vivo. In some embodiments, L is metastable in vivo.

[0210] The targeted polypeptide and M can be linked together by L using standard linkers and procedures known to those skilled in the art. In some aspects, the targeted polypeptide and M are directly condensed and L is a bond. In other aspects, the targeted polypeptide and M are condensed via a linking group L. For example, in some embodiments, the targeted polypeptide and M are linked together via a peptide bond, optionally through a peptide or amino acid spacer. In some embodiments, the targeted polypeptide and M are linked together via chemical conjugation, optionally through a linking group (L). In some embodiments, L is directly conjugated to each of the targeted polypeptide and M.

[0211] Chemical conjugation can occur by reacting a nucleophilic reactive group of one compound with an electrophilic reactive group of another compound. In some embodiments, when L is a bond, the targeting polypeptide conjugates to M by either reacting a nucleophilic reactive moiety on the targeting polypeptide with an electrophilic reactive moiety on Y or reacting an electrophilic reactive moiety on the targeting polypeptide with a nucleophilic reactive moiety on M. In embodiments where L is a group that links the targeting polypeptide and M together, the targeting polypeptide and / or M can conjugate to L by either reacting a nucleophilic reactive moiety on the targeting polypeptide and / or M with an electrophilic reactive moiety on L or reacting an electrophilic reactive moiety on the targeting polypeptide and / or M with a nucleophilic reactive moiety on L. Non-limiting examples of nucleophilic reactive groups include amino, thiol, and hydroxyl. Non-limiting examples of electrophilic reactive groups include carboxyl, acyl chloride, anhydride, ester, succinimide ester, alkyl halide, sulfonate ester, maleimide, haloacetyl, and isocyanate. In embodiments where the targeting polypeptide and M are conjugated together by reacting a carboxylic acid and an amine, an activator can be used to form an activated ester of the carboxylic acid.

[0212] Activating esters of carboxylic acids can be, for example, N-hydroxysuccinimide (NHS), tosylate (Tos), mesylate, triflate, carbodiimide, or hexafluorophosphate. In some embodiments, the carbodiimide is 1,3-dicyclohexylcarbodiimide (DCC), 1,1'-carbonyldiimidazole (CDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), or 1,3-diisopropylcarbodiimide (DICD). In some embodiments, the hexafluorophosphate is selected from the group consisting of hexafluorophosphate benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), and o-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluorophosphate (HBTU).

[0213] In some embodiments, the targeting polypeptide comprises a nucleophilic reactive group (e.g., an amino group, a thiol group, or a hydroxyl group of the side chain of lysine, cysteine, or serine) capable of conjugating to an electrophilic reactive group on M or L. In some embodiments, the targeting polypeptide comprises an electrophilic reactive group (e.g., a carboxylate group of the side chain of Asp or Glu) capable of conjugating to a nucleophilic reactive group on M or L. In some embodiments, the targeting polypeptide is chemically modified to include a reactive group capable of conjugating directly to M or L. In some embodiments, the targeting polypeptide is modified at the N-terminus or C-terminus to include a natural or unnatural amino acid having a nucleophilic side chain. In an exemplary embodiment, the N-terminal or C-terminal amino acid of the targeting polypeptide is selected from the group consisting of lysine, ornithine, serine, cysteine, and homocysteine. For example, the N-terminal or C-terminal amino acid of the targeting polypeptide can be modified to include a lysine residue. In some embodiments, the targeting polypeptide is modified at the N-terminal or C-terminal amino acid to include a natural or unnatural amino acid having an electrophilic side chain, such as Asp and Glu. In some embodiments, the internal amino acids of the targeting polypeptide are substituted with a natural or unnatural amino acid having a nucleophilic side chain as described previously herein. In an exemplary embodiment, the internal amino acid of the targeting polypeptide to be substituted is selected from the group consisting of lysine, ornithine, serine, cysteine, and homocysteine. For example, the internal amino acid of the targeting polypeptide can be substituted with a lysine residue. In some embodiments, the internal amino acids of the targeting polypeptide are substituted with a natural or unnatural amino acid having an electrophilic side chain, such as Asp and Glu.

[0214] In some embodiments, M comprises a targeting polypeptide or a reactive group capable of directly conjugating to L. In some embodiments, M comprises a nucleophilic reactive group (e.g., amine, thiol, hydroxyl) capable of conjugating to an electrophilic reactive group on the targeting polypeptide or L. In some embodiments, M comprises an electrophilic reactive group (e.g., carboxyl group, activated form of the carboxyl group, a compound having a leaving group) capable of conjugating to a nucleophilic reactive group on the targeting polypeptide or L. In some embodiments, M is chemically modified to comprise any of the nucleophilic reactive groups capable of conjugating to an electrophilic reactive group on the targeting polypeptide or L. In some embodiments, M is chemically modified to comprise an electrophilic reactive group capable of conjugating to a nucleophilic reactive group on the targeting polypeptide or L.

[0215] In some embodiments, conjugation can be effected through treatment with an organosilane, such as an aminosilane treated with glutaraldehyde, carbonyl diimidazole (CDI) activation of a silanol group, or utilization of a dendrimer. A variety of dendrimers are known in the art, including poly(amidoamine) (PAMAM) dendrimers synthesized by a branching method starting from an ammonia or ethylenediamine initiator core reagent, a subclass of PAMAM dendrimers based on a tris-aminoethylene-imine core, radial layer poly(amidoamine-organic silicon) dendrimers (PAMAMOS) which are reverse unimolecular micelles consisting of a hydrophilic, nucleophilic polyamidoamine (PAMAM) interior and a hydrophobic organosilicon (OS) exterior, poly(propylene imine) (PPI) dendrimers which are generally poly-alkylamines having a primary amine as a terminal group but with a dendrimer interior consisting of a large number of tertiary tris-propyleneamines, poly(propyleneamine) (POPAM) dendrimers, diaminobutane (DAB) dendrimers, amphiphilic dendrimers, micelle dendrimers which are unimolecular micelles of water-soluble hyperbranched polyphenylene, polylysine dendrimers, and dendrimers based on a poly-benzyl ether hyperbranched backbone.

[0216] In some embodiments, conjugation can be effected through olefin metathesis. In some embodiments, both M and the targeting polypeptide, M and L, or the targeting polypeptide and L include alkene or alkyne moieties capable of undergoing metathesis. In some embodiments, a suitable catalyst (e.g., copper, ruthenium) is used to accelerate the metathesis reaction. Suitable methods for performing olefin metathesis reactions are described in the art. See, for example, Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000), Walensky et al., Science 305:1466-1470 (2004), and Blackwell et al., Angew, Chem., Int. Ed. 37:3281-3284 (1998).

[0217] In some embodiments, the conjugation can be carried out using click chemistry. "Click reactions" are broad in scope, easy to perform, use only readily available reagents, and are insensitive to oxygen and water. In some embodiments, the click reaction is a cycloaddition reaction between an alkynyl group and an azide group to form a triazolyl group. In some embodiments, the click reaction uses a copper or ruthenium catalyst. Suitable methods for performing click reactions are described in the art. See, for example, Kolb et al., Drug Discovery Today 8:1128 (2003), Kolb et al., Angew. Chem. Int. Ed. 40:2004 (2001), Rostovtsev et al., Angew. Chem. Int. Ed. 41:2596 (2002), Tornoe et al., J. Org. Chem. 67:3057 (2002), Manetsch et al., J. Am. Chem. Soc. 126:12809 (2004), Lewis et al., Angew. Chem. Int. Ed. 41:1053 (2002), Speers, J. Am. Chem. Soc. 125:4686 (2003), Chan et al. Org. Lett. 6:2853 (2004), Zhang et al., J. Am. Chem. Soc. 127:15998 (2005), and Waser et al., J. Am. Chem. Soc. 127:8294 (2005).

[0218] Indirect conjugation via high affinity specific binding partners such as streptavidin / biotin or avidin / biotin or lectin / carbohydrate is also contemplated.

[0219] In some embodiments, the targeting polypeptide and / or M is functionalized to include a nucleophilic or electrophilic reactive group having an organic derivatizing agent. This derivatizing agent is capable of reacting with a selected side chain or N-terminal or C-terminal residue of a targeted amino acid on the targeting polypeptide, and a functional group on M. Reactive groups on the targeting polypeptide and / or M include, for example, aldehyde, amino, ester, thiol, a-haloacetyl, maleimide, or hydrazino groups. Examples of derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residue), N-hydroxysuccinimide (through lysine residue), glutaraldehyde, succinic anhydride, or other agents known in the art. Alternatively, the targeting polypeptide and / or M can be indirectly linked to each other through an intermediate carrier such as a polysaccharide or polypeptide carrier. Examples of polysaccharide carriers include amino dextran. Examples of suitable polypeptide carriers for imparting desirable solubility to the resulting loaded carrier include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and mixed polymers of these amino acids and other amino acids such as serine.

[0220] Cysteinyl residues are most commonly reacted with an a-haloacetate (and corresponding amine) such as chloroacetic acid or chloroacetamide to obtain carboxymethyl or carboxamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, alpha-bromo-beta-(5-imidazolyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyldisulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenz-2-oxa-1,3-diazole.

[0221] The histidyl residue is derivatized by reaction with diethyl pyrocarbonate at pH 5.5 - 7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful, and the reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0.

[0222] The lysinyl and amino-terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residue. Other suitable reagents for derivatizing alpha-amino-containing residues include methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and imido esters such as the transaminase-catalyzed reaction with glyoxylate.

[0223] The arginyl residue is modified by reaction with one or several conventional reagents, among others phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of the arginine residue requires carrying out the reaction under alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents can react with the lysine group as well as the arginine epsilon-amino group.

[0224] Specific modification of the tyrosyl residue can be of interest in particular for introducing a spectral label into the tyrosyl residue by reaction with an aromatic diazonium compound or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively.

[0225] Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with a carbodiimide (R-N=C=N-R’), where R and R’ are different alkyl groups such as 1-cyclohexyl-3-(2-morpholin-4-yl-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Further, reaction with ammonium ions converts aspartyl and glutamyl residues to asparaginyl and glutaminyl residues.

[0226] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of a seryl or threonyl residue, methylation of the alpha-amino group of the side chains of lysine, arginine, and histidine (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), deamidation of asparagine or glutamine, acetylation of the N-terminal amine, and / or amidation or esterification of the C-terminal carboxylic acid group.

[0227] Another type of covalent modification involves chemically or enzymatically attaching a glycoside to a peptide. The sugar(s) can be attached to (a) arginine and histidine, (b) a free carboxyl group, (c) a free sulfhydryl group, e.g., that of cysteine, (d) a free hydroxyl group, e.g., that of serine, threonine, or hydroxyproline, (e) an aromatic residue, e.g., that of tyrosine or tryptophan, or (f) the amide group of glutamine. These methods are described in WO1987 / 05330, and Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0228] In some embodiments, L is a linkage. In these embodiments, the targeting polypeptide and M are conjugated together by reacting a nucleophilic reactive moiety on the targeting polypeptide with an electrophilic reactive moiety on M. In alternative embodiments, the targeting polypeptide and M are conjugated together by reacting an electrophilic reactive moiety on the targeting polypeptide with a nucleophilic moiety on M. In an exemplary embodiment, L is an amide bond formed upon reaction of an amine on the targeting polypeptide (e.g., the ε-amine of a lysine residue) with a carboxyl group on M. In alternative embodiments, the targeting polypeptide and / or M are derivatized with a derivatizing agent prior to conjugation.

[0229] In some embodiments, L is a linking group. In some embodiments, L is a bifunctional linker and contains only two reactive groups prior to conjugation to the targeting polypeptide and M. In embodiments where both the targeting polypeptide and M have electrophilic reactive groups, L contains two of the same nucleophilic groups or two different nucleophilic groups (e.g., amine, hydroxyl, thiol) prior to conjugation to the targeting polypeptide and M. In embodiments where both the targeting polypeptide and M have nucleophilic reactive groups, L contains two of the same electrophilic groups or two different electrophilic groups (e.g., carboxyl group, activated form of a carboxyl group, a compound having a leaving group) prior to conjugation to the targeting polypeptide and M. In embodiments where one of the targeting polypeptide or M has a nucleophilic reactive group and the other of the targeting polypeptide or M has an electrophilic reactive group, L contains one nucleophilic reactive group and one electrophilic group prior to conjugation to the targeting polypeptide and M.

[0230] L can be any molecule having at least two reactive groups capable of reacting with each of the targeting polypeptide and M (prior to conjugation to the targeting polypeptide and M). In some embodiments, L has only two reactive groups and is bifunctional. L (prior to conjugation to the peptide) has the formula VI:

[0231]

Chem.

[0232] It can be represented by the formula: in the formula, A and B are independently a nucleophilic or electrophilic reactive group. In some embodiments, both A and B are either both nucleophilic groups or both electrophilic groups. In some embodiments, one of A or B is a nucleophilic group and the other of A or B is an electrophilic group. Non-limiting combinations of A and B are shown in Table 1 below.

[0233]

Table 1

[0234] In some embodiments, A and B may include alkene and / or alkyne functional groups suitable for olefin metathesis reactions. In some embodiments, A and B include moieties suitable for click chemistry (e.g., alkene, alkyne, nitrile, azide). Other non-limiting examples of reactive groups (A and B) include pyridyldithiol, aryl azide, diazirine, carbodiimide, and hydrazide.

[0235] In some embodiments, L is hydrophobic. Hydrophobic linkers are known in the art. See, for example, Bioconjugate Techniques, G.T. Hermanson (Academic Press, San Diego, CA, 1996), which is incorporated herein by reference in its entirety. Suitable hydrophobic linking groups known in the art include, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoic acid. Prior to conjugation of the composition to the peptide, the hydrophobic linking group contains at least two reactive groups (A and B) as described herein and shown below:

[0236]

Chemical formula

[0237] In some embodiments, the hydrophobic linking group contains, as reactive groups, either a maleimide or an iodoacetyl group and either a carboxylic acid or an activated carboxylic acid (e.g., an NHS ester). In these embodiments, the maleimide or iodoacetyl group can bind to a thiol moiety on the targeting polypeptide or M, and the carboxylic acid or activated carboxylic acid can bind to an amine on the targeting polypeptide or M, with or without the use of a coupling reagent. Any coupling agent known to those skilled in the art can be used to couple the carboxylic acid to a free amine, such as DCC, DIC, HATU, HBTU, TBTU, and other activators described herein. In certain embodiments, the hydrophilic linking group contains an aliphatic chain of 2 to 100 methylene groups, and A and B are carboxyl groups or derivatives thereof (e.g., succinic acid). In other specific embodiments, L is iodoacetic acid.

[0238]

Chemical formula

[0239] In some embodiments, the linking group is hydrophilic, such as a polyalkylene glycol. Before conjugation of the composition to the peptide, the hydrophilic linking group contains at least two reactive groups (A and B) as described herein and shown below:

[0240]

Chemical formula

[0241] In certain embodiments, the linking group is polyethylene glycol (PEG). The PEG in certain embodiments has a molecular weight of from about 100 Daltons to about 10,000 Daltons, such as from about 500 Daltons to about 5000 Daltons. The PEG in some embodiments has a molecular weight of from about 10,000 Daltons to about 40,000 Daltons.

[0242] In some embodiments, the hydrophilic linking group contains, as reactive groups, either a maleimide or an iodoacetyl group, and either a carboxylic acid or an activated carboxylic acid (e.g., an NHS ester). In these embodiments, the maleimide or iodoacetyl group can bind to a thiol moiety on the targeting polypeptide or M, and the carboxylic acid or activated carboxylic acid can bind to an amine on the targeting polypeptide or M, with or without the use of a coupling reagent. Any suitable coupling agent known to those skilled in the art can be used to couple the carboxylic acid to an amine, such as DCC, DIC, HATU, HBTU, TBTU, and other activators described herein. In some embodiments, the linking group is maleimide-polymer (20 - 40 kDa)-COOH, iodoacetyl-polymer (20 - 40 kDa)-COOH, maleimide-polymer (20 - 40 kDa)-NHS, or iodoacetyl-polymer (20 - 40 kDa)-NHS.

[0243] In some embodiments, the linking group consists of an amino acid, a dipeptide, a tripeptide, or a polypeptide, and the amino acid, dipeptide, tripeptide, or polypeptide contains at least two activating groups described herein. In some embodiments, the linking group (L) contains a moiety selected from the group consisting of amino, ether, thioether, maleimide, disulfide, amide, ester, thioester, alkene, cycloalkene, alkyne, triazolyl, carbamate, carbonate, cathepsin B cleavable, and hydrazone.

[0244] In some embodiments, L contains a chain of atoms that is 1 to about 60, or 1 to 30 or more, 2 to 5, 2 to 10, 5 to 10, or 10 to 20 atoms in length. In some embodiments, all of the chain atoms are carbon atoms. In some embodiments, the chain atoms in the backbone of the linker are selected from the group consisting of C, O, N, and S. The chain atoms and the linker can be selected based on their expected solubility (hydrophilicity) to provide a more soluble conjugate. In some embodiments, L provides a functional group that is a target for cleavage by an enzyme or other catalyst found in the target tissue or organ or cell or by hydrolysis conditions. In some embodiments, the length of L is long enough to reduce the likelihood of steric hindrance.

[0245] In some embodiments, L is stable in a biological fluid such as blood or a blood fraction. In some embodiments, L is stable in serum for at least 5 minutes, e.g., less than 25%, 20%, 15%, 10%, or 5% of the conjugate is cleaved when incubated in serum for 5 minutes. In other embodiments, L is stable in serum for at least 10, or 20, or 25, or 30, or 60, or 90, or 120 minutes, or 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, or 24 hours. In these embodiments, L does not contain functional groups that are susceptible to hydrolysis in vivo. In some exemplary embodiments, L is stable in serum for at least about 72 hours. Non-limiting examples of functional groups that cannot undergo significant hydrolysis in vivo include amides, ethers, and thioethers. For example, the following compounds do not undergo significant hydrolysis in vivo:

[0246] [Chemical formula]

[0247] In some embodiments, L is hydrolysable in vivo. In these embodiments, L contains functional groups that are susceptible to hydrolysis in vivo. Non-limiting examples of functional groups that are susceptible to hydrolysis in vivo include esters, anhydrides, and thioesters. For example, the following compounds are susceptible to hydrolysis in vivo because they contain ester groups.

[0248] [Chemical formula]

[0249] In some exemplary embodiments, L is unstable and undergoes substantial hydrolysis within 3 hours in plasma at 37°C, and hydrolysis is complete within 6 hours. In some exemplary embodiments, L is not unstable.

[0250] In some embodiments, L is metastable in vivo. In these embodiments, L optionally includes a functional group (e.g., an acid-labile, reduction-labile, or enzyme-labile functional group) that can be chemically or enzymatically cleaved in vivo over a period of time. In these embodiments, L can include, for example, a hydrazone moiety, a disulfide moiety, or a cathepsin-cleavable moiety. While not intending to be bound by any particular theory, the targeted polypeptide-L-M conjugate is stable in the extracellular environment, e.g., in serum over the period described above, but is unstable in the intracellular environment or under conditions mimicking the intracellular environment, and as a result, is cleaved when it enters the cell. In some embodiments, when L is metastable, L is stable in serum for at least about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, or 48 hours, e.g., at least about 48, 54, 60, 66, or 72 hours, or about 24-48, 48-72, 24-60, 36-48, 36-72, or 48-72 hours.

[0251] In another embodiment, the polymer derivative of the present invention has the structure: X-CH2CH2O--(CH2CH2O) n --CH2CH2-O-(CH2) m -W-N=N=N and includes a polymer backbone having the formula, where W is an aliphatic or aromatic linker moiety containing 1 to 10 carbon atoms, n is from 1 to about 4000, X is the functional group described above, and m is from 1 to 10.

[0252] The azide-containing polymer derivatives of the present invention can be prepared by various methods known in the art and / or disclosed herein. In one method shown below, a water-soluble polymer backbone having an average molecular weight of about 800 Da to about 100,000 Da, having a first end bonded to a first functional group and a second end bonded to a suitable leaving group, is reacted with an azide anion (which may be paired with any of several suitable counterions including sodium, potassium, tert-butylammonium, etc.). The leaving group undergoes nucleophilic substitution and is replaced by the azide moiety to obtain the desired azide-containing polymer: X-polymer-LY + N3 - → X-polymer-L N3 As exemplified, a polymer backbone suitable for use in the present invention has the formula X-polymer-LY, wherein the polymer is poly(ethylene glycol), X is a functional group that does not react with the azide group, and Y is a suitable leaving group. Examples of suitable functional groups include, but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl, amine, aminooxy, protected amine, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine, and vinylpyridine, and ketone. Examples of suitable leaving groups include, but are not limited to, chloride, bromide, iodide, mesylate, tresylate, and tosylate.

[0253] In another method for the preparation of the azide-containing polymer derivatives of the present invention, a linker having an azide functional group is contacted with a water-soluble polymer backbone having an average molecular weight of about 800 Da to about 100,000 Da, and the linker has a chemical functional group that selectively reacts with a chemical functional group on the polymer to form an azide-containing polymer derivative product, and the azide is separated from the polymer backbone by a linking group.

[0254] An exemplary reaction scheme is shown below: X-polymer-Y + N-linker-N=N=N → PG-X-polymer-linker-N=N=N, wherein The polymer is poly(ethylene glycol), X is a capping group such as alkoxy or the functional groups described above, and Y is a functional group that is not reactive with an azide functional group but reacts efficiently and selectively with an N-functional group.

[0255] Examples of suitable functional groups include, but are not limited to, when N is an amine, Y is a carboxylic acid, carbonate, or active ester; when N is a hydrazide or aminooxy moiety, Y is a ketone; and when N is a nucleophile, Y is a leaving group. Purification of the crude product can be achieved by known methods including, but not limited to, precipitation of the product followed by chromatography, if necessary.

[0256] More specific examples are shown below. In the case of a polymer diamine, one of the amines is protected by a protecting group moiety such as tert-butyl-Boc, and the resulting mono-protected polymer diamine is reacted with a linking moiety having an azide functional group. BocHN-polymer-NH2 + HO2C-(CH2)3-N=N=N In this case, the amine group can be coupled to the carboxylic acid group using thionyl chloride or a carbodiimide reagent, as well as various activators such as N-hydroxysuccinimide or N-hydroxybenzotriazole to create an amide bond between the monoamine polymer derivative and the azide-bearing linker moiety. After successful formation of the amide bond, the resulting N-tert-butyl-Boc-protected azide-containing derivative can be used directly to modify a bioactive molecule or it can be further elaborated to install other useful functional groups. For example, the N-t-Boc group can be hydrolyzed by treatment with a strong acid to generate an omega-aminopolymer azide. The resulting amine can be used as a synthetic handle for installing other useful functional groups such as maleimide groups, activated disulfides, activated esters, etc. to create useful heterobifunctional reagents.

[0257] Heterobifunctional derivatives are particularly useful when it is desired to attach different molecules to each end of a polymer. For example, an omega-N-amino-N-azide polymer would allow attachment of a molecule having an activated electrophilic group such as an aldehyde, ketone, activated ester, activated carbonate, etc. to one end of the polymer and a molecule having an acetylene group to the other end of the polymer.

[0258] In another embodiment of the invention, A is an aliphatic linker of 1 to 10 carbon atoms, or a substituted aryl ring of 6 to 14 carbon atoms. X is a functional group that does not react with an azide group, and Y is a suitable leaving group.

[0259] The plurality of targeting polypeptides may be joined by a linker polypeptide, which may optionally be 6-14, 7-13, 8-12, 7-11, 9-11, or 9 amino acids in length. Other linkers include, but are not limited to, small polymers such as PEG, which may be of the multi-arm type that allows multiple targeting polypeptide molecules to be linked together. The plurality of targeting polypeptides and modified targeting polypeptides may be linked to each other via their N-termini in a head-to-head arrangement through the use of such linkers or by direct chemical bonds between the respective N-termini of each polypeptide. For example, two targeting polypeptides may be linked to form a dimer by a chemical bond between their N-terminal amino groups or modified N-terminal amino groups. Also, a linking molecule designed to contain multiple chemical functional groups for binding to the N-terminus of each targeting polypeptide may be used to join the plurality of targeting polypeptides at their respective N-termini. In addition, the plurality of targeting polypeptides may be linked through bonds between amino acids other than the N-terminal or C-terminal amino acids. Examples of covalent bonds that may be utilized to form dimers and multimers of the targeting polypeptides described herein include, but are not limited to, disulfide or sulfhydryl or thiol bonds. In addition, a specific enzyme, such as sortase, may be used to form a covalent bond between the targeting polypeptide and the linker at a position containing the N-terminus of the targeting polypeptide.

[0260] The linker may have a wide range of molecular weights or molecular lengths. Linkers of larger or smaller molecular weights may be used to provide a desired spatial relationship or conformation between the targeting polypeptide and the entity to which it is linked, or between the linked entity and its binding partner (if any). Also, linkers having longer or shorter molecular lengths may be used to provide a desired space or flexibility between the targeting polypeptide and the entity to which it is linked, or between the linked entity and its binding partner.

[0261] In some embodiments, the present invention provides a water-soluble bifunctional linker having a dumbbell structure, comprising a) an azide, alkyne, hydrazine, hydrazide, hydroxylamine, or carbonyl-containing moiety on at least a first end of a polymer backbone, and b) at least a second functional group on a second end of the polymer backbone. The second functional group may be the same as or different from the first functional group. In some embodiments, the second functional group is not reactive with the first functional group. The present invention, in some embodiments, provides a water-soluble compound comprising at least one arm of a branched molecular structure. For example, the branched molecular structure may be dendritic.

[0262] In exemplary embodiments, the polymer is linked to a targeted polypeptide or a modified targeted polypeptide through a linker. For example, the linker may comprise one or two amino acids that bind to the polymer (such as an albumin-binding moiety, etc.) at one end and to any available position on the polypeptide backbone at the other end. Additional exemplary linkers include hydrophilic linkers such as chemical moieties containing at least five non-hydrogen atoms, 30-50% of which are either N or O. Additional exemplary linkers that can link the polymer to a targeted polypeptide or a modified targeted polypeptide are disclosed in U.S. 2012 / 0295847 and WO / 2012 / 168430, each of which is hereby incorporated by reference in its entirety.

[0263] Optionally, a plurality of targeting polypeptides or modified targeting polypeptide molecules may be joined by a linker polypeptide, which may optionally have a length of 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12 amino acids, and a length greater than that, and optionally, the N-terminus of one targeting polypeptide is condensed to the C-terminus of the linker polypeptide, and the N-terminus of the linker polypeptide is condensed to the N-terminus of another targeting polypeptide. Further exemplary linker polypeptides that may be utilized are disclosed in WO / 2013 / 004607, which is incorporated herein by reference in its entirety.

[0264] As used herein, terms such as "electrophilic group", "electrophile" refer to an atom or group of atoms that can accept an electron pair to form a covalent bond. The "electrophilic group" as used herein includes, but is not limited to, halides, carbonyls, and epoxide-containing compounds. General electrophiles can be halides such as thiophosgene, glycerin dichlorohydrin, phthaloyl chloride, succinyl chloride, chloroacetyl chloride, chlorosuccinyl chloride, ketones such as chloroacetone, bromoacetone, aldehydes such as glyoxal, isocyanates such as hexamethylene diisocyanate, tolylene diisocyanate, meta-xylylene diisocyanate, cyclohexylmethane-4,4-diisocyanate, and derivatives of these compounds.

[0265] As used herein, terms such as "nucleophilic group", "nucleophile" refer to an atom or group of atoms having an electron pair that can form a covalent bond. This type of group can be an ionizable group that reacts as an anionic group. The "nucleophilic group" as used herein includes, but is not limited to, hydroxyl, primary amine, secondary amine, tertiary amine, and thiol.

[0266] Table 2 provides various starting electrophiles and nucleophiles that can be combined to create the desired functional groups. The information provided is illustrative and not limited to the synthetic techniques described herein.

[0267]

Table 2

[0268] Generally, a carbon electrophile is readily attacked by a complementary nucleophile that includes a carbon nucleophile, and an aggressive nucleophile provides an electron pair to the carbon electrophile in order to form a new bond between the nucleophile and the carbon electrophile.

[0269] Non-limiting examples of carbon nucleophiles include alkyl, alkenyl, aryl, and alkynyl Grignard, organolithium, organozinc, alkyl-, alkenyl-, aryl-, and alkynyl-tin reagents (organostannanes), alkyl-, alkenyl-, aryl-, and alkynyl-borane reagents (organoboranes and organoboronates), but are not limited thereto. These carbon nucleophiles have the advantage of being kinetically stable in water or polar organic solvents. Other non-limiting examples of carbon nucleophiles include phosphorus ylides, enols, and enolate reagents. These carbon nucleophiles have the advantage of being relatively easy to generate from precursors well known to those skilled in synthetic organic chemistry. Carbon nucleophiles, when used in combination with carbon electrophiles, result in the formation of a new carbon-carbon bond between the carbon nucleophile and the carbon electrophile.

[0270] Non-limiting examples of non-carbon nucleophiles suitable for bonding to carbon electrophiles include primary and secondary amines, thiols, thiolates, as well as thioethers, alcohols, alkoxides, azides, semicarbazides, etc., but are not limited thereto. These non-carbon nucleophiles, when used in combination with carbon electrophiles, typically generate a heteroatom linkage (C-X-C), where X is a heteroatom including, but not limited to, oxygen, sulfur, or nitrogen.

[0271] In some cases, the polymer used in the present invention is terminated at one end with hydroxy or methoxy, i.e., X is H or CH3 (“methoxy PEG”). Alternatively, the polymer can be terminated with a reactive group, thereby forming a bifunctional polymer. Typical reactive groups include those generally used to react with functional groups found in the 20 common amino acids (including but not limited to maleimide groups, activated carbonates (including but not limited to p-nitrophenyl esters), activated esters (including but not limited to N-hydroxysuccinimide, p-nitrophenyl esters), and aldehydes), as well as functional groups that are inert to the 20 common amino acids but specifically react with complementary functional groups (including but not limited to azide groups, alkyne groups). Note that the other end of the polymer represented by the above formula by Y is attached to the target polypeptide either directly or indirectly via a naturally occurring or non-natural encoded amino acid. For example, Y can be an amide, carbamate, or urea linkage to an amine group of the polypeptide (including but not limited to the epsilon amine of lysine or the N-terminus). Alternatively, Y can be a maleimide linkage to a thiol group (including but not limited to the thiol group of cysteine). Alternatively, Y can be a linkage to a residue that is not generally accessible via the 20 common amino acids. For example, an azide group on the polymer can be reacted with an alkyne group on the target polypeptide to form a Huisgen [3+2] cycloaddition product. Alternatively, an alkyne group on the polymer can be reacted with an azide group present in the target polypeptide to form a similar product. In some embodiments, where applicable, a strong nucleophile (including but not limited to hydrazine, hydrazide, hydroxylamine, semicarbazide) can be reacted with an aldehyde or ketone group present in the target polypeptide to form a hydrazone, oxime, or semicarbazone, which can in some cases be further reduced by treatment with an appropriate reducing agent.Alternatively, a strong nucleophile can be incorporated into a targeted polypeptide via a non-naturally encoded amino acid and used to preferentially react with a ketone or aldehyde group present in a water-soluble polymer.

[0272] Any molecular weight of the polymer can be used substantially as desired, including but not limited to from about 100 Daltons (Da) to 100,000 Da or more (optionally including but not limited to 0.1 to 50 kDa or 10 to 40 kDa). The molecular weight of the polymer can be in a wide range including but not limited to from about 100 Da to about 100,000 Da or more. The polymer can be from about 100 Da to about 100,000 Da, including but not limited to 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the polymer is from about 100 Da to about 50,000 Da. Also usable are branched-chain polymers, including but not limited to those in which each chain comprises polymer molecules having a molecular weight in the range of 1 to 100 kDa (optionally including but not limited to 1 to 50 kDa or 5 to 20 kDa). The molecular weight of each chain of the branched-chain polymer can be, although not limited, including from about 1,000 Da to about 100,000 Da or more.The molecular weight of each chain of the branched polymer can be from about 1,000 Da to about 100,000 Da, including but not limited to 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecular weight of each chain of the branched polymer is from about 1,000 Da to about 50,000 Da. In some embodiments, the molecular weight of each chain of the branched polymer is from about 1,000 Da to about 40,000 Da. In some embodiments, the molecular weight of each chain of the branched polymer is from about 5,000 Da to about 40,000 Da. In some embodiments, the molecular weight of each chain of the branched polymer is from about 5,000 Da to about 20,000 Da. A wide range of polymer molecules are described in, but not limited to, those described in the catalogs of Shearwater Polymers, Inc., and Nektar Therapeutics (incorporated herein by reference).

[0273] In some embodiments, the present invention provides azide- and acetylene-containing polymer derivatives comprising a water-soluble polymer backbone having an average molecular weight of from about 800 Da to about 100,000 Da. The polymer backbone of the water-soluble polymer can be poly(ethylene glycol). However, a wide variety of water-soluble polymers including, but not limited to, poly(ethylene glycol), as well as other related polymers including poly(dextran) and poly(propylene glycol), are also suitable for use in the practice of the present invention, and it is to be understood that the use of the term PEG or poly(ethylene glycol) is intended to encompass and include all such molecules. The term PEG includes poly(ethylene glycol) in any of its forms, including bifunctional PEG, multi-arm PEG, derivatized PEG, fork PEG, branched PEG, pendant PEG (i.e., PEG or related polymer having one or more functional groups pendant to the polymer backbone), or PEG having a cleavable linkage therein, but is not limited thereto.

[0274] In addition to these forms of polymers, the polymers can also be prepared with weak or cleavable linkages within the backbone. For example, the polymer can be prepared with ester linkages within the polymer backbone that are subject to hydrolysis. As shown below, this hydrolysis cleaves the polymer into low molecular weight fragments. -Polymer-CO2-Polymer- + H2O → Polymer-CO2H + HO-Polymer- Many polymers are also suitable for use in the present invention. In some embodiments, a water-soluble polymer backbone having from 2 to about 300 termini is particularly useful in the present invention. Examples of suitable polymers include other poly(alkylene glycols) such as poly(propylene glycol) (“PPG”), copolymers thereof (including, but not limited to, copolymers of ethylene glycol and propylene glycol), terpolymers thereof, mixtures thereof, and the like, but are not limited thereto. The molecular weight of each chain of the polymer backbone can vary, but is typically in the range of about 800 Da to about 100,000 Da, often in the range of about 6,000 Da to about 80,000 Da. The molecular weight of each chain of the polymer backbone can be about 100 Da to about 100,000 Da, including, but not limited to, about 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is about 100 Da to about 50,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is about 100 Da to about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is about 1,000 Da to about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is about 5,000 Da to about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is about 10,000 Da to about 40,000 Da.

[0275] In one aspect of this embodiment of the invention, an intact polymer conjugate is minimally degraded upon administration such that hydrolysis of the cleavable bond is effective to control the slow release rate of the active targeting polypeptide into the bloodstream prior to hydrolysis, which is in contrast to enzymatic degradation of the targeting polypeptide prior to its release into the systemic circulation.

[0276] Suitable physiologically cleavable linkages include, but are not limited to, esters, carbonates, carbamates, sulfates, phosphates, acyloxyalkyl ethers, acetals, and ketals. Such conjugates should have physiologically cleavable linkages that are stable during storage and upon administration. For example, a targeting polypeptide or modified targeting polypeptide conjugated to a polymer should maintain its integrity during the manufacture of the final pharmaceutical composition, when used, upon dissolution in a suitable delivery vehicle, and upon administration regardless of the route.

[0277] The present invention also includes phosphate-based linkers having adjustable stability for intracellular delivery of drug conjugates disclosed in US2017 / 0182181, which is incorporated herein by reference. The phosphate-based linker includes a phosphate group (monophosphate, diphosphate, triphosphate, or tetraphosphate group) covalently attached to the distal end of a linker arm that includes, from the distal direction to the proximal direction, a regulatory element, optionally a spacer element, and a reactive functional group. The phosphate group of the phosphate-based linker can conjugate to a payload, and the reactive functional group can conjugate to a cell-specific targeting ligand such as an antibody. The general structure of the phosphate-based linker is phosphate group - regulatory element - optional spacer element - functional reactive group. The phosphate-based linker conjugated to a payload has the general structure: payload - phosphate group - regulatory element - optional spacer element - functional reactive group, and when conjugated to a targeting ligand, has the general structure: payload - phosphate group - regulatory element - optional spacer element - targeting ligand. These phosphate-based linkers are distinguished in the blood and have adjustable stability compared to the intracellular environment (e.g., lysosomal compartment). The rate at which the phosphate group is cleaved in the intracellular environment to release the payload in its native or active form is affected by the structure of the regulatory element, which has additional effects mediated by substitution of the phosphate group, and by whether the phosphate group is monophosphate, diphosphate, triphosphate, or tetraphosphate. Furthermore, these phosphate-based linkers provide the ability to construct conjugates such as antibody-drug conjugates in which the conjugate has a reduced tendency to form aggregates compared to conjugates in which the same payload is conjugated to an antibody or targeting ligand using a linker that is not a phosphate-based linker disclosed herein.

[0278] Structure and Synthesis of TLR-Agonist Linker Derivatives: Electrophilic and Nucleophilic Groups TLR agonist derivatives having a linker containing a hydroxylamine (also called aminooxy) group can react with various electrophilic groups to form conjugates (including but not limited to PEG or other water-soluble polymers). Similar to hydrazine, hydrazide, and semicarbazide, the enhanced nucleophilicity of the aminooxy group enables it to react efficiently and selectively with various molecules containing carbonyl or dicarbonyl groups, including but not limited to ketones, aldehydes, or other functional groups with similar chemical reactivity. See, for example, Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995), H. Hang and C. Bertozzi, Acc. Chem. Res. 34(9):727-736 (2001). The result of the reaction with a hydrazine group is the corresponding hydrazone, while oximes generally result from the reaction of an aminooxy group with a carbonyl or dicarbonyl-containing group such as a ketone, aldehyde, or other functional group with similar chemical reactivity. In some embodiments, TLR-agonist derivatives having a linker containing an azide, alkyne, or cycloalkyne enable the ligation of molecules via cycloaddition reactions (e.g., 1,3-dipolar cycloaddition, azide-alkyne Huisgen cycloaddition, etc.). (To the extent of the reaction, as described in U.S. Patent No. 7,807,619, which is incorporated herein by reference).

[0279] Thus, in certain embodiments, a TLR-agonist derivative having a linker comprising a hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, keto-amine, keto-alkyne, and en-dione hydroxylamine group, a hydroxylamine-like group (having reactivity similar to a hydroxylamine group and structurally similar to a hydroxylamine group), a masked hydroxylamine group (which can be readily converted to a hydroxylamine group), or a protected hydroxylamine group (having reactivity similar to a hydroxylamine group upon deprotection) is described herein. In some embodiments, the TLR agonist derivative having a linker comprises an azide, alkyne, or cycloalkyne.

[0280] Such TLR-agonist linker derivatives or targeted polypeptides can be in the form of salts or can be incorporated into non-natural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.

[0281] In certain embodiments, the compounds of formulas (I)-(VII) are stable in aqueous solution for at least one month under weakly acidic conditions. In certain embodiments, the compounds of formulas (I)-(VII) are stable for at least two weeks under weakly acidic conditions. In certain embodiments, the compounds of formulas (I)-(VII) are stable for at least five days under weakly acidic conditions. In certain embodiments, such acidic conditions are pH 2-8.

[0282] The methods and compositions provided and described herein include polypeptides comprising unnatural amino acids having at least one carbonyl or dicarbonyl group, an oxime group, a hydroxylamine group, or a protected or masked form thereof. The introduction of at least one reactive group into a TLR-agonist linker derivative or a targeted polypeptide allows for the application of conjugation chemistry that involves a specific chemical reaction with one or more targeted polypeptides (including but not limited to) without reacting with the commonly present amino acids. Once incorporated, the targeted polypeptide of the TC side chain can also be modified by utilizing the chemical methodologies described herein or can be suitable for specific functional groups or substituents present in the TLR-agonist linker derivative or the targeted polypeptide.

[0283] The TLR-agonist linker derivatives and targeted polypeptides, methods, and compositions described herein provide conjugates of substances having a wide variety of functional groups, substituents, or moieties, including but not limited to polymers, water-soluble polymers, polyethylene glycol derivatives, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, and any combination thereof.

[0284] In certain embodiments, the TLR-agonist linker derivatives, targeted polypeptides, TCs, linkers, and reagents described herein, including compounds of formulas (I)-(VII), are stable in aqueous solution under weakly acidic conditions (including but not limited to pH 2-8). In other embodiments, such compounds are stable for at least one month under weakly acidic conditions. In other embodiments, such compounds are stable for at least two weeks under weakly acidic conditions. In other embodiments, such compounds are stable for at least five days under weakly acidic conditions.

[0285] In another aspect of the compositions, methods, techniques, and strategies, methods are described herein for studying or using any of the foregoing "modified or unmodified" non-natural amino acid-targeted polypeptides. This aspect includes, by way of example only, therapeutic, diagnostic, assay-based, industrial, cosmetic, plant biological, environmental, energy-producing, consumer product, and / or military uses that would benefit from a targeted polypeptide comprising a "modified or unmodified" non-natural amino acid polypeptide or protein.

[0286] The present invention provides a TC molecule comprising at least one non-natural amino acid. In certain embodiments of the present invention, the TC having at least one non-natural amino acid comprises at least one post-translational modification. In one embodiment, the at least one post-translational modification is a label, dye, linker, another TC polypeptide, polymer, water-soluble polymer, derivative of polyethylene glycol, photocrosslinker, radionuclide, cytotoxic compound, drug, affinity label, photoaffinity label, reactive compound, resin, second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelating agent, cofactor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, antisense polynucleotide, sugar, cyclodextrin, inhibitory ribonucleic acid, biomaterial, nanoparticle, spin label, fluorophore, metal-containing moiety, radioactive moiety, novel functional group, group that interacts covalently or non-covalently with other molecules, photocaged moiety, actin radiation-excitable moiety, photo-isomerizable moiety, biotin, biotin derivative, biotin analog, moiety incorporating heavy atoms, chemically cleavable group, photocleavable group, elongated side chain, carbon-linked sugar, redox activator, aminothio acid, toxic moiety, isotope-labeled moiety, biophysical probe, phosphorescent group, chemiluminescent group, electron-dense group, magnetic group, intercalating group, chromophore, energy transfer agent, biologically active agent, detectable label, small molecule, quantum dot, nanocarrier, radioactive nucleotide, radioactive carrier, neutron capture agent, or any combination of the above, or any other desired compound or substance (including a second reactive group for at least one non-natural amino acid, including a first reactive group that utilizes chemical methodologies known to those skilled in the art to be suitable for specific reactive groups), including the binding of molecules, but not limited thereto. For example, the first reactive group is an alkynyl moiety (including but not limited to within the non-natural amino acid p-propargyloxyphenylalanine, where the propargyl group may also be referred to as an acetylene moiety), the second reactive group is an azide moiety, and [3+2] cycloaddition chemistry methodology is utilized.In another embodiment, the first reactive group is an azide moiety (including, but not limited to, the unnatural amino acid p-azido-L-phenylalanine or pAZ, which is sometimes referred to herein), and the second reactive group is an alkynyl moiety. In certain embodiments of the modified TC of the present invention, at least one unnatural amino acid (including, but not limited to, unnatural amino acids containing a keto functional group) containing at least one post-translational modification is used, where at least one post-translational modification includes a sugar moiety. In certain embodiments, the post-translational modification is performed in vivo in eukaryotic or prokaryotic cells. A linker, polymer, water-soluble polymer, or other molecule may bind the molecule to the polypeptide. In a further embodiment, the linker attached to the TC is of sufficient length to allow for dimer formation. The molecule may also be directly linked to the polypeptide.

[0287] In certain embodiments, the TC protein contains at least one post-translational modification performed in vivo by one host cell, where the post-translational modification is not normally performed by another host cell type. In certain embodiments, the protein contains at least one post-translational modification performed in vivo by eukaryotic cells, where the post-translational modification is not normally performed by prokaryotic cells. Examples of post-translational modifications include, but are not limited to, glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linked modification, and the like.

[0288] In some embodiments, the TC comprises one or more non-naturally encoded amino acids for glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitate addition, phosphorylation, or glycolipid-linked modification of the polypeptide. In some embodiments, the TC comprises one or more non-naturally encoded amino acids for glycosylation of the polypeptide. In some embodiments, the TC comprises one or more naturally encoded amino acids for glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitate addition, phosphorylation, or glycolipid-linked modification of the polypeptide. In some embodiments, the TC comprises one or more naturally encoded amino acids for glycosylation of the polypeptide.

[0289] In some embodiments, the TC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation of the polypeptide. In some embodiments, the TC comprises one or more deletions that enhance glycosylation of the polypeptide. In some embodiments, the TC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation at different amino acids in the polypeptide. In some embodiments, the TC comprises one or more deletions that enhance glycosylation at different amino acids in the polypeptide. In some embodiments, the TC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation at non-naturally encoded amino acids in the polypeptide. In some embodiments, the TC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation at naturally encoded amino acids in the polypeptide. In some embodiments, the TC comprises one or more naturally encoded amino acid additions and / or substitutions that enhance glycosylation at different amino acids in the polypeptide. In some embodiments, the TC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation at naturally encoded amino acids in the polypeptide. In some embodiments, the TC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation at non-naturally encoded amino acids in the polypeptide.

[0290] In one embodiment, post-translational modifications include the attachment of oligosaccharides to asparagine by GlcNAc-asparagine linkage (including, but not limited to, cases where the oligosaccharide contains, for example, (GlcNAc-Man)2-Man-GlcNAc-GlcNAc). In another embodiment, post-translational modifications include the attachment of oligosaccharides (including, but not limited to, Gal-GalNAc, Gal-GlcNAc, etc.) to serine or threonine by GalNAc-serine, GalNAc-threonine, GlcNAc-serine, or GlcNAc-threonine linkage. In certain embodiments, the proteins or polypeptides of the invention can include secretion or localization sequences, epitope tags, FLAG tags, polyhistidine tags, GST fusions, and / or the like. Examples of secretion signal sequences include, but are not limited to, prokaryotic secretion signal sequences, eukaryotic secretion signal sequences, eukaryotic secretion signal sequences optimized at the 5' end for bacterial expression, novel secretion signal sequences, pectate lyase secretion signal sequences, OmpA secretion signal sequences, and phage secretion signal sequences. Examples of secretion signal sequences include, but are not limited to, STII (prokaryotic), Fd GIII and M13 (phage), Bgl2 (yeast), and the signal sequence bla derived from a transposon. Any such sequence can be modified to provide the desired result in a polypeptide, including, but not limited to, substituting one signal sequence with a different signal sequence, substituting a leader sequence with a different leader sequence, and the like.

[0291] The protein or polypeptide of interest can contain at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10 or more non-natural amino acids. The non-natural amino acids can be the same or different, and for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different sites in a protein containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different non-natural amino acids can exist. In certain embodiments, at least one, but less than all, of the specific amino acids present in a naturally occurring protein form are replaced with non-natural amino acids.

[0292] The present invention provides methods and compositions based on TCs that include at least one non-naturally encoded amino acid. The introduction of at least one non-naturally encoded amino acid into a TC enables the application of conjugate chemistry that involves specific chemical reactions with one or more, but not limited to, non-naturally encoded amino acids that do not react with the 20 commonly occurring amino acids. In some embodiments, the TC containing the non-naturally encoded amino acid is linked via the side chain of the non-naturally encoded amino acid to a water-soluble polymer such as polyethylene glycol (PEG) or a linker. The present invention provides a highly efficient method for the selective modification of proteins with PEG derivatives or TLR-linker derivatives, which involves selectively incorporating non-gene-encoded amino acids, including but not limited to those containing functional groups or substituents not found in the 20 naturally incorporated amino acids that include, but are not limited to, ketone, azide, or acetylene moieties, into proteins in response to a selector codon, and then modifying those amino acids with a suitable reactive PEG derivative. Once incorporated, the amino acid side chains can then be modified by utilizing chemical methodologies known to those skilled in the art to be suitable for the particular functional groups or substituents present in the non-naturally encoded amino acid. A wide variety of known chemical methodologies are suitable for use in the present invention for incorporating water-soluble polymers into proteins. Such methodologies include, but are not limited to, Huisgen [3+2] cycloaddition reactions with, but not limited to, those containing acetylene or azide derivatives (see, for example, Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991) Ed. Trost, B.M., Pergamon, Oxford, p. 1069-1109 and Huisgen, R. in 1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, New York, p. 1-176).

[0293] The Huisgen [3+2] cycloaddition method involves cycloaddition rather than nucleophilic substitution, so proteins can be modified with extremely high selectivity. The reaction can be carried out at room temperature under aqueous conditions with excellent regioselectivity (1,4>1,5) by adding a catalytic amount of a Cu(I) salt to the reaction mixture. See, for example, Tornoe, et al., (2002) J. Org. Chem. 67:3057-3064 and Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599, as well as WO03 / 101972. Molecules that can be added to the proteins of the present invention through [3+2] cycloaddition include substantially any molecule having a suitable functional group or substituent, including but not limited to azide or acetylene derivatives. These molecules can be added to unnatural amino acids having an acetylene group, including but not limited to p-propargyloxyphenylalanine, or an azide group, including but not limited to p-azido-phenylalanine, respectively.

[0294] The five-membered ring resulting from the Huisgen [3+2] cycloaddition is generally not reversible in a reducing environment and is stable to long-term hydrolysis in an aqueous environment. Thus, the physical and chemical characteristics of a wide variety of substances can be modified under harsh aqueous conditions using the active PEG derivatives or TLR-linker derivatives of the present invention. More importantly, since the azide and acetylene moieties are specific to each other (and do not react with any of the 20 common genetically encoded amino acids, for example), proteins can be modified at one or more specific sites with extremely high selectivity.

[0295] The present invention also provides water-soluble and hydrolytically stable derivatives of PEG derivatives or TLR linker derivatives, and related hydrophilic polymers having one or more acetylene or azide moieties. PEG polymer derivatives containing an acetylene moiety are highly selective for binding to an azide moiety selectively introduced into a protein in response to a selector codon. Similarly, PEG polymer derivatives containing an azide moiety are highly selective for binding to an acetyl moiety selectively introduced into a protein in response to a selector codon. More specifically, the azide moiety includes, but is not limited to, alkyl azides, aryl azides, and derivatives of these azides. Derivatives of alkyl and aryl azides can include other substituents as long as the acetylene-specific reactivity is maintained. The acetylene moiety includes alkyl and aryl acetylenes, and derivatives thereof. Derivatives of alkyl and aryl acetylenes can include other substituents as long as the azide-specific reactivity is maintained.

[0296] The present invention provides conjugates of substances having a variety of functional groups, substituents, or moieties with other substances including, but not limited to, labels, dyes, polymers, water-soluble polymers, derivatives of polyethylene glycol, photo-crosslinking agents, radionuclides, cytotoxic compounds, drugs, affinity labels, photoaffinity labels, reactive compounds, resins, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelating agents, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, antisense polynucleotides, sugars, water-soluble dendrimers, cyclodextrins, inhibitory ribonucleic acids, biomaterials, nanoparticles, spin labels, fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that interact covalently or non-covalently with other molecules, photocaging moieties, actin radiation-excitable moieties, photo-isomerizable moieties, biotin, biotin derivatives, biotin analogs, moieties incorporating heavy atoms, chemically cleavable groups, photocleavable groups, elongated side chains, carbon-linked sugars, redox-active agents, amino acids, toxic moieties, isotope-labeled moieties, biophysical probes, phosphorescent groups, chemiluminescent groups, electron-dense groups, magnetic groups, intercalating groups, chromophores, energy transfer agents, biologically active agents, detectable labels, small molecules, quantum dots, nanotransporters, radioactive nucleotides, radioactive transporters, neutron capture agents, or any combination of the foregoing, or any other desired compounds or substances. The present invention also includes conjugates of substances having an azide or acetylene moiety with a PEG polymer derivative having a corresponding acetylene or azide moiety. For example, a PEG polymer containing an azide moiety can be conjugated to a biologically active molecule at a position within a protein containing a non-genetically encoded amino acid having an acetylene functional group. The linkage to which the PEG and the biologically active molecule are attached includes, but is not limited to, a Huisgen [3+2] cycloaddition product.

[0297] It is well established in the art that the surface of a biomaterial can be modified using PEG (see, for example, U.S. Patent No. 6,610,281, which is incorporated herein by reference, and Mehvar, R., J. Pharm Pharm Sci., 3(1):125-136 (2000)). The present invention also includes a biomaterial comprising a surface having one or more reactive azide or acetylene moieties and one or more of the azide- or acetylene-containing polymers of the present invention attached to the surface via a Huisgen [3+2] cycloaddition ligation. Biomaterials and other substances can also be attached to an azide- or acetylene-activated polymer derivative through linkages other than azide or acetylene linkages, such as linkages containing carboxylic acid, amine, alcohol, or thiol moieties, to make the azide or acetylene moiety available for subsequent reactions.

[0298] The present invention includes a method for synthesizing the azide- and acetylene-containing polymers of the present invention. In the case of azide-containing PEG derivatives, the azide can be directly attached to a carbon atom of the polymer. Alternatively, an azide-containing PEG derivative can be prepared by attaching a linker having an azide moiety at one end to a conventional activated polymer such that the resulting polymer has an azide moiety at its end. In the case of acetylene-containing PEG derivatives, the acetylene can be directly attached to a carbon atom of the polymer. Alternatively, an acetylene-containing PEG derivative can be prepared by attaching a linker having an acetylene moiety at one end to a conventional activated polymer such that the resulting polymer has an acetylene moiety at its end.

[0299] More specifically, in the case of azide-containing PEG derivatives, the water-soluble polymer having at least one active hydroxyl moiety undergoes a reaction to produce a substituted polymer having a more reactive moiety thereon, such as a mesylate, tresylate, tosylate, or halogen leaving group. The preparation and use of PEG derivatives or TLR-linker derivatives containing sulfonyl halides, halogen atoms, and other leaving groups are known to those skilled in the art. The resulting substituted polymer then undergoes a reaction to use a more reactive moiety in place of the azide moiety at the end of the polymer. Alternatively, a water-soluble polymer having at least one active nucleophilic or electrophilic moiety undergoes a reaction with a linker having an azide at one end such that a covalent bond is formed between the PEG polymer and the linker and the azide moiety is positioned at the end of the polymer. Nucleophilic and electrophilic moieties including amines, thiols, hydrazides, hydrazines, alcohols, carboxylates, aldehydes, ketones, thioesters, etc. are known to those skilled in the art.

[0300] More specifically, in the case of acetylene-containing PEG derivatives, the water-soluble polymer having at least one active hydroxyl moiety undergoes a reaction to substitute a halogen or other activated leaving group from a precursor containing an acetylene moiety. Alternatively, a water-soluble polymer having at least one active nucleophilic or electrophilic moiety undergoes a reaction with a linker having an acetylene at one end such that a covalent bond is formed between the PEG polymer and the linker and the acetylene moiety is positioned at the end of the polymer. The use of halogen moieties, activated leaving groups, nucleophilic and electrophilic moieties in the context of organic synthesis and the preparation and use of PEG derivatives or TLR-linker derivatives are well established to those skilled in the art.

[0301] The present invention also provides a method for the selective modification of proteins for addition to proteins modified with other substances, including but not limited to water-soluble polymers such as PEG and PEG derivatives or TLR-linker derivatives, linkers, or another TC polypeptide containing an azide or acetylene moiety. Azide- and acetylene-containing PEG derivatives or TLR-linker derivatives can be used to modify the properties of surfaces and molecules where lack of biocompatibility, stability, solubility, and immunogenicity are important, while at the same time providing a more selective means of conjugating PEG derivatives or TLR-linker derivatives to proteins than previously known in the art.

[0302] General recombinant nucleic acid methods for use in the present invention In many embodiments of the invention, nucleic acids encoding a targeting polypeptide of interest of a TC are isolated, cloned, and often modified using recombinant methods. Such embodiments include, but are not limited to, use for protein expression or in the generation of variants, derivatives, expression cassettes, or other sequences derived from the targeting polypeptide of the TC. In some embodiments, the sequences encoding the polypeptides of the invention are operably linked to a heterologous promoter.

[0303] The nucleotide sequence encoding a targeted polypeptide of TC containing a non-naturally encoded amino acid is synthesized based on the amino acid sequence of the parent polypeptide and can then be altered to effect the introduction (i.e., incorporation or substitution) or removal (i.e., deletion or substitution) of the relevant amino acid residue(s). The nucleotide sequence can be conveniently modified by site-directed mutagenesis by conventional methods. Alternatively, the nucleotide sequence may be prepared by chemical synthesis including, but not limited to, the use of an oligonucleotide synthesizer, designing the oligonucleotide based on the amino acid sequence of the desired polypeptide, and preferably selecting those codons that are preferred in the host cell in which the recombinant polypeptide is to be produced. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and assembled by PCR, ligation, or ligation chain reaction. See, e.g., Barany, et al., Proc. Natl. Acad. Sci. 88:189-193 (1991), U.S. Patent No. 6,521,427, which is incorporated herein by reference.

[0304] The present invention utilizes conventional techniques in the field of recombinant genetics. Basic texts disclosing general methods of use in the present invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd ed. 2001), Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990) and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994).

[0305] The present invention also relates to eukaryotic host cells, prokaryotic host cells, and organisms for in vivo incorporation of unnatural amino acids via orthogonal tRNA / RS pairs. The host cells are genetically engineered (including, but not limited to, transformation, transduction, or transfection) with a construct comprising a polynucleotide of the present invention, which can be, for example, a cloning vector or an expression vector, of the present invention.

[0306] Several well-known methods are available for introducing a target nucleic acid into a cell, and any of these may be used in the present invention. These include fusion of recipient cells with bacterial protoplasts containing DNA, electroporation, particle bombardment, and infection with viral vectors (discussed further below). Bacterial cells can be used to amplify the number of plasmids containing the DNA constructs of the present invention. The bacteria are grown to the log phase, and the plasmids within the bacteria can be isolated by various methods known in the art (see, for example, Sambrook). In addition, kits are commercially available for the purification of plasmids from bacteria (see, for example, EasyPrep™, FlexiPrep™ (both Pharmacia Biotech), StrataClean™ (Stratagene), and QIAprep™ (Qiagen)). The isolated and purified plasmids are then further manipulated to produce other plasmids for use in transfecting cells or for incorporation into related vectors for infecting organisms. Typical vectors contain transcriptional and translational terminators, transcriptional and translational initiation sequences, and promoters useful for regulating the expression of a particular target nucleic acid. Vectors optionally contain at least one independent terminator sequence, a general expression cassette containing sequences that allow for replication of the cassette in eukaryotes or prokaryotes, or both (including but not limited to shuttle vectors), and selectable markers for both prokaryotic and eukaryotic systems. Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or both. See Gillam & Smith, Gene 8:81 (1979), Roberts, et al., Nature, 328:731 (1987), Schneider, E., et al., Protein Expr. Purif. 6(1):10-14 (1995), Ausubel, Sambrook, Berger (all supra).Catalogs of bacteria and bacteriophages useful for cloning are provided, for example, by ATCC, for example, The ATCC Catalogue of Bacteria and Bacteriophage (1992) Gherna et al. (eds). Additional basic procedures for molecular biology sequencing, cloning, and other aspects, as well as underlying theoretical considerations, can also be found in Watson et al. (1992) Recombinant DNA Second Edition Scientific American Books, NY. In addition, essentially any nucleic acid (and substantially any labeled nucleic acid, whether standard or non-standard) can be specially ordered or ordered standard from any of a variety of commercial sources, such as Midland Certified Reagent Company (Midland, TX, available on the World Wide Web at mcrc.com), The Great American Gene Company (Ramona, CA, available on the World Wide Web at genco.com), ExpressGen Inc. (Chicago, IL, available on the World Wide Web at expressgen.com), Operon Technologies Inc. (Alameda, CA), and many other various commercial suppliers.

[0307] Selector codon The selector codons of the present invention expand the genetic codon framework of the protein biosynthesis mechanism. For example, selector codons include, but are not limited to, unique three-base codons, nonsense codons such as stop codons (amber codon (UAG), ochre codon, or opal codon (UGA)), non-natural codons, codons of four or more bases, rare codons, etc. Those skilled in the art will readily appreciate that the number of selector codons that can be introduced into a desired gene or polynucleotide has a wide range including, but not limited to, one or more, two or more, three or more, four, five, six, seven, eight, nine, ten or more in a single polynucleotide encoding at least a portion of TC.

[0308] In one embodiment, the method involves the use of a selector codon that is a stop codon for incorporating one or more non-natural amino acids in vivo. For example, an O-tRNA that recognizes a stop codon including, but not limited to, UAG is produced and aminoacylated by an O-RS having the desired non-natural amino acid. This O-tRNA is not recognized by the naturally occurring host aminoacyl-tRNA synthetase. Conventional site-directed mutagenesis can be used to introduce a stop codon including, but not limited to, TAG into a site of interest within the polypeptide of interest. See, for example, Sayers, J.R., et al. (1988), 5'-3' Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res, 16:791-802. When the nucleic acids encoding the O-RS, O-tRNA, and the polypeptide of interest are combined in vivo, the non-natural amino acid is incorporated in response to the UAG codon, and a polypeptide containing the non-natural amino acid at a specific position is obtained.

[0309] In vivo incorporation of non-natural amino acids can be carried out without significant perturbation of eukaryotic host cells. For example, the suppression efficiency of the UAG codon depends on the competition between an O-tRNA, including but not limited to an amber suppressor tRNA, and a eukaryotic release factor (including but not limited to eRF) (which binds to the stop codon and initiates the release of the growing peptide from the ribosome), so the suppression efficiency can be regulated by, including but not limited to, increasing the expression level of the O-tRNA and / or the suppressor tRNA.

[0310] Non-natural amino acids can also be encoded by rare codons. For example, when the arginine concentration in an in vitro protein synthesis reaction is decreased, the rare arginine codon AGG has been shown to be efficient for the insertion of Ala by a synthetic tRNA acylated with alanine. See, for example, Ma et al., Biochemistry, 32:7939 (1993). In this case, the synthetic tRNA competes with the naturally occurring tRNAArg, which is present as a minor species in Escherichia coli. Some organisms do not use all of the triplet codons. The unassigned codon AGA in Micrococcus luteus has been utilized for amino acid insertion in an in vitro transcription / translation extract. See, for example, Kowal and Oliver, Nucl. Acid. Res., 25:4685 (1997). The components of the present invention can be generated for use of these rare codons in vivo.

[0311] The selector codons also include, but are not limited to, extension codons that include four or more base codons, such as four, five, six or more base codons. Examples of four-base codons include, but are not limited to, AGGA, CUAG, UAGA, CCCU, etc. Examples of five-base codons include, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC, etc. A feature of the present invention includes using extension codons based on frameshift suppression. Four or more base codons, including but not limited to one or more non-natural amino acids, can be inserted into the same protein. For example, in the presence of a mutant O-tRNA, including but not limited to a special frameshift suppressor tRNA having an anticodon loop, such as an anticodon loop of at least 8-10 nt, four or more base codons are read as a single amino acid. In other embodiments, anticodon loops including, but not limited to, at least four-base codons, at least five-base codons, or at least six or more base codons can be decoded. Since there are 256 possible four-base codons, multiple non-natural amino acids can be encoded in the same cell using four or more base codons. See Anderson et al., (2002) Exploring the Limits of Codon and Anticodon Size, Chemistry and Biology, 9:237-244, Magliery, (2001) Expanding the Genetic Code: Selection of Efficient Suppressors of Four-base Codons and Identification of “Shifty” Four-base Codons with a Library Approach in Escherichia coli, J. Mol. Biol. 307:755-769.

[0312] For example, four-base codons have been used to incorporate unnatural amino acids into proteins using in vitro biosynthetic methods. See, for example, Ma et al., (1993) Biochemistry, 32:7939, and Hohsaka et al., (1999) J. Am. Chem. Soc., 121:34. Using CGGG and AGGU, two chemically acylated frameshift suppressor tRNAs were used to simultaneously incorporate 2-naphthylalanine and an NBD derivative of lysine into streptavidin in vitro. See, for example, Hohsaka et al., (1999) J. Am. Chem. Soc., 121:12194. In in vivo studies, Moore et al. examined the ability of a tRNALeu derivative with an NCUA anticodon to suppress UAGN codons (where N can be U, A, G, or C) and found that quadruple UAGA has little decoding in the 0 or -1 frame and can be decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13-26%. See Moore et al., (2000) J. Mol. Biol., 298:195. In one embodiment, elongation codons based on rare codons or nonsense codons can be used in the present invention, which can reduce missense readthrough and frameshift suppression at other unwanted sites.

[0313] For a given system, the selector codon can also include one of the natural three-base codons, and the endogenous system does not use (or hardly uses) natural base codons. For example, this includes systems lacking tRNAs that recognize natural three-base codons and / or systems where the three-base codon is a rare codon.

[0314] The selector codons optionally include unnatural base pairs. These unnatural base pairs further expand the existing genetic alphabet. One extra base pair increases the number of triplet codons from 64 to 125. Characteristics of the third base pair include stable selective base pairing, efficient enzymatic incorporation into DNA with high fidelity by polymerase, and efficient continued primer extension after synthesis of the nascent unnatural base pair. Descriptions of unnatural base pairs that can be adapted to methods and compositions include, for example, Hirao, et al., (2002) An unnatural base pair for incorporating amino acid analogues into protein, Nature Biotechnology, 20:177-182. See also Wu, Y., et al., (2002) J. Am. Chem. Soc. 124:14626-14630. Other related publications are listed below.

[0315] For in vivo use, the unnatural nucleosides are membrane permeable and phosphorylated to form the corresponding triphosphates. In addition, the increased genetic information is stable and not destroyed by cellular enzymes. Previous attempts by Benner et al. utilize hydrogen bonding patterns different from those of the canonical Watson-Crick pairs, the most notable example being the iso-C:iso-G pair. See, for example, Switzer et al., (1989) J. Am. Chem. Soc., 111:8322 and Piccirilli et al., (1990) Nature, 343:33, Kool, (2000) Curr. Opin. Chem. Biol., 4:602. These bases generally mispair to some extent with natural bases and cannot be replicated by enzymes. Kool and colleagues have shown that hydrophobic packing interactions between bases can drive base pair formation by replacing hydrogen bonds. See Kool, (2000) Curr. Opin. Chem. Biol., 4:602 and Guckian and Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. For the purpose of developing unnatural base pairs that meet all of the above requirements, Schultz, Romesberg, and colleagues systematically synthesized and studied a series of unnatural hydrophobic bases. PICS:PICS self-pairs have been found to be more stable than natural base pairs and can be efficiently incorporated into DNA by the Klenow fragment of Escherichia coli DNA polymerase I (KF). See, for example, McMinn et al., (1999) J. Am. Chem. Soc., 121:11585-6 and Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274. 3MN:3MN self-pairs can be synthesized by KF with sufficient efficiency and selectivity for biological function. See, for example, Ogawa et al., (2000) J. Am. Chem. Soc., 122:8803. However, both bases act as chain terminators for further replication. A mutant DNA polymerase that can be used to replicate PICS self-pairs has recently been devised. In addition, 7AI self-pairs can be replicated.For example, see Tae et al., (2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py, which forms a stable pair when bound to Cu(II), has also been developed. See Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Since the extended codons and non-natural codons are essentially orthogonal to the natural codons, the method of the present invention can utilize this property to generate orthogonal tRNAs for them.

[0316] Non-natural amino acids can also be incorporated into a desired polypeptide using a translational bypass system. In a translational bypass system, a large sequence is incorporated into the gene but not translated into the protein. This sequence contains a structure that functions as a cue to induce the ribosome to skip the sequence and resume translation downstream of the insertion.

[0317] Nucleic acid molecules encoding proteins of interest, such as a TC-targeted polypeptide, can be readily mutated to introduce cysteine at any desired position of the polypeptide. Cysteine is widely used to introduce reactive molecules, water-soluble polymers, proteins, or a wide variety of other molecules onto the protein of interest. Suitable methods for incorporating cysteine at a desired position of a polypeptide are those described in U.S. Patent No. 6,608,183, which is incorporated herein by reference, and standard mutagenesis techniques known to those of skill in the art.

[0318] III. Non-Naturally Encoded Amino Acids A very wide variety of non-naturally encoded amino acids are suitable for use in the present invention. Any number of non-naturally encoded amino acids can be introduced into the TC. Generally, the introduced non-naturally encoded amino acids are substantially chemically inert with respect to the 20 common genetically encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). In some embodiments, the non-naturally encoded amino acids include side chain functional groups that react efficiently and selectively with functional groups (including, but not limited to, azide, ketone, aldehyde, and aminooxy groups) not found in the 20 common amino acids to form stable conjugates. For example, a targeted polypeptide of TC containing a non-naturally encoded amino acid containing an azide functional group can be reacted with a polymer (including, but not limited to, poly(ethylene glycol), or alternatively, a second polypeptide or linker containing an alkyne moiety) to form a stable conjugate resulting from the selective reaction of the azide and alkyne functional groups and form a Huisgen [3+2] cycloaddition product.

[0319] The general structure of an alpha-amino acid is shown as follows (Formula I):

[0320]

Chemical formula

[0321] Non-naturally encoded amino acids typically have any structure with the formula listed above, where the R group is any substituent other than those used in the 20 natural amino acids and may be suitable for use in the present invention. The non-naturally encoded amino acids of the present invention typically differ from natural amino acids only in the structure of the side chain, so non-naturally encoded amino acids form amide bonds in the same manner as formed within naturally occurring polypeptides, including but not limited to other amino acids, whether natural or non-naturally encoded. However, non-naturally encoded amino acids have side chain groups that distinguish them from natural amino acids. For example, R optionally includes alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynyl, ether, thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino groups, etc., or any combination thereof. Other non-naturally occurring amino acids of interest that may be suitable for use in the present invention include amino acids containing a photoactivatable crosslinker, spin-labeled amino acids, fluorescent amino acids, metal-binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that interact covalently or non-covalently with other molecules, photo-caged and / or photo-isomerizable amino acids, amino acids containing biotin or biotin analogs, glycosylated amino acids such as sugar-substituted serine, other carbohydrate-modified amino acids, keto-containing amino acids, amino acids containing polyethylene glycol or polyether, heavy atom-substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids having an elongated side chain compared to natural amino acids (including but not limited to those containing more than about 5 or more than about 10 carbons, including but not limited to polyethers or long-chain hydrocarbons), carbon-linked sugar-containing amino acids, redox-active amino acids, amino acid-containing amino acids, and amino acids containing one or more toxin moieties, but are not limited thereto.

[0322] Exemplary non-naturally encoded amino acids that may be suitable for use in the present invention and are useful for reaction with water-soluble polymers include, but are not limited to, those having carbonyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide, and alkyne reactive groups. In some embodiments, the non-naturally encoded amino acid includes a sugar moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine. Examples of such amino acids also include those in which the naturally occurring N- or O-linkage between the amino acid and the sugar is replaced by a covalent bond that is not generally found in nature, including but not limited to alkenes, oximes, thioethers, amides, etc. Examples of such amino acids also include saccharides not generally found in naturally occurring proteins, such as 2-deoxy-glucose, 2-deoxygalactose, etc.

[0323] Many of the unnatural encoded amino acids provided in this specification are commercially available, for example, from Sigma-Aldrich (St. Louis, MO, USA), Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), or Peptech (Burlington, MA, USA). Those that are not commercially available are optionally synthesized as provided herein or using standard methods known to those skilled in the art. For organic synthesis techniques, see, for example, Organic Chemistry by Fessendon and Fessendon, (1982, Second Edition, Willard Grant Press, Boston Mass.), Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York), and Advanced Organic Chemistry by Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York). See also U.S. Patent Nos. 7,045,337 and 7,083,970, which are incorporated herein by reference. In addition to unnatural amino acids containing novel side chains, unnatural amino acids that may be suitable for use in the present invention optionally have the structures of Formulas II and III:

[0324]

Chemical formula

[0325]

Chemical formula

[0326] (wherein Z typically includes OH, NH2, SH, NH-R’, or S-R’, X and Y may be the same or different, typically include S or O, and R and R’ may optionally be the same or different and are typically selected from the same list of components of the R group described above for unnatural amino acids having Formula I and hydrogen) includes, but is not limited to, modified backbone structures including these. For example, the unnatural amino acids of the present invention optionally include substitutions on the amino or carboxyl groups as shown by Formulas II and III. This type of unnatural amino acid includes, but is not limited to, α-hydroxy acids, α-thio acids, α-aminothiocarboxylates having side chains corresponding to the common 20 natural amino acids or unnatural side chains. In addition, substitutions at the α-carbon optionally include, but are not limited to, α-α-disubstituted amino acids such as L, D, or D-glutamate, D-alanine, D-methyl-O-tyrosine, and aminobutyric acid. Other structural alternatives include cyclic amino acids such as proline analogs, and β and γ amino acids such as 3, 4, 6, 7, 8, and 9-membered ring proline analogs, substituted β-alanine, and γ-aminobutyric acid.

[0327] Many unnatural amino acids are based on natural amino acids such as tyrosine, glutamine, phenylalanine, etc. and are suitable for use in the present invention. Examples of tyrosine analogs include, but are not limited to, para-substituted tyrosine, ortho-substituted tyrosine, and meta-substituted tyrosine, where the substituted tyrosine has a keto group (including, but not limited to, an acetyl group), a benzoyl group, an amino group, hydrazine, hydroxylamine, a thiol group, a carboxyl group, an isopropyl group, a methyl group, C6-C 20It includes, but is not limited to, linear or branched hydrocarbons, saturated or unsaturated hydrocarbons, O-methyl groups, polyether groups, nitro groups, alkynyl groups, etc. In addition, multiple substituted aryl rings are also contemplated. Glutamine analogs that may be suitable for use in the present invention include, but are not limited to, α-hydroxy derivatives, γ-substituted derivatives, cyclic derivatives, and amide-substituted glutamine derivatives. Exemplary phenylalanine analogs that may be suitable for use in the present invention include, but are not limited to, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine, where the substituents include, but are not limited to, hydroxy groups, methoxy groups, methyl groups, allyl groups, aldehydes, azides, iodine, bromine, keto groups (including but not limited to acetyl groups), benzoyl, alkynyl groups, etc. Specific examples of unnatural amino acids that may be suitable for use in the present invention include p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine p-amino-L-phenylalanine, isopropyl-L-phenylalanine, and p-propargyloxy-phenylalanine, etc., but are not limited to these. Examples of the structures of various unnatural amino acids that may be suitable for use in the present invention are provided, for example, in WO2002 / 085923 entitled "In vivo incorporation of unnatural amino acids".For additional methionine analogs, see also Kiick et al., (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation, PNAS 99:19-24, which is incorporated herein by reference. The international application No. PCT / US06 / 47822 entitled "Compositions Containing, Methods Involving, and Uses of Non-natural Amino Acids and Polypeptides", which is incorporated herein by reference, describes, among other things, the reductive alkylation of aromatic amine moieties including p-amino-phenylalanine and reductive amination.

[0328] In another embodiment of the invention, the TC polypeptide having one or more non-naturally encoded amino acids is covalently modified. Selective chemical reactions that are orthogonal to a variety of functional groups in biological systems are recognized as important tools in chemical biology. As a relatively new addition to the synthetic chemistry repertoire, these bioorthogonal reactions have stimulated new strategies for compound library synthesis, protein engineering, functional proteomics, and chemical remodeling of the cell surface. Azides have secured an important role as unique chemical handles for bioconjugates. The Staudinger ligation has been used with phosphines to tag azide sugars introduced metabolically into cellular glycoconjugates. The Staudinger ligation can be carried out in living animals without physiological harm. Nevertheless, the Staudinger reaction is not without disadvantages. The required phosphines are prone to air oxidation, and their optimization for improved water solubility and increased reaction rates has proven synthetically difficult.

[0329] The azide group has an alternative mode of bioorthogonal reactivity, the [3+2] cycloaddition with alkynes described by Huisgen. In its classical form, this reaction is limited in its applicability to biological systems due to the requirement for high temperature (or pressure) for reasonable reaction rates. Sharpless and colleagues overcame this obstacle by developing a copper(I)-catalyzed version called "click chemistry" that proceeds readily at physiological temperature and in a sufficiently functionalized biological environment. This discovery enabled the selective modification of virus particles, nucleic acids, and proteins from complex tissue lysates. Unfortunately, the essential copper catalyst is toxic to both bacterial and mammalian cells and thus precludes applications where cells must survive. Catalyst-free Huisgen cycloadditions of alkynes activated by electron-withdrawing substituents have been reported to occur at ambient temperature. However, these compounds undergo Michael reactions with biological nucleophiles.

[0330] In one embodiment, provided is a composition of a targeted polypeptide of a TC comprising a non-natural amino acid such as p-(propargyloxy)-phenylalanine. Also provided are various compositions including, but not limited to, proteins and / or cells, comprising p-(propargyloxy)-phenylalanine. In one aspect, a composition comprising the p-(propargyloxy)-phenylalanine non-natural amino acid further comprises an orthogonal tRNA. The non-natural amino acid can be bound (including, but not limited to, covalently) to the orthogonal tRNA, including covalent binding to the orthogonal tRNA through an amino-acyl bond, covalent binding to the 3'OH or 2'OH of the terminal ribose sugar of the orthogonal tRNA, and the like.

[0331] Chemical moieties via non-natural amino acids that can be incorporated into proteins provide various advantages and manipulations of the proteins. For example, the inherent reactivity of keto functional groups enables selective modification of proteins using any of several hydrazine or hydroxylamine-containing reagents, both in vitro and in vivo. Heavy atom non-natural amino acids can be useful, for example, for phasing X-ray structural data. Site-specific introduction of heavy atoms using non-natural amino acids also provides selectivity and flexibility in choosing the position of the heavy atoms. Photoreactive non-natural amino acids (including but not limited to amino acids having benzophenone and aryl azide (including but not limited to phenyl azide) side chains) enable, for example, efficient photocrosslinking of proteins both in vivo and in vitro. Examples of photoreactive non-natural amino acids include, but are not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine. A protein having a photoreactive non-natural amino acid can then be optionally crosslinked by excitation of the photoreactive group, providing temporal control. In one embodiment, the methyl group of the non-natural amino can be replaced with an isotope-labeled one, including but not limited to, the use of nuclear magnetic resonance and vibrational spectroscopy, as a probe of local structure and dynamics. For example, alkynyl or azide functional groups enable selective modification of proteins by molecules through [3+2] cycloaddition reactions.

[0332] A non-natural amino acid incorporated into the amino-terminal polypeptide may be composed of an R group that is any substituent other than the substituents used for the 20 natural amino acids, and a second reactive group different from the NH2 group normally present in alpha-amino acids. Similar non-natural amino acids can be incorporated at the C-terminus having a second reactive group different from the COOH group normally present in alpha-amino acids.

[0333] The non-natural amino acids of the present invention can be selected or designed to provide additional features not available with the 20 natural amino acids. For example, non-natural amino acids can be optionally designed or selected, for example, to modify the biological properties of the proteins into which they are incorporated. For example, the following properties can be optionally modified by including non-natural amino acids in proteins: toxicity, biodistribution, solubility, stability, for example, resistance to heat, hydrolysis, oxidation, enzymatic degradation, etc., ease of purification and processing, structural properties, spectroscopic properties, chemical and / or photochemical properties, catalytic activity, redox potential, half-life, the ability to react with other molecules, for example, covalent or non-covalent, etc.

[0334] In some embodiments, the present invention provides TC linked to a water-soluble polymer, such as PEG, by an oxime bond. Many types of non-naturally encoded amino acids are suitable for the formation of oxime bonds. These include, but are not limited to, non-naturally encoded amino acids containing a carbonyl, dicarbonyl, or hydroxylamine group. Such amino acids are described in U.S. Patent Publication Nos. 2006 / 0194256, 2006 / 0217532, and 2006 / 0217289, and WO2006 / 069246 entitled "Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides", which are hereby incorporated by reference in their entirety. Non-naturally encoded amino acids are also described in U.S. Patent Nos. 7,083,970 and 7,045,337, which are hereby incorporated by reference in their entirety.

[0335] Some embodiments of the present invention utilize a TC polypeptide substituted at one or more positions with para-acetylphenylalanine amino acids. The synthesis of p-acetyl-(+ / -)-phenylalanine and m-acetyl-(+ / -)-phenylalanine is described in Zhang, Z., et al., Biochemistry 42:6735-6746 (2003) (incorporated by reference). Other carbonyl or dicarbonyl-containing amino acids can be similarly prepared by those skilled in the art. Further, non-limiting exemplary syntheses of non-natural amino acids included herein are presented in U.S. Patent No. 7,083,970 (incorporated herein by reference in its entirety).

[0336] Amino acids having an electrophilic reactive group enable various reactions for linking molecules, particularly via nucleophilic addition reactions. Such electrophilic reactive groups include a carbonyl group (including a keto group and a dicarbonyl group), a carbonyl-like group (having reactivity similar to a carbonyl group (including a keto group and a dicarbonyl group) and being structurally similar to a carbonyl group), a masked carbonyl group (which can be easily converted to a carbonyl group (including a keto group and a dicarbonyl group)), or a protected carbonyl group (having reactivity similar to a carbonyl group (including a keto group and a dicarbonyl group) upon deprotection). Such amino acids include amino acids having the structure of formula (IV):

[0337]

Chemical formula

[0338] 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, aralkylen, or substituted aralkylen, B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(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) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) k N(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’)-(each R’ is independently H, alkyl, or substituted alkyl) and is a linker selected from the group consisting of J is

[0339]

Chemical formula

[0340] and is R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, each R” is independently H, alkyl, substituted alkyl, or a protecting group, or when two or more R” groups are present, two R”s optionally form a heterocycloalkyl, R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, R2 is optional, and when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, Each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4, or two R3 groups, optionally form cycloalkyl or heterocycloalkyl, or the -A-B-J-R group together forms a bicyclic or tricyclic cycloalkyl or heterocycloalkyl containing at least one carbonyl group containing a dicarbonyl group, a protected carbonyl group containing a protected dicarbonyl group, or a masked carbonyl group containing a masked dicarbonyl group, or the -J-R group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl containing at least one carbonyl group containing a dicarbonyl group, a protected carbonyl group containing a protected dicarbonyl group, or a masked carbonyl group containing a masked dicarbonyl group, provided that when A is phenylene and each R3 is H, B is present; when A is -(CH2)4- and each R3 is H, B is not -NHC(O)(CH2CH2)-; and when A and B are absent and each R3 is H, R is not methyl.

[0341] In addition, those having the structure of formula (V) are included:

[0342]

Chemical formula

[0343] 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 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) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) k N(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’)-(each R’ is independently H, alkyl, or substituted alkyl) and is a linker selected from the group consisting of R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, R2 is optional, and when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, provided that when A is phenylene, B is present; when A is -(CH2)4-, B is not -NHC(O)(CH2CH2)-; and when A and B are absent, R is not methyl.

[0344] In addition, an amino acid having the structure of formula (VI) is included:

[0345]

Chemical formula

[0346] wherein B is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) ka linker selected from the group consisting of -N(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')- (each R' is independently H, alkyl, or substituted alkyl), R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, R2 is optional, and when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, each R a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) k R' (each R' is independently H, alkyl, or substituted alkyl).

[0347] In addition, the following amino acids are included:

[0348]

Chemical formula

[0349] (wherein such compounds are optionally an amino protecting group, carboxyl protected, or a salt thereof). In addition, any of the following non-natural amino acids can be incorporated into the non-natural amino acid polypeptide.

[0350] In addition, the following amino acids having the structure of formula (VII) are included:

[0351]

Chemical formula

[0352] In the formula, B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(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) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) k N(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’)-(each R’ is independently H, alkyl, or substituted alkyl), and is a linker selected from the group consisting of R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, Each R a is independently 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’ (each R’ is independently selected from the group consisting of H, alkyl, or substituted alkyl), and n is from 0 to 8, provided that when A is -(CH2)4-, B is not -NHC(O)(CH2CH2)-.

[0353] In addition, the following amino acids are included:

[0354]

Chemical formula

[0355] (wherein such a compound is optionally amino-protected, optionally carboxyl-protected, optionally amino- and carboxyl-protected, or a salt thereof). In addition, any of these non-natural amino acids and the following non-natural amino acids can be incorporated into the non-natural amino acid polypeptide.

[0356] In addition, the following amino acids having the structure of formula (VIII) are included:

[0357]

Chemical formula

[0358] 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, aralkylen, or substituted aralkylen, B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(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) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) k a linker selected from the group consisting of -N(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-(each R’ is independently H, alkyl, or substituted alkyl), R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0359] In addition, the following amino acids having the structure of formula (IX) are included:

[0360]

Chemical formula

[0361] B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(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) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) k N(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’)-(each R’ is independently H, alkyl, or substituted alkyl) and is a linker selected from the group consisting of R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, each R a is independently 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) kEach R’ is independently selected from the group consisting of H, alkyl, or substituted alkyl.

[0362] In addition, the following amino acids are included:

[0363]

Chemical formula

[0364] (wherein such a compound is optionally amino-protected, optionally carboxyl-protected, optionally amino- and carboxyl-protected, or a salt thereof). In addition, any of these non-natural amino acids and the following non-natural amino acids can be incorporated into a non-natural amino acid polypeptide.

[0365] In addition, the following amino acids having the structure of formula (X) are included:

[0366]

Chemical formula

[0367] wherein B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(k is 1, 2, or 3), -S(O) k(Alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) k A linker selected from the group consisting of -N(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)- (each R’ is independently H, alkyl, or substituted alkyl), R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, Each R a is independently H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O) k R’ (k is 1, 2, or 3), -C(O)N(R’)2, -OR’, and -S(O) k R’ (each R’ is independently H, alkyl, or substituted alkyl), and n is from 0 to 8.

[0368] In addition, the following amino acids are included:

[0369]

Chemical formula

[0370] (wherein such a compound is optionally amino-protected, optionally carboxyl-protected, optionally amino- and carboxyl-protected, or a salt thereof). In addition, any of these non-natural amino acids and the following non-natural amino acids can be incorporated into the non-natural amino acid polypeptide.

[0371] In addition to the monocarbonyl structure, the non-natural amino acids described herein may include groups such as dicarbonyl, dicarbonyl-like, masked dicarbonyl, and protected dicarbonyl groups.

[0372] For example, the following amino acids having the structure of formula (XI) are included:

[0373]

Chemical formula

[0374] 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, aralkylen e, or substituted aralkylene, B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(k is 1, 2, or 3), -S(O) k(Alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) k A linker selected from the group consisting of -N(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)- (each R’ is independently H, alkyl, or substituted alkyl), R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, R2 is optional, and when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0375] In addition, the following amino acids having the structure of formula (XII) are included:

[0376]

Chemical formula

[0377] B is optional, and when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k-(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) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) k a linker selected from the group consisting of -N(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)- (each R’ is independently H, alkyl, or substituted alkyl), R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, R2 is optional, and when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, each R a is 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) k R’ (each R’ is independently H, alkyl, or substituted alkyl).

[0378] In addition, the following amino acids are included:

[0379]

Chemical formula

[0380] (wherein such a compound is optionally amino-protected, optionally carboxyl-protected, optionally amino- and carboxyl-protected, or a salt thereof). In addition, any of these non-natural amino acids and the following non-natural amino acids can be incorporated into the non-natural amino acid polypeptide.)

[0381] In addition, the following amino acids having the structure of formula (XIII) are included:

[0382]

Chemical formula

[0383] wherein B is optional, and when present, 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 -(k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) ka linker selected from the group consisting of -N(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')- (each R' is independently H, alkyl, or substituted alkyl); R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional, and when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; each R a is 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) k R' (each R' is independently H, alkyl, or substituted alkyl), and n is from 0 to 8.

[0384] In addition, the following amino acids are included:

[0385]

Chemical formula

[0386] (wherein such compounds are optionally amino-protected, optionally carboxyl-protected, optionally amino- and carboxyl-protected, or salts thereof). In addition, any of these non-natural amino acids and the following non-natural amino acids can be incorporated into non-natural amino acid polypeptides.

[0387] In addition, the following amino acids having the structure of formula (XIV) are included:

[0388]

Chemical formula

[0389] In the formula, A is arbitrary, and when present, it 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, aralkylen, or substituted aralkylen, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is arbitrary, and when present, it is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, R2 is arbitrary, and when present, it is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, X1 is C, S, or S(O), and L is alkylene, substituted alkylene, N(R’)(alkylene), or N(R’)(substituted alkylene), where R’ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0390] In addition, the following amino acids having the structure of formula (XIV-A) are included:

[0391]

Chemical formula

[0392] In the formula, 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, aralkylen, or substituted aralkylen, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, L is alkylene, substituted alkylene, N(R’)(alkylene), or N(R’)(substituted alkylene), where R’ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0393] In addition, the following amino acids having the structure of formula (XIV-B) are included:

[0394]

Chemical formula

[0395] In the formula, 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, aralkylen, or substituted aralkylen, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, L is alkylene, substituted alkylene, N(R’)(alkylene), or N(R’)(substituted alkylene), where R’ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0396] In addition, the following amino acids having the structure of formula (XV) are included:

[0397]

Chemical formula

[0398] In the formula, 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, aralkylen e, or substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, X1 is C, S, or S(O), n is 0, 1, 2, 3, 4, or 5, and each CR 8 R 9 on each R 8 and R 9is independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 and R 9 can together form =O or cycloalkyl, or any adjacent R 8 groups can together form cycloalkyl.

[0399] In addition, the following amino acids having the structure of formula (XV-A) are included:

[0400]

Chemical formula

[0401] 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, aralkylen, or substituted aralkylen, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, R2 is optional, and when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, n is 0, 1, 2, 3, 4, or 5, and each R 8 R 9 on each CR 8 and R 9 is independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 and R 9both can form =O or cycloalkyl, or any adjacent R 8 groups together can form cycloalkyl.

[0402] In addition, the following amino acids having the structure of formula (XV - B) are included:

[0403]

Chemical formula

[0404] 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, aralkylen, or substituted aralkylen, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, R2 is optional, and when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, n is 0, 1, 2, 3, 4, or 5, and each CR 8 R 9 on each R 8 and R 9 is independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 and R 9 both can form =O or cycloalkyl, or any adjacent R 8 groups together can form cycloalkyl.

[0405] In addition, the following amino acids having the structure of formula (XVI) are included:

[0406] [Chemical formula]

[0407] 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, aralkylen, or substituted aralkylen, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is optional and, when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, R2 is optional and, when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, X1 is C, S, or S(O), and L is alkylene, substituted alkylene, N(R’)(alkylene), or N(R’)(substituted alkylene), where R’ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0408] In addition, the following amino acids having the structure of formula (XVI-A) are included:

[0409] [Chemical formula]

[0410] 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, aralkylen, or substituted aralkylen, R is H, alkyl, substituted alkyl, cyclo...

Claims

1. A composition comprising one or more targeted polypeptides having one or more incorporated non-natural amino acids, wherein at least one of said polypeptides is linked to a TLR agonist molecule via a linker covalently attached to said non-natural amino acid of said targeted polypeptide, said linker being a bond, The TLR agonist is a TLR agonist comprising the following structure, or a salt thereof, the composition. 【Chemical Formula 1】

2. (i) the one or more targeted polypeptides are the same targeted polypeptide, or (ii) the one or more targeted polypeptides bind to a cell surface target or a tumor cell target, or (iii) the one or more targeted polypeptides are monospecific targeted polypeptides, the composition according to claim 1.

3. The composition according to claim 1, wherein the one or more targeted polypeptides are bispecific or multispecific targeted polypeptides.

4. The composition according to claim 2 or 3, wherein the monospecific, bispecific or multispecific targeted polypeptide comprises a drug conjugate or a checkpoint inhibitor.

5. The composition according to claim 1, wherein the one or more targeted polypeptides comprise an antibody or an antibody fragment.

6. The one or more targeted polypeptides are an antibody or an antibody fragment that binds to an antigen of a cell, or The composition according to claim 5, wherein the one or more targeted polypeptides are antibodies or antibody fragments that bind to a target selected from the group consisting of HER2, HER3, PD-1, PDL-1, EGFR, TROP2, PSMA, VEGFR, CTLA-4, EpCAM, MUC1, MUC16, c-met, GPC3, ENPP3, TIM-1, FOLR1, STEAP1, mesothelin, 5T4, CEA, CA9, cadherin 6, ROR1, SLC34A2, SLC39A6, SLC44A4, LY6E, DLL3, ePhA2, GPNMB, SLITRK6, CD3, CD19, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD47, CD52, CD56, CD70, CD96, CD97, CD99, CD117, CD123, CD179, CD223, and CD276.

7. The composition according to claim 6, wherein the one or more targeted polypeptides comprise an antibody or antibody fragment that binds to HER2.

8. The composition according to claim 7, wherein the one or more targeted polypeptides is trastuzumab.

9. (i) the antibody or antibody fragment comprises at least one amino acid sequence of SEQ ID NOs: 1-13; (ii) the antibody or antibody fragment comprises at least two amino acid sequences of SEQ ID NOs: 1-13; (iii) the antibody or antibody fragment comprises a) SEQ ID NO: 1 or 2, and b) any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13; or (iv) the antibody or antibody fragment comprises a) SEQ ID NO: 2, and b) any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13; The composition according to claim 7.

10. The composition according to claim 7, wherein the antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO:

3.

11. The antibody or antibody fragment is IgG, Fab, (Fab’)2, Fv, or single-chain Fv (scFv), one or more Fab, (Fab’)2, Fv, or single-chain Fv (scFv) mutations, one or more Fc mutations, or, one to six Fc mutations, The composition according to claim 5, comprising

12. The composition according to claim 5, wherein the antibody or antibody fragment comprises a heavy chain and / or a light chain, and the one or more non-natural amino acids are incorporated into the heavy chain, the light chain, or both the heavy chain and the light chain.

13. The one or more targeted polypeptides include one or more non-natural amino acids selected from the group consisting of para-acetylphenylalanine, p-nitrophenylalanine, p-sulfotyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-boronylphenylalanine, o-boronylphenylalanine, m-boronylphenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-OMe phenylalanine, o-OMe phenylalanine, m-OMe phenylalanine, p-sulfophenylalanine, o-sulfophenylalanine, m-sulfophenylalanine, 5-nitroHis, 3-nitroTyr, 2-nitroTyr, nitro-substituted Leu, nitro-substituted His, nitro-substituted De, nitro-substituted Trp, 2-nitroTrp, 4-nitroTrp, 5-nitroTrp, 6-nitroTrp, 7-nitroTrp, 3-aminotyrosine, 2-aminotyrosine, O-sulfotyrosine, 2-sulfooxyphenylalanine, 3-sulfooxyphenylalanine, o-carboxyphenylalanine, m-carboxyphenylalanine, p-acetyl-L-phenylalanine, p-propargyl-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propargyloxy-L-phenylalanine, 4-azido-L-phenylalanine, para-azidoethoxyphenylalanine, and para-azidomethyl-phenylalanine, Optionally, the unnatural amino acid is selected from the group consisting of p - propargyloxy - L - phenylalanine, para - acetyl - phenylalanine, 4 - azido - L - phenylalanine, para - azidoethoxyphenylalanine, or para - azidomethyl - phenylalanine, and / or, Optionally, the unnatural amino acid is site - specifically incorporated into the one or more targeted polypeptides, the composition according to claim 1.

14. The composition according to claim 13, wherein the unnatural amino acid is para - acetylphenylalanine.

15. The composition according to claim 1, wherein the one or more targeted polypeptides are conjugated to one or more polymers or biologically active molecules.

16. (i) the one or more polymers are linear, (ii) the one or more polymers are bifunctional or polyfunctional polymers, or, (iii) the one or more polymers are water - soluble polymers, the composition according to claim 15.

17. The one or more polymers are water - soluble polymers, and the water - soluble polymer is polyethylene glycol (PEG), Optionally, the PEG has a molecular weight of 0.1 kDa to 100 kDa, or a molecular weight of 0.1 kDa to 50 kDa, the composition according to claim 16.

18. The at least one polymer or biologically active molecule is linked to at least one unnatural amino acid, the composition according to claim 15.

19. The unnatural amino acid is reactive with respect to a polymer or biologically active molecule that would otherwise be non - reactive with respect to any of the 20 common amino acids within the polypeptide, Optionally, the unnatural amino acid contains a carbonyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group, Optionally, the unnatural amino acid contains a carbonyl group, the composition according to claim 1.

20. The composition according to claim 1, wherein the one or more targeted polypeptides are linked to a cytotoxic agent or an immunostimulatory agent.

21. A TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist, wherein the TLR agonist is covalently bound via a linker to one or more unnatural amino acids incorporated into the anti-HER2 antibody or antibody fragment, and the linker is a bond, The TC, wherein the TLR agonist is a TLR agonist having the following structure, or a salt thereof. 【Chemical Formula 2】

22. The anti-HER2 antibody or antibody fragment comprises a heavy chain and / or a light chain, and the one or more unnatural amino acids are incorporated into the heavy chain, the light chain, or both the heavy chain and the light chain, Optionally, the anti-HER2 antibody or antibody fragment comprises an Fc region, and the anti-HER2 antibody or antibody fragment further comprises one or more mutations in the Fc region, the TC according to claim 21.

23. The one or more non-natural amino acids are selected from the group consisting of para-acetylphenylalanine, p-nitrophenylalanine, p-sulfotyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-boronylphenylalanine, o-boronylphenylalanine, m-boronylphenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-OMe phenylalanine, o-OMe phenylalanine, m-OMe phenylalanine, p-sulfophenylalanine, o-sulfophenylalanine, m-sulfophenylalanine, 5-nitro His, 3-nitro Tyr, 2-nitro Tyr, nitro-substituted Leu, nitro-substituted His, nitro-substituted De, nitro-substituted Trp, 2-nitro Trp, 4-nitro Trp, 5-nitro Trp, 6-nitro Trp, 7-nitro Trp, 3-aminotyrosine, 2-aminotyrosine, O-sulfotyrosine, 2-sulfooxyphenylalanine, 3-sulfooxyphenylalanine, o-carboxyphenylalanine, m-carboxyphenylalanine, p-acetyl-L-phenylalanine, p-propargyl-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propargyloxy-L-phenylalanine, 4-azido-L-phenylalanine, para-azidoethoxyphenylalanine, and para-azidomethyl-phenylalanine, the TC according to claim 21.

24. The TC according to claim 23, wherein the unnatural amino acid is p - propargyloxy - L - phenylalanine, para - acetyl - phenylalanine, 4 - azido - L - phenylalanine, para - azidomethyl - phenylalanine, or para - azidoethoxyphenylalanine.

25. The TC according to claim 24, wherein the unnatural amino acid is para - acetyl - phenylalanine.

26. (i) The anti - HER2 antibody or antibody fragment contains at least one amino acid sequence among SEQ ID NOs: 1 to 13, (ii) The anti - HER2 antibody or antibody fragment contains at least two amino acid sequences among SEQ ID NOs: 1 to 13, (iii) The anti - HER2 antibody or antibody fragment contains a) SEQ ID NO: 1 or 2, and b) any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13, (iv) The anti - HER2 antibody or antibody fragment contains a) SEQ ID NO: 2, and b) any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13, or (v) The TC according to any one of claims 21 to 25, wherein the anti - HER2 antibody or antibody fragment contains SEQ ID NO: 2 and SEQ ID NO:

3.

27. The TC according to claim 21, further comprising a chemotherapeutic agent or an immunotherapeutic agent.

28. The anti - HER2 antibody or antibody fragment contains SEQ ID NO: 2 and SEQ ID NO: 3, and The TC according to any one of claims 21 to 27, wherein the unnatural amino acid is para - acetyl - phenylalanine.

29. A pharmaceutical composition comprising a therapeutically effective amount of the composition according to any one of claims 1 to 20 or the TC according to any one of claims 21 to 28, and a pharmaceutically acceptable carrier or excipient.

30. Use of the composition according to any one of claims 1 to 20 or 29 in the manufacture of a medicament.

31. An immunostimulatory antibody conjugate (ISAC) comprising a TLR agonist conjugate (TC) according to any one of claims 21 to 28.

32. A compound having the following structure or a salt thereof. 【Formula 3】

33. A pharmaceutical composition comprising the compound according to claim 32 or a salt thereof, Optionally further comprising a pharmaceutically acceptable excipient.

34. Use of the compound according to claim 32 or a salt thereof in the manufacture of a medicament, or use of the pharmaceutical composition according to claim 33.

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