Compositions comprising antibody-TLR agonist conjugate, methods and use thereof

Abstract

To provide trastuzumab-conjugated TLR agonist derivative analogs containing at least one non-natural amino acid, as well as methods for preparing such non-natural amino acids and polypeptides.SOLUTION: The invention relates to targeting polypeptides having one or more non-naturally encoded amino acids conjugated to agonist compounds of TLRs including but not limited to TLR7 and / or TLR8. TLR-agonist conjugates of the present invention include targeting biological molecules or polypeptides and TLR agonist compounds conjugated together using non-naturally encoded amino acids by site-specific conjugation to produce novel biological TLR-agonist conjugates. The targeting biological molecules or polypeptides can be a tumor targeting biological molecules or polypeptides.SELECTED DRAWING: Figure 3

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 February 12, 2019, each entitled "Compositions Containing, Methods And Uses Of Antibody-TLR Agonist Conjugates," the contents of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application has been submitted in ASCII format via EFS-Web and contains a Sequence Listing, which is incorporated herein 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 present disclosure relates to TLR agonist compounds and TLR agonist conjugates (TCs) and their uses. The present invention further relates to pharmaceutical compositions containing the (TCs) therapeutically or prophylactically.

[0004] [Background technology] Targeting molecules or polypeptides, such as antibodies and fragments thereof, and TLR agonist compounds can be conjugated together using non-naturally encoded amino acids by site-specific conjugation to produce novel TLR-agonist conjugates (TCs). The novel TCs can be constructed in such a way that circulating TCs can target the TLR agonist to tumor sites during systemic treatment and stimulate a local beneficial immune response, thereby minimizing systemic cytokine release syndrome.

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

[0006] In a further embodiment, the present invention 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 tumor or cancer growth, comprising contacting a tumor with an effective amount of a TC of the present invention to stimulate the patient's immune system in proximity to the tumor. The present invention also provides a method for inhibiting or reducing tumor or cancer growth, comprising contacting a tumor with an effective amount of a PEGylated TC of the present invention, or a stable dimer or multimer of a 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. In one embodiment, the TC has two or more and / or the same TLR agonist molecules attached thereto. Another embodiment of the present invention provides a method for using the TC of the present invention to regulate an immune response to 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 may be selected from the group consisting of temozolomide, gemcitabine, doxorubicin, cyclophosphamide, paclitaxel, cisplatin, fluoropyrimidines, taxanes, anthracyclines, lapatinib, capecitabine, letrozole, pertuzumab, docetaxel, IFN-α. In another embodiment of the 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 a light chain and at least one CDR of a 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 (Fab'). In some embodiments, the ABP comprises two or more (Fab'). 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 light chain variable region and a heavy chain variable region. 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 portions thereof. In some embodiments, the ABP comprises any combination of the above embodiments. In some embodiments, the ABP comprises a homodimer, heterodimer, homomultimer, or heteromultimer of any 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 is associated 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 a key role in sensing invading pathogens and subsequently initiating innate immune responses. Ten members of the TLR family are known in humans. They 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 in 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 agonist binding to TLR7 or TLR8, the dimerized form of the receptor is thought to undergo a conformational change that leads to the subsequent recruitment of adaptor proteins in its cytoplasmic domain, including myeloid differentiation primary response gene 88 (MyD88). After initiation of 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 primarily expressed on plasmacytoid cells and B cells. Altered immune cell responsiveness may contribute to reduced innate immune responses in cancer patients. Therefore, agonist-induced activation of TLR7 and / or TLR8 conjugated to targeting moieties such as antibodies or their fragments may represent a novel approach for the treatment of cancer. Treatment with TLR7 or TLR8 agonists represents a promising solution, offering greater efficacy with better tolerability.Suitable TLR7 and / or TLR8 agonists for use in the present invention to generate TCs can be 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, and U.S. Patent No. 8,673,932.

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

[0011] In some embodiments, one or more engineered bonds are made with one or more unnatural amino acids. Intramolecular bonds can be made in a number of ways, including, but not limited to, reaction between two amino acids in a protein under suitable conditions (one or both amino acids can be unnatural amino acids), reaction with two amino acids (each of which can be naturally encoded or unnaturally encoded) with a linker, polymer, or other molecule under suitable conditions, etc.

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

[0013] In some embodiments, the non-naturally encoded amino acid comprises 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 comprises a carbonyl group. In some embodiments, the non-naturally encoded amino acid has the structure:

[0015] [ka]

[0016] where n is 0-10; R1 is alkyl, aryl, substituted alkyl, or substituted aryl; R2 is H, alkyl, aryl, substituted alkyl, and substituted aryl; R3 is H, an amino acid, a polypeptide, or an amino terminus modification group; and R4 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.

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

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

[0019] [ka]

[0020] where n is 0-10; R1 is alkyl, aryl, substituted alkyl, substituted aryl, or not present; X is O, N, S, or not present; m is 0-10; R2 is H, an amino acid, a polypeptide, or an amino terminus modification group; and R3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.

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

[0022] [ka]

[0023] where n is 0-10; R1 is alkyl, aryl, substituted alkyl, or substituted aryl; X is O, N, S, or absent; m is 0-10; R2 is H, an amino acid, a polypeptide, or an amino terminus modification group; and R3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.

[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 biologically active molecules.

[0025] The present invention also provides isolated nucleic acids comprising polynucleotides encoding targeting polypeptides of TCs, and the present invention provides isolated nucleic acids comprising polynucleotides that hybridize to polynucleotides under stringent conditions. The present invention also provides isolated nucleic acids comprising polynucleotides encoding targeting polypeptides, wherein the polynucleotides comprise at least one selector codon. It will be readily apparent to one of skill in the art that several different polynucleotides can encode any polypeptide of the present 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 methods for producing TC polypeptides linked to water-soluble polymers or to one or more TC polypeptides to form homodimers or homomultimers. In some embodiments, the methods involve contacting an isolated TC polypeptide comprising a non-naturally encoded amino acid with a water-soluble polymer or linker comprising a moiety reactive 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 unreactive 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 unreactive with any of the 20 common amino acids.

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

[0030] In some embodiments, a TC polypeptide linked to a water-soluble polymer or linker is made by reacting a TC containing an alkyne-containing amino acid with a poly(ethylene glycol) molecule containing 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, a TC polypeptide linked to a water-soluble polymer or linker is made by reacting a TC polypeptide containing an azide-containing amino acid with a poly(ethylene glycol) molecule containing 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 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, a hydrazine group, 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 compositions 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 a water soluble polymer.

[0034] The invention also provides cells comprising a polynucleotide encoding a targeting polypeptide of a 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 into the targeting polypeptide of the TC.

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

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

[0037] The present invention also provides methods 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 non-glycosylated.

[0038] The present invention also provides a TC comprising a water-soluble polymer or linker covalently linked 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 attached to the water-soluble polymer or linker is a non-naturally encoded amino acid present in a targeting polypeptide of the TC.

[0039] The present invention provides TC polypeptides 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 that is ribosomally incorporated into the targeting polypeptide of the TC. In TC conjugates, PEG or other water-soluble polymers, another TC, polypeptide, or biologically active molecule can be directly conjugated to the TC via a linker. In one embodiment, the linker is long enough to allow flexibility and enable dimer formation. In one embodiment, the linker is at least 3 amino acids or 18 atoms in length to allow dimer formation. In some embodiments, the polypeptide is linked to the linker to allow multimer formation. In some embodiments, the linker is a bifunctional linker. In some embodiments, the compositions and / or TCs of the present invention can include multiple linkers. In other embodiments, each linker can include one or more compounds attached thereto. Linkers can also include alkylene, alkenylene, alkynylene, polyether, polyester, polyamide group(s), as well as polyamino acids, polypeptides, cleavable peptides, or aminobenzyl carbamates. In some embodiments, the linkers can be the same or different linkers. 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 or endosomal proteases. Cleavable linkers can include valine-citrulline linkers or valine-alanine peptides. In some embodiments, the linker can be a dipeptide linker, such as a valine-citrulline or phenylalanine-lysine linker. The valine-citrulline or valine-alanine-containing linker can contain a maleimide or succinimide group. The valine-citrulline or valine-alanine containing linker may contain a para-aminobenzyl alcohol (PABA) group or a para-aminobenzyl carbamate (PABC).Other suitable linkers include linkers that are hydrolyzable at a pH below 5.5, such as hydrazone linkers. Further suitable cleavable linkers include disulfide linkers. In some embodiments, the cleavable linker may include a linker that is cleaved in the tumor microenvironment, such as by tumor-infiltrating T cells. In some embodiments, the non-cleavable linker includes, but is not limited to, a maleimidocaproyl linker. The maleimidocaproyl linker may include, but is not limited to, N-maleimidomethylcyclohexane-1-carboxylate, a succinimide group, a pentafluorophenyl group, and / or one or more PEG molecules. In some embodiments, any one of the compositions, compounds, or salts thereof of the present invention may be linked to a polypeptide by a linker. In some embodiments, any one of the compounds disclosed herein, or salts thereof, in Tables 3, 4, 5, 6, and 7, may be linked to a polypeptide by a linker. In some embodiments, the polypeptide is a targeting polypeptide, a biological targeting polypeptide, or a tumor-targeting polypeptide. In some embodiments, the targeting polypeptide is an antibody or an antibody fragment.

[0040] In some embodiments, the TC polypeptide is monoPEGylated. The invention also provides a TC comprising a linker, polymer, or biologically active molecule attached to one or more non-naturally encoded amino acids, where the non-naturally encoded amino acids are ribosomally incorporated into the polypeptide at a preselected site.

[0041] In some embodiments, the invention provides compositions comprising one or more targeting 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-naturally encoded 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 polypeptide comprises a drug conjugate or a checkpoint inhibitor. Any suitable immune checkpoint inhibitor is contemplated for use with the compositions or TCs of the present invention. 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-inducible TNFR-related protein (GITR), inducible T-cell costimulatory (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, or 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 antibody fragment, hi other embodiments, the targeting polypeptide is an antibody or antibody fragment that binds to a cellular antigen. In another embodiment, the target polypeptide is an antibody or 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 antibody fragment that binds to HER2, hi another embodiment, the targeting polypeptide is trastuzumab.

[0045] In another embodiment, the antibody or antibody fragment comprises an 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 one to six 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 chain and the light chain. 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 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 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 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 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, the 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 further comprises at least six Fc mutations.

[0047] In another embodiment, the targeting polypeptide is 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, 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-sulfooxyphenyl Alanine, 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-GlcNAc β-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido p-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 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 targeting polypeptides.

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

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

[0050] In some embodiments, the one or more linkers are between 0.1 kDa and 50 kDa. In other embodiments, the one or more linkers are between 0.1 kDa and 10 kDa. In other embodiments, the one or more linkers or polymers are linear, branched, multimeric, or dendrimeric. In other embodiments, the one or more linkers or polymers are bifunctional or multifunctional linkers or bifunctional or multifunctional polymers.

[0051] In other embodiments, 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) molecule has a molecular weight of about 0.1 kDa to about 100 kDa. In some embodiments, the poly(ethylene glycol) molecule has a molecular weight of 0.1 kDa to 50 kDa. In some embodiments, the poly(ethylene glycol) has a molecular weight of 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) molecule is a branched PEG. In some embodiments, the poly(ethylene glycol) molecule is a branched 5K PEG. In some embodiments, the poly(ethylene glycol) molecule is a branched 10K PEG. In some embodiments, the poly(ethylene glycol) molecule is a branched 20K PEG. In some embodiments, the poly(ethylene glycol) molecule is a linear PEG. In some embodiments, the poly(ethylene glycol) molecule is a linear 5K PEG. In some embodiments, the poly(ethylene glycol) molecule is a linear 10K PEG. In some embodiments, the poly(ethylene glycol) molecule is a 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 an average molecular weight. In certain embodiments, the average molecular weight is a 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 PEG having a molecular weight of 0.1 kDa to 50 kDa. In other embodiments, the linker is PEG having a molecular weight of 0.1 kDa to 40 kDa. In other embodiments, the linker is PEG having a molecular weight of 0.1 kDa to 30 kDa. In other embodiments, the linker is PEG having a molecular weight of 0.1 kDa to 20 kDa. In other embodiments, the linker is PEG having a molecular weight of 0.1 kDa to 10 kDa. In other embodiments, the linker is PEG having a molecular weight of 0.1 kDa to 5 kDa.

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

[0054] In another embodiment, the non-naturally encoded amino acid is reactive to a linker, polymer, or biologically active molecule that is otherwise unreactive to any of the 20 common amino acids in polypeptides. 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 targeting polypeptide is linked to a cytotoxic agent or an immunostimulatory agent. In another embodiment, a TC or BTC of the invention is linked to a cytotoxic agent or an immunostimulatory agent. In another embodiment, the targeting polypeptide comprises a cytotoxic agent or an immunostimulatory agent. In another embodiment, a TC or BTC of the invention comprises a cytotoxic agent or an immunostimulatory agent.

[0056] In another embodiment, the invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist comprising a structure set forth in any of the structures in Figure 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 dual TLR7 / TLR8 agonist. In another embodiment, the TLR agonist comprises a structure set forth in Structure 1 in Figure 1.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-7 11、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-726 C-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-759 760, 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] The 1,000-year-old TLR 》ストは、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、A XC-689、AXC-690、AXC-691、AXC-697、AXC-699、AXC-700、AXC-701、AX C-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-7 38、AXC-739、AXC-740、AXC-741、AXC-743、AXC-742、AXC-747、AXC-74 8、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、A XC-800、AXC-801、AXC-802、AXC-803、AXC-804、AXC-805、AXC-806、AX C-807, AXC-808, AXC-809, AXC-810, AXC-831, and AXC-910 TL R、AXC-801、AXC-802、AXC-8 31.AXC-910-AXC-80-AXC-800-AXC-80-AXC-AXC-80000000000202000 31、AXC-910 Ready-to-Wear 1.1. The TLR, the TLR, the Chinese, the Chinese, the Chinese.

[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, the light chain, or both the heavy and light chains. In another embodiment, the one or more non-naturally encoded amino acids are 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. Lanine, 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- It is selected from the group consisting of 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 multifunctional linkers.

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

[0062] In another embodiment, the TLR agonist comprises the structure set forth in Structure 3 in Figure 1. In another embodiment, the TLR agonist comprises the structure set forth in Structure 3, which is an AXC-837 or AXC-847 compound. In another embodiment, the TLR agonist comprises the structure set forth in an AXC-837 or AXC-847 compound, further comprising a linker. In another embodiment, the TLR agonist comprises the structure set forth in an AXC-847 compound, further comprising a linker. In another embodiment, the TLR agonist comprises the structure set forth in Structure 3, further comprising a linker.

[0063] In another embodiment, the TLR agonist comprises a structure set forth in Structure 4 in Figure 1. In another embodiment, the TLR agonist comprising a structure set forth 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 a structure set forth 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 a structure set forth in Structure 4 further comprising a linker.

[0064] In another embodiment, the TLR agonist comprises a structure set forth in structure 5 of Figure 1. In another embodiment, the TLR agonist comprising a structure set forth in structure 5 is selected from the group consisting 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-901, AXC-902, AXC-903, AXC-904, AXC-905, AXC-906, AXC-907, AXC-908, AXC-909, AXC-910, AXC-911, AXC-912, AXC-913, AXC-914, AXC-915, AXC-916, AXC-917, AXC-918, AXC-919, AXC-920, AXC-921, AXC-922, AXC-923, AXC-924, AXC-925, AXC-926, AXC-927, AXC-928, AXC-929, AXC-930, AXC-931, AXC-932, AXC-933, AXC-934, AXC-935, AXC-9 -889, AXC-890, AXC-891, AXC-892, AXC-893, AXC-895, AXC-896, AXC-897, AXC-898, AXC-901, AXC-903, AXC-904, AXC-9 05, 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 set forth 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, 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 In another embodiment, the TLR agonist comprises the structure set forth in any one of 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 comprising a linker. In another embodiment, the TLR agonist comprises the structure set forth in Structure 5, further comprising a linker.

[0065] In another embodiment, the anti-HER2 antibody or antibody fragment comprises at least one amino acid sequence of SEQ ID NOs: 1-13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises at least two amino acid sequences of SEQ ID NOs: 1-13. 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) a heavy chain of 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) 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 invention provides an anti-HER2 antibody or antibody fragment, wherein a non-naturally encoded amino acid is specifically incorporated at position 114 according to the Kabat numbering.

[0066] In another embodiment, the invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist comprising a structure set forth in any of the structures in Figure 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 or immunotherapeutic agent. In another embodiment, the 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 in 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 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 in 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 or immunotherapeutic agent.

[0067] In another embodiment, the invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist comprising a structure set forth in any of the structures in Figure 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 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 in 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 in 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 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 a 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 for treating a subject or patient with cancer or a disease or condition, comprising administering to the subject or patient a therapeutically effective amount of a composition or TC of the present invention, further comprising a chemotherapeutic or immunotherapeutic agent. In certain embodiments, the TC is co-administered with at least one chemotherapeutic agent. The chemotherapeutic agent may be selected from the group consisting of temozolomide, gemcitabine, doxorubicin, cyclophosphamide, paclitaxel, cisplatin, fluoropyrimidine, taxane, anthracycline, lapatinib, capecitabine, letrozole, pertuzumab, docetaxel, and 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 a composition or TC of the present invention, which further comprises an antibody drug conjugate, a cytotoxic agent, or a checkpoint inhibitor.

[0071] In another embodiment, the present invention provides a method for killing a cell, comprising contacting the cell with a TC of the present invention. In other embodiments, the cell is a tumor or cancer cell. In certain embodiments, the tumor or cancer cell is a colon, ovarian, breast, melanoma, lung, glioblastoma, prostate, bladder, cervical, pancreatic, kidney, esophageal, vaginal, stomach, or leukemia cancer cell. 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 for inhibiting or reducing tumor or cancer growth, comprising contacting a tumor with an effective amount of a 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 tumor or cancer growth, comprising contacting a tumor with an effective amount of a PEGylated TC of the present invention, or a stable dimer or multimer of a 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 regulate an immune response to tumor cells.

[0073] In some embodiments, the present invention provides methods of using TCs to treat cancer. In some embodiments, the TCs of the present invention can be used to treat or prevent cancer-related diseases, disorders, and conditions, including conditions directly or indirectly associated with cancer, e.g., precancerous conditions 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, but is not limited to, 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. Carcinoma is a cancer that begins in epithelial cells, which are the cells that cover the surfaces of the body, produce hormones, and make up glands. Non-limiting examples of 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, vulva cancer, uterine cancer, oral cancer, penile cancer, testicular cancer, esophageal cancer, skin cancer, cancer of the fallopian tubes, head and neck cancer, gastrointestinal stromal cancer, adenocarcinoma, cutaneous or intraocular melanoma, cancer of the anal region, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, cancer of the urethra, cancer of the renal pelvis, cancer of the ureter, cancer of the endometrium, cancer of the cervix, cancer of the pituitary gland, tumors of the central nervous system (CNS), primary CNS lymphoma, brain stem glioma, and spinal axis tumor. In some cases, the cancer is a skin cancer such as basal cell carcinoma, squamous cell carcinoma, melanoma, non-melanoma, or solar (solar) keratosis. In some embodiments, the present invention also relates to a method for treating acute leukemia in a mammal, comprising administering a therapeutically effective amount of a TC of the present invention to the mammal. The present invention also provides a method for inhibiting the proliferation of acute leukemia blast cells, comprising administering a therapeutically effective amount of a TC of the present invention to a mammal suffering from acute leukemia.

[0074] In another embodiment, the TCs disclosed herein can be used to regulate immune responses. Regulating immune responses can include stimulating, activating, increasing, enhancing, or upregulating immune responses. Regulating immune responses can include suppressing, inhibiting, preventing, reducing, or downregulating immune responses.

[0075] In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a 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 a 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 set forth in any one of the structures in Figure 1. In another embodiment, the present invention provides an immunostimulatory antibody conjugate (ISAC) comprising a TLR agonist set forth in any one of the compounds in Tables 3, 4, 5, 6, and 7. In another embodiment, the present invention provides an ISAC wherein the TLR agonist comprises a compound selected from the group of 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 compounds.

[0078] In another embodiment, the present invention provides a salt of any one of the compounds having the structure set forth in Figure 1. In another embodiment, the present invention provides a salt of any one of the compounds in Tables 3, 4, 5, 6, and 7. In another embodiment, the present invention provides a pharmaceutical composition or salt thereof according to the compositions, compounds, and TCs 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 TLR agonists suitable for use in the present invention.

[0080] [Figure 2] Structures of various TC conjugates.

[0081] [Figure 3] Structures of additional TC conjugates are shown.

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

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

[0084] FIG. 6 shows the TLR7 activity of various TLR7 agonists conjugated to linkers.

[0085] FIG. 7 shows the TLR7 activity of additional TLR7 agonists and TLR7 agonists linked to linkers.

[0086] FIG. 8 shows the TLR7 activity of additional TLR7 agonists and TLR7 agonists linked to linkers.

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

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

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

[0090] Figure 12 compares the tumor-dependent ISAC activity of additional payload linkers conjugated to anti-HER2 antibodies in the SKBR3 HER2-high expressing tumor cell line (A) and the HCC1806 HER2-ultra-low expressing tumor cell line (B).

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

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

[0093] Figure 15: Comparison of 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-ultra-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 invention may comprise a TLR agonist linked to an unnatural amino acid in the targeting moiety. Also included are methods for making such TCs comprising an unnatural amino acid incorporated into the targeting moiety polypeptide.

[0095] In certain embodiments, a pharmaceutical composition is provided that includes any of the described compounds and a pharmaceutically acceptable carrier, excipient, or binder.

[0096] In 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 heterocycle-containing non-natural amino acid, wherein the resulting heterocycle-containing non-natural amino acid polypeptide modulates the immunogenicity of the polypeptide relative to a homologous naturally occurring amino acid polypeptide.

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

[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 otherwise defined, 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. Although 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, the preferred methods, devices, and materials are now described.

[0100] All publications and patents mentioned herein are incorporated by reference in their entirety for the purpose of describing and disclosing, for example, the constructs and methodology described in the publications, which may be used in connection with the presently described invention. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should 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 ligation" or "mixed aldol-based ligation" refers to the acid- or base-catalyzed condensation of one carbonyl compound with the enolate / enol of another carbonyl compound, which may or may not be the same, to produce a β-hydroxycarbonyl compound-aldol.

[0102] As used herein, the term "affinity label" refers to a label that reversibly or irreversibly binds to another molecule to either modify it, destroy it, or form a compound with it. Examples of affinity labels include enzymes and their substrates, or antibodies and their antigens.

[0103] The terms "alkoxy," "alkylamino," and "alkylthio" (or thioalkoxy) are used in their conventional sense to refer to those 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, means, unless otherwise stated, a straight-chain or branched-chain, or cyclic hydrocarbon radical, or combinations thereof, which may be fully saturated, mono- or polyunsaturated, and having the specified number of carbon atoms (i.e., C1-C6). 10(meaning 1 to 10 carbons) may include divalent and polyvalent radicals. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated alkyl groups are those containing one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers. The term "alkyl," unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail herein, such as "heteroalkyl," "haloalkyl," and "homoalkyl."

[0105] The term "alkylene" by itself or as part of another molecule means (-CH2-) n where n can be from 1 to about 24. By way of example only, such groups include, but are not limited to, groups having 10 or fewer carbon atoms, such as the structures -CH2CH2- and -CH2CH2CH2CH2-. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having 8 or fewer carbon atoms. The term "alkylene" is also meant to include those groups described herein as "heteroalkylene," unless otherwise specified.

[0106] The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid 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 pyrrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, by way of example only: an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs may have modified R groups (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 methylsulfonium.

[0107] Amino acids may be referred to herein by either their name, 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 commonly accepted one-letter codes.

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

[0109] As used herein, the term "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by all or part of antibody genes. Immunoglobulin genes include, but are not limited to, the kappa, lambda, alpha, gamma (IgG1, IgG2, IgG3, and IgG4), delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. As used herein, antibody is meant to encompass full-length antibodies and antibody fragments, and to include naturally occurring antibodies in any organism or engineered antibodies (e.g., variants).

[0110] "Antibody fragment" refers to any form of an antibody other than the full-length form. Antibody fragments, as used herein, include smaller components of a full-length antibody and engineered antibodies. Antibody fragments include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, and variable 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 immunoglobulin heavy and light chains covalently linked by a peptide linker (Sz Hu et al., 1996, Cancer Research, 56, 3055-3061). These small (Mr 25,000) proteins generally retain specificity and affinity for an antigen in a single polypeptide and can provide convenient building blocks for larger antigen-specific molecules. Unless otherwise stated, the specification and claims using the term "antibody(s)" specifically include "antibody fragment(s)."

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

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

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

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

[0115] A "bifunctional polymer," also referred to as a "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 may 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 may be linked to the bifunctional linker or bifunctional polymer may be the same or different. By way of example only, a bifunctional linker may have a functional group reactive with a group on a first peptide and another functional group reactive with a group on a second peptide, thereby forming a conjugate comprising the first peptide, the bifunctional linker, and the second peptide. Many procedures and linker molecules are known for attaching various compounds to peptides. See, for example, European Patent Application No. 188,256, U.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784, 4,680,338, and 4,569,789, which are incorporated herein by reference in their entireties. A "multifunctional polymer," also referred to as a "multifunctional linker," refers to a polymer containing two or more functional groups capable of reacting with other moieties. Such moieties may include, but are not limited to, natural or unnatural amino acids, or side groups on peptides containing such natural or unnatural amino acids, for forming covalent or non-covalent bonds (including, but not limited to, amino acid side groups). Bifunctional or multifunctional polymers may be of any desired length or molecular weight and may be selected to provide a 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 portion is delivered from a pharmaceutical dosage form and becomes available at the site of action or in general circulation. Increased bioavailability refers to the rate and extent to which a substance or its active portion is delivered from a pharmaceutical dosage form and becomes available at the site of action or in general circulation. For example, increased bioavailability can be shown as an increase in the concentration of a substance or its active portion in the blood when compared with other substances or active portions. Methods for assessing increased bioavailability are known in the art and can be used to assess the bioavailability of any polypeptide.

[0117] As used herein, the terms "biologically active molecule," "biologically active moiety," or "biologically active agent" refer to any substance that can affect any physical or biochemical property of a biological system, pathway, molecule, or interaction in an organism, including, but not limited to, a virus, bacteria, bacteriophage, transposon, prion, insect, fungus, plant, animal, or human. In particular, as used herein, a biologically active molecule includes, but is not limited to, any substance intended for the diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or for otherwise 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, antivirals, anti-inflammatory agents, anti-tumor agents, cardiovascular medications, anti-anxiety agents, hormones, growth factors, steroids, microbial toxins, and the like.

[0118] "Modulating biological activity" means increasing or decreasing the reactivity of a polypeptide, altering the selectivity of a polypeptide, or enhancing or decreasing the substrate preference of a polypeptide. Analysis of modified biological activity can be performed by comparing the biological activity of the non-naturally occurring polypeptide with the biological activity of the naturally occurring polypeptide.

[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 capable of detecting or monitoring conformational changes in a molecule. Such molecules include, but are not limited to, proteins, and "biophysical probes" can be used to detect or monitor interactions of proteins 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 utilizing a translation system (cellular or non-cellular) that includes the use of at least one of the following components: polynucleotides, codons, tRNAs, and ribosomes. By way of example, unnatural amino acids can be "biosynthetically incorporated" into unnatural amino acid polypeptides using the methods and techniques described in WO2002 / 085923, which is incorporated herein by reference in its entirety. In addition, methods for selecting useful unnatural amino acids that can be "biosynthetically incorporated" into unnatural amino acid polypeptides are described in WO2002 / 085923, which is incorporated herein by reference in its entirety.

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

[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 a linear, branched, or cyclic molecule.

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

[0125] As used herein, the term "chemically cleavable group," also referred to as "chemically labile," refers to a group that decomposes or cleaves upon exposure to acids, bases, oxidizing agents, reducing agents, chemical initiators, or radical initiators.

[0126] As used herein, "cofolding" refers to a refolding process, reaction, or method using at least two molecules that interact with each other and result in the conversion of an unfolded or improperly folded molecule into a properly folded molecule. By way of example only, "cofolding" uses at least two polypeptides that interact with each other and result in the conversion of an unfolded or improperly folded polypeptide into a native, properly folded polypeptide. Such polypeptides may contain natural amino acids and / or at least one unnatural amino acid.

[0127] As used herein, a "conjugate" refers to a polypeptide linked, e.g., covalently attached, either directly or via a linker, to a compound or compound-linker described herein, e.g., a compound or salt of any one of the structures set forth in Figure 1, or any one of the structures in Tables 3-7. A "targeting moiety" refers to a structure having selective affinity for a target molecule compared to other non-target molecules. Targeting moieties of the present invention bind to a target molecule. Targeting moieties can include, for example, antibodies, peptides, ligands, receptors, or binding portions thereof. The target biological molecule can be a biological receptor or other structure on a cell, such as a tumor antigen. As used herein, the terms "conjugate of the present invention," "targeting moiety conjugate," "targeting conjugate," "targeting moiety-active molecule conjugate," or "TC" refer to a targeting polypeptide, or a portion, analog, or derivative thereof, that binds to a target present on a cell or its subunit conjugated to a biologically active molecule, portion, or analog thereof, including, but not limited to, a TLR7 and / or TLR8 agonist. As used herein, the terms "tumor targeting moiety conjugate," "tumor targeting moiety-biologically active molecule conjugate," or "BTC" refer to a tumor-targeting polypeptide, or a portion, analog, or derivative thereof, that binds to a target present on a tumor cell or a subunit thereof, conjugated to a biologically active molecule, portion, or analog thereof, including, but not limited to, a TLR7 and / or TLR8 agonist. Unless otherwise indicated, the terms "compound of the invention" and "composition of the invention" are used interchangeably with the term "conjugate of the invention."

[0128] The term "conservatively modified variants" applies to both natural and non-natural amino acids, as well as natural and non-natural nucleic acid sequences, and combinations thereof. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to those natural and non-natural nucleic acids that encode identical or essentially identical natural and non-natural amino acid sequences, or that do not encode natural and non-natural amino acid sequences for essentially identical natural and non-natural amino acid sequences. For example, due to the degeneracy of the genetic code, a large 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 alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Thus, by way of example, all natural or non-natural nucleic acid sequences herein that encode natural or non-natural polypeptides also describe all possible silent variations of the natural or non-natural nucleic acid. Those skilled in the art will recognize that each codon in a natural or non-natural nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Accordingly, each silent variation of natural and non-natural nucleic acids that encode natural and non-natural polypeptides is implicit in each described sequence.

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

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

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

[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" may cause reversible unfolding of a protein. The strength of a denaturing agent or denaturant is determined by both the properties and concentration of the 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 may include, but are not limited to, strong detergents such as sodium dodecyl sulfate, or polyoxyethylene ethers (e.g., Tween or Triton detergents), sarkosyl, mild nonionic detergents (e.g., digitonin), mild cationic detergents such as N->2,3-(dioleoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents (e.g., sodium cholate or sodium deoxycholate), or zwitterionic detergents (which may include, but are not limited to, sulfobetaine (Zwittergent), 3-(3-chloroamidopropyl)dimethylammonio-1-propanesulfate (CHAPS), and 3-(3-chloroamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO)). Non-limiting examples of organic water-miscible solvents that can be used as denaturants include, but are not limited to, acetonitrile, a lower alkanol (particularly a C2-C4 alkanol such as ethanol or isopropanol), or a lower alkanediol (a C2-C4 alkanediol such as ethylene glycol).Non-limiting examples of phospholipids include, but are not limited to, naturally occurring phospholipids, such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol, or synthetic phospholipid derivatives or variants, such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

[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, amidine, imine, 1,1-diamine, 1,2-diamine, 1,3-diamine, and 1,4-diamine groups. Additionally, such groups can be part of linear, branched, or cyclic molecules.

[0135] As used herein, the term "detectable label" refers to a label that may be observable using analytical techniques, including, but not limited to, fluorescence, chemiluminescence, electron-spin resonance, ultraviolet / visible absorbance 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, 1,3-dicarbonyl, and 1,4-dicarbonyl groups, as well as 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 dicarbonyl groups include diketones, ketoaldehydes, ketoacids, ketoesters, and ketothioesters. In addition, such groups can be part of linear, branched, or cyclic molecules. The two moieties in a dicarbonyl group can be the same or different, and either of the two moieties can contain substituents that produce esters, ketones, aldehydes, thioesters, or amides, by way of example only.

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

[0138] As used herein, the term "effective amount" refers to a sufficient quantity of an administered agent or compound to relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be 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 include, but is not limited to, a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-amino acid polypeptide. Compositions containing such natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides can be administered for prophylactic, augmentative, and / or therapeutic treatments. An appropriate "effective" amount in any individual case can be determined using techniques such as a dose escalation study.

[0139] The terms "enhance" or "enhancing" mean to increase or prolong either the potency or duration of a desired effect. By way of example, "enhancing" the effect of a therapeutic agent refers to the ability to increase or prolong, in either potency or duration, the effect of the therapeutic agent 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 the therapeutic agent in treating a disease, disorder, or condition. When used in a patient, amounts effective for this use will depend on the severity and course of the disease, disorder, or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.

[0140] As used herein, the term "eukaryote" refers to organisms belonging to the phylogenetic domain Eukarya, 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 a carboxylic acid having a hydrocarbon side chain of about C6 or longer.

[0142] As used herein, the term "fluorophore" refers to a molecule that emits a photon when excited, thereby being 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 the portion or unit of a molecule at which a chemical reaction occurs. These terms are somewhat synonymous in the chemical arts and are used herein to refer to the portion of a molecule that performs 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)CH, -C(O)CF, -C(O)CHOCH, and the like.

[0146] As used herein, the term "haloalkyl" refers to an alkyl group containing a halogen moiety, including -CF3, -CH2CF3, and the like.

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

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

[0149] Similarly, the term "heteroalkylene" refers to a divalent radical derived from heteroalkyl, exemplified by, but not limited to, -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-. For heteroalkylene groups, the same or different heteroatoms can also occupy either or both of the chain termini (including, but not limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. By way of example, the formula -C(O)R'- represents both -C(O)R'- and -R'C(O)-.

[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, where the nitrogen and sulfur atoms can be optionally oxidized and the nitrogen atom(s) can be optionally quaternized. A heteroaryl group can be substituted or unsubstituted. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.

[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 sequences or subsequences that are the same. Additionally, as used herein, the term "substantially identical" refers to two or more sequences that have the same percentage of sequence units when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using a comparison algorithm or by manual alignment and visual inspection. By way of example only, two or more sequences may be "substantially identical" if the sequence 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 the designated region. Such percentages describe the "percent identity" of two or more sequences. Sequence identity can exist over a region that is at least about 75-100 sequence units in length, over a region that is about 50 sequence units in length, or, if not specified, over the entire sequence. This definition also refers to the complementarity of a test sequence. 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 that is at least about 75 to about 100 amino acids in length, over a region that is about 50 amino acids in length, or, if not specified, over the entire sequence of the polypeptide sequences. Additionally, 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. The identity can exist over a region that is at least about 75 to about 100 nucleic acids in length, over a region that is about 50 nucleic acids in length, or, if not specified, over the entire sequence of the polynucleotide sequence.

[0153] For sequence comparison, typically, one sequence serves as the reference sequence with which test sequence is compared.When using sequence comparison algorithm, test and reference sequences are input into computer, and if necessary, subsequence coordinates are designated, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence to reference sequence based on program parameters.

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

[0155] As used herein, the term "isolated" refers to the separation and removal of a component of interest from components not of interest. An isolated material can be in either 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. Furthermore, a component of interest is described herein as substantially purified if it is isolated and is the predominant species present in a preparation. As used herein, the term "purified" can refer to a component of interest that is at least 85% pure, at least 90% pure, at least 95% pure, or at least 99% or more pure. By way of example only, a nucleic acid or protein is "isolated" when such nucleic acid or protein is free from at least some of the cellular components with which it is naturally associated, or when the nucleic acid or protein is concentrated to a level greater 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 which flank the gene and 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 so that its physical distribution can be detected and / or monitored.

[0157] As used herein, the term "linkage" or "linker" refers to a bond or chemical moiety formed from a chemical reaction between a functional group of a linker and another molecule. Such bonds can include, but are not limited to, covalent and non-covalent bonds. In turn, such chemical moieties can include, but are not limited to, esters, carbonates, imine phosphate esters, 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 for extended periods of time, perhaps even permanently, at useful pH values, including, but not limited to, under physiological conditions. A hydrolytically unstable or degradable linkage means that the linkage is degradable in water or aqueous solutions, including, for example, blood. An enzymatically unstable or degradable linkage means that the linkage can be degraded by one or more enzymes. By way of example only, PEG and related polymers may contain degradable linkages within the polymer backbone or within the 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 acids or activated PEG carboxylic acids with alcohol groups on the biologically active agent; such ester groups typically hydrolyze under physiological conditions to release the biologically active agent. Other hydrolyzable linkages include, but are not limited to, carbonate linkages, imine linkages resulting from the reaction of an amine with an aldehyde, phosphate linkages formed by the reaction of an alcohol with a phosphate group, hydrazone linkages which are the reaction product of a hydrazide with an aldehyde, acetal linkages which are the reaction product of an aldehyde with an alcohol, orthoester linkages which are the reaction product of a formate with an alcohol, peptide linkages formed by an amine group, including but not limited to, the terminus of a polymer such as PEG, and a carboxyl group of a peptide, and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, the terminus of a polymer, and a 5' hydroxyl group of an oligonucleotide.Linkers include, but are not limited to, short linear, branched, multi-armed, or dendrimeric molecules, such as polymers. In some embodiments of the present invention, the linker may be branched. In other embodiments, the linker may be a bifunctional linker. In some embodiments, the linker may be a trifunctional linker. Several different cleavable linkers are known to those of skill in the art. See U.S. Pat. Nos. 4,618,492, 4,542,225, and 4,625,014. Mechanisms for releasing drugs from these linker groups include, for example, irradiation and acid-catalyzed hydrolysis of photolabile bonds. For example, U.S. Pat. No. 4,671,958 includes a description of immunoconjugates containing linkers that are cleaved at target sites in vivo by proteolytic enzymes of the patient's complement system. The length of the linker can be predetermined or selected depending on the desired spatial relationship between the polypeptide and the molecule linked to it. Given that numerous methods have been reported for conjugating various radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and other agents to antibodies, one of skill 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 an alteration to a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. Such an alteration or modification can be obtained by post-synthetic modification of a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide, or by co-translational or post-translational modification of a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. The form "modified or unmodified" means that the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide being discussed is optionally modified, i.e., the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide being discussed can be modified or unmodified.

[0159] As used herein, the term "adjusted 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, non-natural amino acids, natural amino acid polypeptides, or non-natural amino acid polypeptides. For example, serum half-life is measured by taking blood samples at various time points after administration of a biologically active molecule or a modified biologically active molecule and determining the concentration of the molecule in each sample. The correlation between serum concentration and time allows for the calculation of serum half-life. For example, an adjusted serum half-life can be an increase in serum half-life, which can enable improved dosing regimens or avoid toxic effects. Such an increase in serum half-life 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 the increase in serum half-life of any polypeptide are well known to those skilled 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, non-natural amino acids, natural amino acid polypeptides, or non-natural amino acid polypeptides. For example, therapeutic half-life is measured by measuring the pharmacokinetic and / or pharmacodynamic properties of the molecule at various time points after administration. An increase in therapeutic half-life may enable a particular beneficial dosing regimen, a particular beneficial total dose, or avoid undesirable effects. For example, an increase in therapeutic half-life may result from increased efficacy, increased or decreased 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, increased or decreased degradation of the molecule by enzymes such as proteases. Methods for assessing the increased therapeutic half-life of any polypeptide are well known to those skilled in the art.

[0161] "Unnatural amino acid" refers to an amino acid that is not one of the 20 common amino acids, or pyrrolysine or selenocysteine. Other terms that may be used synonymously with the term "unnatural amino acid" are "non-naturally encoded amino acid," "unnatural amino acid," "non-naturally occurring amino acid," 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 pyrrolysine and selenocysteine), but are not themselves incorporated into a growing polypeptide chain by the translation complex. Examples of such amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine. In addition, the term "unnatural amino acid" includes, but is not limited to, amino acids that do not occur in nature and can be obtained synthetically or by modification of an unnatural amino acid. In some embodiments, the unnatural amino acid comprises a lysine analog, such as N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, or allyloxycarbonyl lysine. In some embodiments, the unnatural amino acid comprises a sugar moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine. Examples of such amino acids also include those in which the naturally occurring N- or O-linkage between the amino acid and the sugar is replaced with a covalent bond not commonly found in nature, including, but not limited to, an alkene, oxime, thioether, amide, etc. Examples of such amino acids also include sugars not commonly found in naturally occurring proteins, such as 2-deoxy-glucose and 2-deoxygalactose.Specific examples of unnatural amino acids include p-acetyl-L-phenylalanine, p-propargyloxyphenylalanine, 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 In some embodiments, the unnatural amino acid includes, but is not limited to, para-acetyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propargyloxy-L-phenylalanine, 4-azido-L-phenylalanine, para-azidoethoxyphenylalanine, and para-azidomethyl-phenylalanine. In some embodiments, the unnatural amino acid is selected from the group consisting of para-acetyl-phenylalanine, 4-azido-L-phenylalanine, para-azidoethoxyphenylalanine, or para-azidomethyl-phenylalanine.

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

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

[0164] As used herein, the term "pharmaceutically acceptable" refers to a material, including but not limited to, salts, carriers, or diluents, that does not abrogate the biological activity or properties of the compound and that is relatively non-toxic, i.e., the material can be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0165] As used herein, the terms "polyalkylene glycol" or "poly(alkene glycol)" refer to linear or branched polymeric polyether polyols. Such polyalkylene glycols include, but are not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof. Other exemplary embodiments are listed in commercial supplier catalogs, such as, for example, Shearwater Corporation's catalog "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001). By way of example only, such polymeric polyether polyols have an average molecular weight of about 0.1 kDa to about 100 kDa. By way of example, such polymeric polyether polyols include, but are not limited to, those having molecular weights of about 100 Da to about 100,000 Da or more. The molecular weight of the polymer may be 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, The molecular weight of the polymer may be from about 100 Da to about 100,000 Da, including, but not limited to, 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, hi some embodiments, the molecular weight of the polymer is from about 2,000 to about 50,000 Da.In some embodiments, the molecular weight of the polymer is about 5,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the polymer is about 10,000 Da to about 40,000 Da. In some embodiments, the poly(ethylene glycol) molecule is a branched polymer. The molecular weight of the branched PEG is 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, or about 30,000 Da. The molecular weight of the branched PEG may be from about 1,000 Da to about 100,000 Da, including, but not limited to, about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, and about 1,000 Da. In some embodiments, the molecular weight of the branched PEG is from about 1,000 Da to about 50,000 Da. In some embodiments, the molecular weight of the branched PEG is from about 1,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the branched PEG is from about 5,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the branched PEG is from about 5,000 Da to about 20,000 Da, while in other embodiments, the molecular weight of the branched 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, including, but not limited to, a polypeptide, a polynucleotide, or a polysaccharide or polyalkylene glycol.

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

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

[0169] As used herein, the term "prodrug" or "pharmaceutically acceptable prodrug" refers to an agent that is converted into the parent drug in vivo or in vitro, does not abrogate the biological activity or properties of the drug, and is relatively non-toxic; i.e., the material can be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition containing it. Prodrugs are generally drug precursors that, after administration to a subject and subsequent absorption, are converted into an active or more active species through some process, such as conversion by a metabolic pathway. Some prodrugs have chemical groups present on the prodrug that reduce the activity of the prodrug and / or confer solubility or some other property to the drug. When the chemical group is cleaved and / or modified from the prodrug, the active drug is generated. Prodrugs are converted into active drugs in the body through enzymatic or non-enzymatic reactions. Prodrugs may provide improved physiochemical properties, such as better solubility, enhanced delivery characteristics, such as specific targeting to particular 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 orally bioavailable, whereas the parent drug is not, and (iii) the prodrug may also have improved solubility in pharmaceutical compositions compared to the parent drug. Prodrugs include pharmacologically inactive or reduced-activity derivatives of active drugs. Prodrugs can be designed to regulate the amount of a drug or biologically active molecule that reaches a desired site of action through manipulation of the drug's properties, e.g., physicochemical, biopharmaceutical, or pharmacokinetic properties. Examples of prodrugs include, but are not limited to, non-natural amino acid polypeptides ("prodrugs") administered as esters to facilitate transport across cell membranes, where water solubility is detrimental to mobility but which are subsequently metabolically hydrolyzed to the active substance carboxylic acid once inside the cell, where water solubility is beneficial.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 an amount of a composition containing at least one non-natural amino acid polypeptide or at least one modified non-natural amino acid polypeptide administered prophylactically to a patient that will relieve to some extent one or more of the symptoms of the disease, condition, or disorder being treated. In such prophylactic applications, such amount may depend on the patient's health, weight, and the like. It is considered well within the skill of one in the art for one to determine such prophylactically effective amounts 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 reaction of a chemically reactive functional group under certain reaction conditions. The protecting group varies depending on the type of chemically reactive group being protected. By way of example only, (i) if the chemically reactive group is an amine or hydrazide, the protecting group can be selected from tert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc), (ii) if the chemically reactive group is a thiol, the protecting group can be orthopyridyl disulfide, and (iii) if 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, blocking / protecting groups are selected from:

[0173] [ka]

[0174] Further protecting groups include, but are not limited to, photolabile groups such as Nvoc and MeNvoc, as well as other protecting groups known in the art. Other protecting groups are described in Greene and Wuts, Protective Groups in Organic Synthesis, Vol. 1, No. 1, pp. 111-114, 1997. Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, which is incorporated herein by reference in its entirety.

[0175] The term "recombinant host cell," also referred to as "host cell," refers to a cell that contains an exogenous polynucleotide; methods used to insert the exogenous polynucleotide into a cell include, but are not limited to, direct uptake, transduction, f-mating, or other methods known in the art for generating recombinant host cells. By way of example only, such exogenous polynucleotides may be non-integrative vectors, including, but not limited to, plasmids, or may 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 reducing or oxidizing the redox active agent. Examples of redox active agents include ferrocene, quinones, Ru, 2+ / 3+ Complex, Co 2+ / 3+ Complex, and Os 2+ / 3+ These include, but are not limited to, complexes.

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

[0178] As used herein, "refolding" describes any process, reaction, or method that converts an improperly folded or unfolded state into a native or properly folded conformation. By way of example only, refolding transforms a disulfide bond-containing polypeptide from an improperly folded or unfolded state into 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 term "safety" or "safety profile" refers to the side effects that may be associated with the administration of a drug relative to the number of times the drug is administered. For example, a drug that is administered multiple times and has mild or no side effects is said to have a good safety profile. Methods used to evaluate the safety profile of any polypeptide are known in the art.

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

[0181] The phrase "stringent hybridization conditions" refers to the hybridization of sequences of DNA, RNA, PNA, or other nucleic acid mimics, or combinations thereof, under conditions of low ionic strength and high temperature. For example, under stringent conditions, a probe will hybridize to its target sequence in a complex mixture of nucleic acids (including, but not limited to, whole cell or library DNA or RNA), but will not hybridize to other sequences in the complex mixture. Stringent conditions are sequence-dependent and are different under different conditions. For example, longer sequences hybridize specifically at higher temperatures. Stringent hybridization conditions include (i) about 5-10°C lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength and pH; (ii) a salt concentration of about 0.01M to about 1.0M at about pH 7.0 to about pH 8.3, and a temperature of at least about 30°C for short probes (including, but not limited to, about 10 to about 50 nucleotides) and at least about 60°C for long probes (including, but not limited to, more than 50 nucleotides); (iii) the addition of a destabilizing agent, including, but not limited to, formamide; and (iv) 50% formamide, 5x SSC, and 1% SDS, incubation at 42°C, or 5x SSC, about 1% SDS, incubation at 65°C, washing with 0.2x SSC, and about 0.1% SDS, incubation at 65°C for about 5 minutes to about 120 minutes. By way of example only, detection of selective or specific hybridization includes, but is not limited to, a positive signal of at least two times background. An extensive guide to nucleic acid hybridization is provided by 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 object of treatment, observation, or experiment. By way of example only, a subject may be a mammal, including, but not limited to, a human.

[0183] As used herein, the term "substantially purified" refers to a component of interest that may be substantially or essentially free from other components that normally accompany or interact with the component of interest prior to purification. By way of example only, a component of interest may be "substantially purified" if a preparation of the component of interest contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating components. Thus, a "substantially purified" component of interest may have a purity level of about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater. By way of example only, a natural or non-natural amino acid polypeptide may be purified from a natural cell, or, in the case of a recombinantly produced natural or non-natural amino acid polypeptide, from a host cell. By way of example, a preparation of natural or non-natural amino acid polypeptides can be "substantially purified" if the preparation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating materials. By way of example, when a natural or non-natural amino acid polypeptide is recombinantly produced by a host cell, the natural or non-natural amino acid polypeptide can be present at no more than about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% by dry weight of the cells. By way of example, when a natural or non-natural amino acid polypeptide is recombinantly produced by a host cell, the natural or non-natural amino acid polypeptide can be present in the culture 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 or non-natural amino acid polypeptide can have a purity level of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater as determined by suitable methods, including, but not limited to, SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0184] The term "substituents," also referred to as "non-interfering substituents," refers to groups that can be used to replace another group on a molecule. Such groups include halo, C-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, toluolyl, xylenyl, biphenyl, C2-C 12 Alkoxyalkyl, C5-C 12 Alkoxyaryl, C5-C 12 Aryloxyalkyl, C7-C 12 Oxyaryl, C1-C6 alkylsulfinyl, C1-C 10 Alkylsulfonyl, -(CH2) m -O-(C1-C 10 alkyl) (where m is 1 to 8), aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic radical, nitroalkyl, —NO, —CN, —NRC(O)—(C-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 Examples of R groups include, but are not limited to, —C(O)NR, —C(S)NR, —SONR, —NRC(O)NR, —NRC(S)NR, salts thereof, and the like. Each R group in the foregoing list includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl, or alkaryl. Where substituents are specified by their conventional chemical formula written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., —CHO— is equivalent to —OCH—.

[0185] By way of example only, substituents for 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, —NR, —SR, -halogen, —SiR, —OC(O)R, —C(O)R, —COR, —CONR, —OC(O)NR, —NRC(O)R, —NRC(O)NR, —NR(O)R, —NR—C(NR)═NR, —S(O)R, —S(O)R, —S(O)NR, —NRSOR, —CN, and —NO. Each R group in the preceding list includes, but is not limited to, hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (including, but not limited to, aryl substituted with 1 to 3 halogens), substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or aralkyl groups. When two R groups are bonded to the same nitrogen atom, they can combine with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR2 is meant to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl.

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

[0187] As used herein, the term "therapeutically effective amount" refers to an amount of a composition containing at least one non-natural amino acid polypeptide and / or at least one modified non-natural amino acid polypeptide administered to a patient already suffering from a disease, condition, or disorder sufficient to cure, or at least partially arrest, or alleviate to some extent, one or more of the symptoms of the disease, disorder, or condition being treated. The effectiveness of such a composition will depend on factors including, but not limited to, the severity and course of the disease, disorder, or condition, previous therapy, the patient's health status and response to drugs, and the judgment of the treating physician. By way of example only, a therapeutically effective amount may be determined by routine experimentation, including, but not limited to, a dose escalation clinical trial.

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

[0189] As used herein, the term "toxic moiety" or "toxic group" refers to a compound that can cause harm, injury, or death. Toxic moieties include auristatins, DNA minor groove binders, DNA minor groove alkylators, enediynes, lexitropsins, duocarmycins, taxanes, puromycins, TLR agonists, maytansinoids, vinca alkaloids, AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, TLR-agonist-10, echinomycin, and combretus. statin, calicheamicin, maytansine, DM-1, netropsin, podophyllotoxin (e.g., etoposide, teniposide, etc.), baccatin and its derivatives, antitubulin agents, cryptophysin, combretastatin, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, epothilone A, epothilone B, nocodazole, colchicine, colcimide, estramustine, cemadotin, discodermolide, maytansine, eleutherobin, mechlorethamine , cyclophosphamide, melphalan, carmustine, lomustine, semustine, streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide, itarabine, cytosine arabinoside, fluorouracil, floxuridine, 6-thioguanine, 6-mercaptopurine, pentostatin, 5-fluorouracil, methotrexate, 10-propanol Gyl-5,8-dideazafolate, 5,8-dideazatetrahydrofolic acid, leucovorin, fludarabine phosphate, pentostatin, gemcitabine, Ara-C, paclitaxel, docetaxel, deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine, brequinar, antibiotics (e.g., anthracyclines, gentamicin, cephalothin, vancomycin, telavancin, daptomycin, azithromycin, erythromycin, roxthromycin, furazolidone, amoxicillin,ampicillin, carbenicillin, flucloxacillin, 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, fosphonet, gaclovir, etc.), cyclovir, ibacitabine, immunovir, idoxuridine, inosine, lopinavir, methisazone, nexavir, nevirapine, oseltamivir, penciclovir, stavudine, trifluridine, truvada, valacyclovir, zanamivir, etc.), daunorubicin hydrochloride, daunomycin, rubidomycin, cerubicin, idarubicin, doxorubicin, epirubicin, and morpholino derivatives, phenoxyzolidinone cyclopeptides (e.g., dactinomycin), basic glycopeptides amides (e.g., bleomycin), anthraquinone glycosides (e.g., plicamycin, mithramycin), anthracenediones (e.g., mitoxantrone), azirinopyrroloindoleziones (e.g., mitomycin), macrocyclic immunosuppressants (e.g., cyclosporine, FK-506, tacrolimus, prograf, rapamycin, etc.), navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, Taxanes include, but are not limited to, roloxafine, allocolchicine, halichondrin B, colchicine, colchicine derivatives, maytansine, rhizoxin, paclitaxel, paclitaxel derivatives, docetaxel, thiocolchicine, tritylcysterol, vinblastine sulfate, vincristine sulfate, cisplatin, carboplatin, hydroxyurea, N-methylhydrazine, epidophyllotoxin, procarbazine, mitoxantrone, leucovorin, and tegafur. "Taxane" includes paclitaxel as well as any active taxane derivative or prodrug.

[0190] As used herein, the terms "treat," "treating," or "treatment" include alleviating, reducing, 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, relieving the disease or condition, causing regression of the disease or condition, alleviating conditions caused by the disease or condition, or halting the symptoms of the disease or condition. The terms "treat," "treating," or "treatment" include, but are not limited to, prophylactic and / or therapeutic treatments.

[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-C6 10Examples of suitable polyols include, but are not limited to, alkoxy or aryloxy derivatives thereof (as described in U.S. Pat. No. 5,252,714, which is 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 copolymers, polyoxyethylated polyols, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives (including, but not limited to, methylcellulose and carboxymethylcellulose), serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycol and derivatives thereof, polyvinyl ethyl ether, and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide], and the like, or mixtures thereof. By way of example only, the attachment of such water-soluble polymers to natural amino acid polypeptides or non-natural polypeptides may result in changes, compared to the unmodified form, including, but not limited to, increased water solubility, increased or modulated serum half-life, increased or modulated therapeutic half-life, increased bioavailability, modulated biological activity, extended circulation time, modulated immunogenicity, modulated physical association characteristics, including, but not limited to, aggregation and multimerization, altered receptor binding, activity modulator or other targeting polypeptide binding, altered binding to one or more binding partners, and altered dimerization or multimerization of targeting polypeptide receptors. In addition, such water-soluble polymers may or may not have their own biological activity and may be utilized as linkers to attach targeting polypeptides to other substances, including, but not limited to, one or more targeting polypeptides or one or more biologically active molecules.

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

[0193] The compounds presented herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides, and reagents for producing the foregoing compounds) include isotopically labeled compounds that are identical to those listed in the various formulas and structures presented herein, but in which one or more atoms are replaced with an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that may be incorporated into the present compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, e.g., 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 35 S, 18 F, 36 Certain isotopically labeled compounds described herein, for example, 3 H and 14 Compounds into which radioactive isotopes such as C are incorporated are useful in drug and / or substrate tissue distribution assays. Additionally, deuterium, i.e., 2 Substitution with isotopes such as H may offer certain therapeutic advantages due to greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements.

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

[0195] In further or additional embodiments, the compounds described herein (including but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, and reagents for producing the foregoing compounds) are used in the form of prodrugs. In further or additional embodiments, the compounds described herein (including but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, and reagents for producing the foregoing compounds) are metabolized upon administration to an organism in need of producing the metabolic products, which are then used to produce a desired effect, including a desired therapeutic effect. In further or additional embodiments, the compounds are active metabolites of the 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 the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides. In certain embodiments, the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides can exist as tautomers. All tautomers are included within the scope of the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides presented herein. In addition, the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides described herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents, such as water, ethanol, and the like. The solvated forms of the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides presented herein are also considered to be disclosed herein.

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

[0198] Some of the compounds herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides, as well as reagents for producing any of the foregoing compounds) may be acidic and may form salts with pharmaceutically acceptable cations. Some of the compounds 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) may be basic and may therefore form salts with pharmaceutically acceptable anions. All such salts, including disalts, are within the scope of the compositions described herein and can be prepared by conventional methods. For example, salts can be prepared by contacting acidic and basic materials in either aqueous, non-aqueous, or partially aqueous media. 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, in the case of aqueous solutions, lyophilization.

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

[0200] Types of pharmaceutically acceptable salts include, but are not limited to, the following: (1) salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with 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- Acid addition salts formed with 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, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion, alkaline earth ion, or aluminum ion, or coordinated with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.

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

[0203] Screening and characterization of polymorphs and / or solvates of pharmaceutically acceptable salts of non-natural amino acid polypeptides can be accomplished using a variety of techniques, including, but not limited to, thermal analysis, X-ray diffraction, spectroscopy, vapor sorption, and microscopy. Thermal analysis methods address thermochemical decomposition or thermophysical processes, including, but not limited to, polymorphic transitions, and such methods are used to analyze relationships between 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. Various spectroscopic techniques used include, but are not limited to, Raman, FTIR, UVIS, and NMR (liquid and solid state). Various microscopy techniques include, but are not limited to, polarized light microscopy, scanning electron microscopy (SEM) with energy dispersive X-ray analysis (EDX), environmental scanning electron microscopy with EDX (in a gas or water vapor atmosphere), IR microscopy, and Raman microscopy.

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

[0205] TLR-agonist linker derivatives Described herein are tools (methods, compositions, techniques) for making and using targeting polypeptides for TCs or analogs containing at least one unnatural amino acid or modified unnatural amino acid bearing a carbonyl, dicarbonyl, oxime, or hydroxylamine group. Such targeting polypeptides for TCs containing unnatural amino acids may contain additional functional groups, including, but not limited to, polymers, water-soluble polymers, polyethylene glycol derivatives, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, and any combination thereof. Note that the various aforementioned functional groups do not imply that members of one functional group cannot be classified as members of another functional group. In fact, there may be overlap depending on the specific situation. As just one example, water-soluble polymers overlap in scope with polyethylene glycol derivatives, but the overlap is not complete, and therefore both functional groups are cited above.

[0206] In one aspect, methods for selecting and designing modified TLR-agonist linker derivatives and targeting polypeptides using the methods, compositions, and techniques described herein are provided. By way of example only, novel TLR-agonist linker derivatives and targeting polypeptides can be designed de novo as part of a high-throughput screening process (where large numbers of polypeptides can be designed, synthesized, characterized, and / or tested) or de novo based on a researcher's interest. Novel TLR-agonist linker derivatives and targeting polypeptides can also be designed based on the structure of known or partially characterized polypeptides. By way of example only, TLR-agonists are 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 replace and / or modify are described separately herein. The selection of which modifications to use is also described herein and can be used to meet the needs of the experimenter or end user. Such needs include, but are not limited to, manipulating the therapeutic effectiveness of the polypeptide, improving the safety profile of the polypeptide, adjusting the pharmacokinetics, pharmacology, and / or pharmacodynamics of the polypeptide, for example, by way of example only, increasing water solubility, bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending circulation time. Additionally, such modifications include, by way of example only, providing the polypeptide with additional functional groups, incorporating antibodies, and any combination of the foregoing modifications.

[0207] Also described herein are TLR-agonist linker derivatives and targeting polypeptides that have or can be modified to contain oxime, carbonyl, dicarbonyl, or hydroxylamine groups, including methods for producing, purifying, characterizing, and using such TLR-agonist linker derivatives and targeting polypeptides.

[0208] The TLR-agonist linker derivative or targeting 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 ten or more of a carbonyl or dicarbonyl group, an oxime group, a hydroxylamine group, or a protected form thereof. The TLR-agonist linker derivative or targeting polypeptide may be the same or different, e.g., there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different sites in a derivative that contains 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.

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

[0210] The targeting polypeptide and M can be linked together by L using standard linking agents and procedures known to those of skill in the art. In some embodiments, the targeting polypeptide and M are directly fused and L is a bond. In other embodiments, the targeting polypeptide and M are fused via a linking group L. For example, in some embodiments, the targeting polypeptide and M are linked together via a peptide bond, optionally through a peptide or amino acid spacer. In some embodiments, the targeting 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 targeting 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 is conjugated to M by either reacting a nucleophilic reactive moiety on the targeting polypeptide with an electrophilic reactive moiety on Y, or by reacting an electrophilic reactive moiety on the targeting polypeptide with a nucleophilic reactive moiety on M. In embodiments where L is a group linking the targeting polypeptide and M together, the targeting polypeptide and / or M can be conjugated to L by either reacting a nucleophilic reactive moiety on the targeting polypeptide and / or M with an electrophilic reactive moiety on L, or by reacting an electrophilic reactive moiety on the targeting polypeptide and / or M with a nucleophilic reactive moiety on L. Non-limiting examples of nucleophilic reactive groups include amino, thiol, and hydroxyl. Non-limiting examples of electrophilic reactive groups include carboxyl, acyl chloride, anhydride, ester, succinimide ester, alkyl halide, sulfonate ester, maleimide, haloacetyl, and isocyanate. In embodiments where the targeting polypeptide and M are conjugated together by reacting a carboxylic acid with an amine, an activating agent can be used to form an activated ester of the carboxylic acid.

[0212] The activated ester of a carboxylic acid 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 on the side chain of lysine, cysteine, or serine) that can be conjugated to an electrophilic reactive group on M or L. In some embodiments, the targeting polypeptide comprises an electrophilic reactive group (e.g., a carboxylate group on the side chain of Asp or Glu) that can be conjugated to a nucleophilic reactive group on M or L. In some embodiments, the targeting polypeptide is chemically modified to include a reactive group that can be directly conjugated to M or L. In some embodiments, the targeting polypeptide is modified at the N- or C-terminus to include a natural or unnatural amino acid having a nucleophilic side chain. In exemplary embodiments, the N- or C-terminal amino acid of the targeting polypeptide is selected from the group consisting of lysine, ornithine, serine, cysteine, and homocysteine. For example, the N- or C-terminal amino acid of the targeting polypeptide can be modified to include a lysine residue. In some embodiments, targeting polypeptides are modified at the N- or C-terminal amino acid to include natural or unnatural amino acids with electrophilic side chains, such as, for example, Asp and Glu. In some embodiments, internal amino acids of targeting polypeptides are substituted with natural or unnatural amino acids with nucleophilic side chains, as described previously herein. In exemplary embodiments, the substituted internal amino acids of targeting polypeptides are selected from the group consisting of lysine, ornithine, serine, cysteine, and homocysteine. For example, internal amino acids of targeting polypeptides can be substituted with lysine residues. In some embodiments, internal amino acids of targeting polypeptides are substituted with natural or unnatural amino acids with electrophilic side chains, such as, for example, Asp and Glu.

[0214] In some embodiments, M comprises a reactive group capable of being directly conjugated to the targeting polypeptide or 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., a carboxyl group, an activated form of a carboxyl group, a compound with 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 any of the nucleophilic reactive groups capable of conjugating to an electrophilic reactive group on the targeting polypeptide or L.

[0215] In some embodiments, conjugation can be achieved through the use of organosilanes, such as aminosilanes treated with glutaraldehyde, carbonyldiimidazole (CDI) activation of silanol groups, or dendrimers. Various dendrimers are known in the art, including poly(amidoamine) (PAMAM) dendrimers synthesized by a branching method starting from ammonia or ethylenediamine initiator core reagents; a subclass of PAMAM dendrimers based on a tris-aminoethylene-imine core; radial-layer poly(amidoamine-organosilicon) dendrimers (PAMAMOS), which are inverse unimolecular micelles consisting of a hydrophilic, nucleophilic polyamidoamine (PAMAM) interior and a hydrophobic organosilicon (OS) exterior; poly(propyleneimine) (PPI) dendrimers, which are generally poly-alkylamines with primary amines as terminal groups but with multiple tertiary tris-propyleneamines in the dendrimer interior; poly(propyleneamine) (POPAM) dendrimers; diaminobutane (DAB) dendrimers; amphiphilic dendrimers; micellar dendrimers, which are unimolecular micelles of water-soluble hyperbranched polyphenylenes; polylysine dendrimers; and dendrimers based on poly-benzyl ether hyperbranched backbones.

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

[0217] In some embodiments, conjugation can be carried out using click chemistry. The "click reaction" is broad in scope, easy to perform, uses only readily available reagents, and is insensitive to oxygen and water. In some embodiments, the click reaction is a cycloaddition reaction between an alkynyl group and an azide group to form a triazolyl group. In some embodiments, the click reaction uses a copper or ruthenium catalyst. Suitable methods for performing the click reaction have been described in the art. For example, Kolb et al., Drug Discovery Today 8:1128(2003), Kolb et al., Angew.Chem.Int.Ed.40:2004(2001), Rostovtsev et al., Angew.Chem.Int.Ed.41:2596(2002), Tornoe et al. al., J.Org.Chem.67:3057(2002), Manetsch et al., J.Am.Chem.Soc.126:12809(2004), Lewis et al. al.,Angew.Chem.Int.Ed.41:1053(2002), Speers,J.Am.Chem.Soc.125:4686(2003),Chan et al.Org.Lett.6:2853(2004),Zhang See, e.g., 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 contain nucleophilic or electrophilic reactive groups with an organic derivatizing agent. This derivatizing agent is capable of reacting with selected side chains of targeted amino acids on the targeting polypeptide or with N- or C-terminal residues and functional groups on M. Reactive groups on the targeting polypeptide and / or M include, for example, aldehyde, amino, ester, thiol, α-haloacetyl, maleimide, or hydrazino groups. Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine ​​residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, or other agents known in the art. Alternatively, the targeting polypeptide and / or M can be indirectly linked to each other through an intermediate carrier, such as a polysaccharide or polypeptide carrier. An example of a polysaccharide carrier is aminodextran. Examples of suitable polypeptide carriers to impart the desired 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 most commonly are reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetylphosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0221] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful, and the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

[0222] 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 residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactions with glyoxylate.

[0223] Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues must be performed under alkaline conditions due to the high pKa of the guanidine functional group. Furthermore, these reagents can react with the lysine groups as well as the arginine epsilon-amino group.

[0224] The specific modification of tyrosyl residues may be made, particularly with interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole 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 carbodiimides (RN=C=N-R'), where R and R' are different alkyl groups such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. 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 seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), deamidation of asparagine or glutamine, acetylation of N-terminal amines, and / or amidation or esterification of C-terminal carboxylic acid groups.

[0227] Another type of covalent modification involves chemically or enzymatically attaching glycosides to peptides. Sugar(s) can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as those of cysteine, (d) free hydroxyl groups, such as those of serine, threonine, or hydroxyproline, (e) aromatic residues, such as those of tyrosine or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 1987 / 05330 and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

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

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

[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] [ka]

[0232] where A and B are independently nucleophilic or electrophilic reactive groups. In some embodiments, 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] JPEG2025131777000008.jpg216169 JPEG2025131777000009.jpg195169 JPEG2025131777000010.jpg209169 JPEG2025131777000011.jpg209169 JPEG2025131777000012.jpg90169

[0234] In some embodiments, A and B may comprise alkene and / or alkyne functional groups suitable for olefin metathesis reactions. In some embodiments, A and B comprise moieties suitable for click chemistry (e.g., alkenes, alkynes, nitriles, azides). Other non-limiting examples of reactive groups (A and B) include pyridyldithiols, aryl azides, diazirines, carbodiimides, and hydrazides.

[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 by reference in its entirety. Suitable hydrophobic linking groups known in the art include, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoic acid. Prior to conjugation of the composition to the peptide, the hydrophobic linking group comprises at least two reactive groups (A and B), as described herein and shown below:

[0236] [ka]

[0237] In some embodiments, the hydrophobic linking group comprises either a maleimide or iodoacetyl group and either a carboxylic acid or an activated carboxylic acid (e.g., NHS ester) as the reactive group. In these embodiments, the maleimide or iodoacetyl group can be attached to a thiol moiety on the targeting polypeptide or M, and the carboxylic acid or activated carboxylic acid can be attached 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 activating agents described herein. In certain embodiments, the hydrophilic linking group comprises 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] [ka]

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

[0240] [ka]

[0241] In certain embodiments, the linking group is polyethylene glycol (PEG). In certain embodiments, PEG has a molecular weight of about 100 daltons to about 10,000 daltons, e.g., about 500 daltons to about 5000 daltons. In some embodiments, PEG has a molecular weight of about 10,000 daltons to about 40,000 daltons.

[0242] In some embodiments, the hydrophilic linking group comprises either a maleimide or an iodoacetyl group and either a carboxylic acid or an activated carboxylic acid (e.g., an NHS ester) as the reactive group. In these embodiments, the maleimide or iodoacetyl group can be attached to a thiol moiety on the targeting polypeptide or M, and the carboxylic acid or activated carboxylic acid can be attached to an amine on the targeting polypeptide or M, with or without the use of a coupling reagent. Any suitable coupling agent known to those of skill in the art can be used to couple the carboxylic acid to the amine, such as DCC, DIC, HATU, HBTU, TBTU, and other activating agents 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 comprises an amino acid, a dipeptide, a tripeptide, or a polypeptide, wherein the amino acid, dipeptide, tripeptide, or polypeptide comprises at least two activating groups described herein. In some embodiments, the linking group (L) comprises a moiety selected from the group consisting of amino, ether, thioether, maleimide, disulfide, amide, ester, thioester, alkene, cycloalkene, alkyne, trizoyl, carbamate, carbonate, cathepsin B cleavable, and hydrazone.

[0244] In some embodiments, L comprises a chain of 1 to about 60 atoms, or 1 to 30 or more atoms, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms in 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. Chain atoms and linkers can be selected for their expected solubility (hydrophilicity) to provide a more soluble conjugate. In some embodiments, L provides a functional group that is subject to cleavage by enzymes or other catalysts or hydrolysis conditions found in target tissues, organs, or cells. In some embodiments, the length of L is long enough to reduce the possibility of steric hindrance.

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

[0246] [ka]

[0247] In some embodiments, L is hydrolyzable in vivo. In these embodiments, L comprises a functional group capable of undergoing hydrolysis in vivo. Non-limiting examples of functional groups capable of undergoing hydrolysis in vivo include esters, anhydrides, and thioesters. For example, the following compound contains an ester group and is therefore capable of undergoing hydrolysis in vivo:

[0248] [ka]

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

[0250] In some embodiments, L is metastable in vivo. In these embodiments, L optionally includes a functional group that can be chemically or enzymatically cleaved in vivo over a period of time (e.g., an acid-labile, reduction-labile, or enzyme-labile functional group). 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, L is metastable, and the targeting polypeptide-LM conjugate is stable in an extracellular environment, e.g., serum, over the period of time described above, but is unstable in an intracellular environment or conditions that mimic an intracellular environment, such that it is cleaved upon entry into the cell. In some embodiments, when L is metastable, L is stable in serum for at least about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, or 48 hours, e.g., at least about 48, 54, 60, 66, or 72 hours, or for about 24-48, 48-72, 24-60, 36-48, 36-72, or 48-72 hours.

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

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

[0253] In another method for preparing the azide-containing polymer derivatives of the present invention, a linking agent having an azide functionality is contacted with a water-soluble polymer backbone having an average molecular weight of from about 800 Da to about 100,000 Da, the linking agent having chemical functionality that selectively reacts with chemical functionality on the polymer to form the azide-containing polymer derivative product, and the azide is separated from the polymer backbone by the 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 the azide functional group but reacts efficiently and selectively with the 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, if necessary, can be achieved by known methods, including, but not limited to, precipitation of the product followed by chromatography.

[0256] A more specific example is shown below, where in the case of a polymeric diamine, one of the amines is protected with a protecting group moiety such as tert-butyl-Boc, and the resulting monoprotected polymeric diamine is reacted with a linking moiety bearing an azide functionality: BocHN-polymer-NH2+HO2C-(CH2)3-N=N=N In this case, amine groups can be attached to carboxylic acid groups using thionyl chloride or carbodiimide reagents, as well as various activators such as N-hydroxysuccinimide or N-hydroxybenzotriazole, to create amide bonds between the monoamine polymer derivative and the azide-bearing linker moiety. After successful amide bond formation, the resulting N-tert-butyl-Boc-protected azide-containing derivative can be used directly to modify bioactive molecules or further elaborated to install other useful functional groups. For example, the Nt-Boc group can be hydrolyzed by treatment with strong acid to generate an omega-amino polymer azide. The resulting amine can be used as a synthetic handle to install other useful functional groups, such as maleimide groups, activated disulfides, or activated esters, to create valuable 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 for the attachment of a molecule bearing an activated electrophilic group, such as an aldehyde, ketone, activated ester, or activated carbonate, to one end of the polymer and a molecule bearing an acetylene group to the other end of the polymer.

[0258] In another embodiment of the present 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 the azide group, and Y is a suitable leaving group.

[0259] Multiple 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 multi-armed, allowing multiple targeting polypeptide molecules to be linked together. Multiple targeting polypeptides and modified targeting polypeptides may be linked to each other through their N-terminus in a head-to-head configuration through the use of such linkers or by direct chemical bonding between the respective N-terminus of each polypeptide. For example, two targeting polypeptides may be linked to form a dimer through chemical bonding between their N-terminal amino groups or modified N-terminal amino groups. Multiple targeting polypeptides may also be joined at their respective N-termini using a linking molecule designed to contain multiple chemical functional groups for binding to the N-terminus of each targeting polypeptide. Additionally, multiple 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 can be used to form dimers and multimers of the targeting polypeptides described herein include, but are not limited to, disulfide, sulfhydryl, or thiol bonds. In addition, certain enzymes, such as sortases, can be used to form covalent bonds between the targeting polypeptide and a linker at positions including the N-terminus of the targeting polypeptide.

[0260] Linkers can have a wide range of molecular weights or molecular lengths. Linkers with larger or smaller molecular weights can be used to provide the desired spatial relationship or conformation (if any) between the targeting polypeptide and the linked entity, or between the linked entity and its binding partner. Linkers with longer or shorter molecular lengths can also be used to provide the desired space or flexibility between the targeting polypeptide and the linked entity, 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 the 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. In some embodiments, the present invention 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 the targeting polypeptide or modified targeting polypeptide through a linker. For example, the linker may comprise one or two amino acids attached at one end to the polymer (e.g., an albumin-binding moiety) and at the other end to any available position on the polypeptide backbone. 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 may link the polymer to the targeting polypeptide or modified targeting polypeptide are disclosed in US2012 / 0295847 and WO / 2012 / 168430, each of which is incorporated herein by reference in its entirety.

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

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

[0265] As used herein, the terms "nucleophilic group," "nucleophile," and the like refer to an atom or group of atoms having an electron pair capable of forming a covalent bond. This type of group can be an ionizable group that reacts as an anionic group. As used herein, "nucleophilic group" 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 desired functional groups. The information provided is exemplary and not limiting to the synthetic techniques described herein.

[0267] [Table 2] JPEG2025131777000019.jpg160169

[0268] Generally, carbon electrophiles are susceptible to attack by complementary nucleophiles, including carbon nucleophiles, where the attacking nucleophile donates an electron pair to the carbon electrophile to form a new bond between the nucleophile and the carbon electrophile.

[0269] Non-limiting examples of carbon nucleophiles include alkyl, alkenyl, aryl, and alkynyl Grignards, organolithium, organozinc, alkyl-, alkenyl-, aryl-, and alkynyl-tin reagents (organostannanes), and alkyl-, alkenyl-, aryl-, and alkynyl-borane reagents (organoboranes and organoborons). 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 ylide, enol, and enolate reagents. These carbon nucleophiles have the advantage of being relatively easy to generate from precursors well known to those skilled in the art of synthetic organic chemistry. When used in conjunction with a carbon electrophile, a carbon nucleophile generates a new carbon-carbon bond between the carbon nucleophile and the carbon electrophile.

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

[0271] In some cases, the polymers used in the present invention are 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. Exemplary reactive groups include those commonly used to react with functional groups found on the 20 common amino acids (including, but not limited to, maleimide groups, activated carbonates (including, but not limited to, p-nitrophenyl esters), activated esters (including, but not limited to, N-hydroxysuccinimide, p-nitrophenyl esters), and aldehydes), as well as functional groups that are inert toward the 20 common amino acids but specifically react with complementary functional groups (including, but not limited to, azide groups, alkyne groups). Note that the other end of the polymer, represented in the above formula by Y, is attached either directly or indirectly to a targeting polypeptide via a naturally occurring or non-naturally 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 through the 20 common amino acids. For example, an azide group on a polymer can react with an alkyne group on a targeting polypeptide to form a Huisgen [3 + 2] cycloaddition product. Alternatively, an alkyne group on a polymer can react with an azide group present in a targeting polypeptide to form an analogous product. In some embodiments, where applicable, strong nucleophiles (including, but not limited to, hydrazine, hydrazide, hydroxylamine, semicarbazide) can react with aldehyde or ketone groups present in a targeting polypeptide to form hydrazones, oximes, or semicarbazones, which can optionally be further reduced by treatment with an appropriate reducing agent.Alternatively, strong nucleophiles can be incorporated into the targeting polypeptide via a non-naturally encoded amino acid and used to preferentially react with ketone or aldehyde groups present in the water-soluble polymer.

[0272] Any molecular weight of the polymer can be used as desired, including but not limited to, from about 100 Daltons (Da) to 100,000 Da or more (including but not limited to, in some cases, 0.1 to 50 kDa or 10 to 40 kDa). The molecular weight of the polymer can range widely, including but not limited to, from about 100 Da to about 100,000 Da or more. Polymers are available in the following sizes: 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10 The molecular weight of the polymer may be from about 100 Da to about 100,000 Da, including, but not limited to, 1,000 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. Branched chain polymers can also be used, including, but not limited to, polymer molecules in which each chain has a molecular weight in the range of 1 to 100 kDa (including, but not limited to, 1 to 50 kDa or 5 to 20 kDa). The molecular weight of each chain of the branched polymer can include, but is not limited to, from about 1,000 Da to about 100,000 Da or more.The molecular weight of each chain of the branched polymer is 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. The molecular weight of each branched polymer chain may be from about 1,000 Da to about 100,000 Da, including, but not limited to, 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 branched polymer chain is from about 1,000 Da to about 50,000 Da. In some embodiments, the molecular weight of each branched polymer chain is from about 1,000 Da to about 40,000 Da. In some embodiments, the molecular weight of each branched polymer chain is from about 5,000 Da to about 40,000 Da. In some embodiments, the molecular weight of each chain of the branched polymer is from about 5,000 Da to about 20,000 Da. A wide range of polymer molecules are described, including but not limited to, the Shearwater Polymers, Inc. catalog, the Nektar Therapeutics catalog (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 about 800 Da to about 100,000 Da. The polymer backbone of the water-soluble polymer can be poly(ethylene glycol). However, it should be understood that a wide variety of water-soluble polymers, including but not limited to poly(ethylene glycol), 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 the use of the term PEG or poly(ethylene glycol) is intended to encompass and include all such molecules. The term PEG includes, but is not limited to, poly(ethylene glycol) in any of its forms, including bifunctional PEG, multi-armed PEG, derivatized PEG, forked PEG, branched PEG, pendant PEG (i.e., PEG or related polymers having one or more functional groups pendant to the polymer backbone), or PEG having degradable linkages therein.

[0274] In addition to these forms of polymers, polymers can also be prepared with weak or degradable linkages in the backbone. For example, polymers can be prepared with ester linkages in the polymer backbone that undergo hydrolysis. This hydrolysis breaks the polymer into lower molecular weight fragments, as shown below: -Polymer-CO2-Polymer-+H2O → Polymer-CO2H+HO-Polymer- Many polymers are also suitable for use in the present invention. In some embodiments, water-soluble polymer backbones having from 2 to about 300 termini are particularly useful in the present invention. Examples of suitable polymers include, but are not limited to, 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. The molecular weight of each chain of the polymer backbone can vary but typically ranges from about 800 Da to about 100,000 Da, often from about 6,000 Da to about 80,000 Da. The molecular weight of each chain of the polymer backbone is approximately 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, and The molecular weight of each chain of the polymer backbone can be from about 100 Da to about 100,000 Da, including, but not limited to, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is from about 100 Da to about 50,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is from about 100 Da to about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is from about 1,000 Da to about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is from about 5,000 Da to about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is from about 10,000 Da to about 40,000 Da.

[0275] In one feature of this embodiment of the invention, the 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, as opposed 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 bonds that are stable during storage and administration. For example, the targeting polypeptide or modified targeting polypeptide linked to a polymer should maintain its integrity during the preparation of the final pharmaceutical composition, when used, when dissolved in an appropriate delivery vehicle, and when administered by any route.

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

[0278] Structure and synthesis of TLR-agonist linker derivatives: electrophilic and nucleophilic groups TLR agonist derivatives with linkers 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 hydrazines, hydrazides, and semicarbazides, the enhanced nucleophilicity of the aminooxy group allows it to react efficiently and selectively with a variety of 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 reaction with a hydrazine group results in 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 with linkers containing azides, alkynes, or cycloalkynes allow for the linking of molecules via cycloaddition reactions (e.g., 1,3-dipolar cycloaddition, azide-alkyne Huisgen cycloaddition, etc.). (Reactions to this extent are described in U.S. Pat. No. 7,807,619, incorporated herein by reference.)

[0279] Thus, in certain embodiments, TLR-agonist derivatives are described herein having linkers containing hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, keto-amine, keto-alkyne, and ene-dione hydroxylamine groups, hydroxylamine-like groups (which have reactivity similar to and are structurally similar to hydroxylamine groups), masked hydroxylamine groups (which can be readily converted to hydroxylamine groups), or protected hydroxylamine groups (which have reactivity similar to hydroxylamine groups upon deprotection). In some embodiments, the TLR agonist derivatives having linkers include azides, alkynes, or cycloalkynes.

[0280] Such TLR-agonist linker derivatives or targeting polypeptides may be in the form of a salt or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or polynucleotide, and may optionally be post-translationally modified.

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

[0282] The methods and compositions provided and described herein include polypeptides containing unnatural amino acids bearing at least one carbonyl or dicarbonyl group, oxime group, hydroxylamine group, or protected or masked forms thereof. The introduction of at least one reactive group into a TLR-agonist linker derivative or targeting polypeptide can allow for the application of conjugation chemistries that do not react with commonly occurring amino acids, but involve specific chemical reactions with one or more targeting polypeptide(s), including, but not limited to, those described herein. Once incorporated, the targeting polypeptide TLR side chains can also be modified by utilizing the chemical methodologies described herein or can be adapted to specific functional groups or substituents present on the TLR-agonist linker derivative or targeting polypeptide.

[0283] The TLR-agonist linker derivatives and targeting polypeptides, methods, and compositions described herein provide for conjugates of substances having a wide variety of functional groups, substituents, or moieties with other substances, including, but not limited to, polymers, water-soluble polymers, polyethylene glycol derivatives, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, and any combination thereof.

[0284] In certain embodiments, the TLR-agonist linker derivatives, targeting polypeptides, TCs, linkers, and reagents described herein, including compounds of Formulae (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 under weakly acidic conditions for at least one month. In other embodiments, such compounds are stable under weakly acidic conditions for at least two weeks. In other embodiments, such compounds are stable under weakly acidic conditions for at least five days.

[0285] In another aspect of the compositions, methods, techniques, and strategies, described herein are methods for studying or using any of the aforementioned "modified or unmodified" non-natural amino acid targeting 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 targeting polypeptides comprising "modified or unmodified" non-natural amino acid polypeptides or proteins.

[0286] The present invention provides TC molecules comprising at least one unnatural amino acid. In certain embodiments of the present invention, the TC with at least one unnatural amino acid comprises at least one post-translational modification. In one embodiment, the at least one post-translational modification is a label, a dye, a linker, another TC polypeptide, a polymer, a water-soluble polymer, a derivative of polyethylene glycol, a photocrosslinker, a radionuclide, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound, a resin, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, DNA, RNA, an antisense polynucleotide, a sugar, a cyclodextrin, an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that interacts covalently or non-covalently with other molecules, a photocaged moiety, an actin radiation-exciting moiety, a photoisomerizable moiety, or a combination thereof. The present invention also includes the attachment of molecules including, but not limited to, a moiety, biotin, a biotin derivative, a biotin analog, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, an elongated side chain, a carbon-linked sugar, a redox-active agent, an aminothioacid, a toxic moiety, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chemiluminescent group, an electron-dense group, a magnetic group, an intercalating group, a chromophore, an energy transfer agent, a biologically active agent, a detectable label, a small molecule, a quantum dot, a nanotransmitter, a radionucleotide, a radiotransmitter, a neutron capture agent, or any combination of the above, or any other desired compound or substance (including a first reactive group and a second reactive group to at least one unnatural amino acid containing a first reactive group utilizing chemical methodology known to those of skill in the art to be suitable for the particular reactive group). For example, the first reactive group is an alkynyl moiety (including but not limited to, in the unnatural amino acid p-propargyloxyphenylalanine, where the propargyl group is sometimes also referred to as an acetylene moiety) and the second reactive group is an azide moiety, and [3+2] cycloaddition chemistry methodology is utilized.In another example, the first reactive group is an azido moiety (including, but not limited to, in the unnatural amino acid p-azido-L-phenylalanine, or pAZ, sometimes referred to herein), and the second reactive group is an alkynyl moiety. In certain embodiments of the modified TCs of the present invention, at least one unnatural amino acid (including, but not limited to, an unnatural amino acid containing a keto functional group) is used that includes at least one post-translational modification, where the at least one post-translational modification includes a sugar moiety. In certain embodiments, the post-translational modification occurs in vivo in a eukaryotic cell or a non-eukaryotic cell. A linker, polymer, water-soluble polymer, or other molecule can attach the molecule to the polypeptide. In further embodiments, the linker attached to the TC is long enough to allow for dimer formation. The molecule can also be directly linked to the polypeptide.

[0287] In certain embodiments, the TC protein comprises at least one post-translational modification carried out in vivo by one host cell, where the post-translational modification is not normally carried out by another host cell type. In certain embodiments, the protein comprises at least one post-translational modification carried out in vivo by a eukaryotic cell, where the post-translational modification is not normally carried out by a non-eukaryotic cell. Examples of post-translational modifications include, but are not limited to, glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-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 the non-naturally encoded amino acid in the polypeptide.

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

[0291] A protein or polypeptide of interest can 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 ten or more unnatural amino acids. The unnatural amino acids can be the same or different, for example, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different sites in the protein that contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different unnatural amino acids. In certain embodiments, at least one, but fewer than all, of a particular amino acid present in the naturally occurring form of the protein is substituted with an unnatural amino acid.

[0292] The present invention provides methods and compositions based on TCs containing at least one non-naturally encoded amino acid. The introduction of at least one non-naturally encoded amino acid into a TC allows for the application of conjugation chemistry involving specific chemical reactions with one or more non-naturally encoded amino acids, including, but not limited to, those that are unreactive with the 20 commonly occurring amino acids. In some embodiments, TCs containing a non-naturally encoded amino acid are linked to a water-soluble polymer, such as polyethylene glycol (PEG), or a linker via the side chain of the non-naturally encoded amino acid. 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-genetically encoded amino acids, including, but not limited to, those amino acids containing functional groups or substituents not found in the 20 naturally incorporated amino acids, including, but not limited to, ketone, azide, or acetylene moieties, into proteins in response to a selector codon, followed by modification of those amino acids with a suitable reactive PEG derivative. Once incorporated, the amino acid side chain can then be modified to suit the specific functional group or substituent present on the non-naturally encoded amino acid by utilizing chemical methodologies known to those skilled in the art. A wide variety of known chemical methodologies are suitable for use in the present invention for incorporating water-soluble polymers into proteins, including, but not limited to, the Huisgen [3+2] cycloaddition reaction with, but not limited to, acetylene or azide derivatives, respectively (see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991) Ed. Trost, B.M., Pergamon, Oxford, pp. 1069-1109 and Huisgen, R. in 1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, New York, pp. 1-176).

[0293] Because the Huisgen [3 + 2] cycloaddition method involves cycloaddition rather than nucleophilic substitution, proteins can be modified with extremely high selectivity. The reaction can be carried out at room temperature in aqueous conditions with excellent regioselectivity (1,4 > 1,5) by adding a catalytic amount of Cu(I) salt to the reaction mixture. See, for example, Tornoe, et al., (2002) J. Org. Chem. 67:3057-3064 and Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599, and WO 03 / 101972. Molecules that can be added to proteins of the present invention via [3 + 2] cycloaddition include virtually any molecule with suitable functional groups or substituents, including, but not limited to, azide or acetylene derivatives. These molecules can be added to unnatural amino acids bearing an acetylene group, including but not limited to, p-propargyloxyphenylalanine, or an azide group, including but not limited to, p-azido-phenylalanine, respectively.

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

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

[0296] The present invention relates to a label, a dye, a polymer, a water soluble polymer, a derivative of polyethylene glycol, a photocrosslinker, a radionuclide, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound, a resin, a second protein or a polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, DNA, RNA, an antisense polynucleotide, a sugar, a water soluble dendrimer, a cyclodextrin, an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that interacts covalently or non-covalently with other molecules, a photocaging moiety, an actin radiation exciting moiety, a photoisomerizable moiety, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that interacts covalently or non-covalently with other molecules, a photocaging moiety, a photoisomerizable ... The present invention provides conjugates of substances bearing a wide variety of functional groups, substituents, or moieties with other substances, including, but not limited to, cleavable moieties, biotin, biotin derivatives, biotin analogs, moieties incorporating heavy atoms, chemically cleavable groups, photocleavable groups, elongated side chains, carbon-linked sugars, redox-active agents, aminothioacids, toxic moieties, isotopically 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, nanotransmitters, radionucleotides, radiotransmitters, neutron capture agents, or any combination of the above, or any other desired compound or substance. The present invention also includes conjugates of substances bearing azide or acetylene moieties with PEG polymer derivatives bearing the corresponding acetylene or azide moieties. For example, PEG polymers containing azide moieties can be attached to biologically active molecules at positions within proteins containing non-genetically encoded amino acids with acetylene functional groups. Linkages connecting PEG and biologically active molecules include, but are not limited to, Huisgen [3+2] cycloaddition products.

[0297] It is well established in the art that PEG can be used to modify the surface of biomaterials (see, e.g., U.S. Pat. No. 6,610,281; Mehvar, R., J. Pharm Pharm Sci., 3(1):125-136 (2000)), which are incorporated herein by reference. The present invention also includes biomaterials 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 linkage. Biomaterials and other substances can also be attached to azide- or acetylene-activated polymer derivatives 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 reaction.

[0298] The present invention includes methods 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 bonded to a carbon atom of the polymer. Alternatively, azide-containing PEG derivatives can be prepared by attaching a linking agent having an azide moiety at one terminus to a conventional activated polymer, such that the resulting polymer has an azide moiety at its terminus. In the case of acetylene-containing PEG derivatives, the acetylene can be directly bonded to a carbon atom of the polymer. Alternatively, acetylene-containing PEG derivatives can be prepared by attaching a linking agent having an acetylene moiety at one terminus to a conventional activated polymer, such that the resulting polymer has an acetylene moiety at its terminus.

[0299] More specifically, in the case of azide-containing PEG derivatives, a water-soluble polymer bearing at least one activated hydroxyl moiety is reacted to produce a substituted polymer bearing a more reactive moiety thereon, such as a mesylate, tresylate, tosylate, or halogen leaving group. The preparation and use of PEG derivatives or TLR-linker derivatives containing sulfonyl acid halides, halogen atoms, and other leaving groups are known to those skilled in the art. The resulting substituted polymer is then reacted to substitute the more reactive moiety for the azide moiety at the end of the polymer. Alternatively, a water-soluble polymer bearing at least one activated nucleophilic or electrophilic moiety is reacted with a linking agent bearing an azide at one end, such that a covalent bond is formed between the PEG polymer and the linking agent, 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, and the like, are known to those skilled in the art.

[0300] More specifically, in the case of acetylene-containing PEG derivatives, a water-soluble polymer having at least one activated hydroxyl moiety undergoes a reaction to displace a halogen or other activated leaving group from a precursor containing an acetylene moiety. Alternatively, a water-soluble polymer having at least one activated nucleophilic or electrophilic moiety undergoes a reaction with a linking agent having an acetylene at one terminus, such that a covalent bond is formed between the PEG polymer and the linking agent, and the acetylene moiety is positioned at the terminus 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 is well established in the art.

[0301] The present invention also provides methods for the selective modification of proteins to add other substances to the modified protein, including, but not limited to, water-soluble polymers such as PEG and PEG derivatives or TLR-linker derivatives, linkers, or other TLR polypeptides containing azide or acetylene moieties. The azide and acetylene-containing PEG derivatives or TLR-linker derivatives can be used to modify surface and molecular properties where biocompatibility, stability, solubility, and lack of immunogenicity are important, while providing a more selective means of attaching PEG derivatives or TLR-linker derivatives to proteins than previously known in the art.

[0302] General Recombinant Nucleic Acid Methods for Use in the Invention In many embodiments of the present invention, nucleic acids encoding TC targeting polypeptides of interest 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 TC targeting polypeptides. In some embodiments, the sequences encoding the polypeptides of the present invention are operably linked to a heterologous promoter.

[0303] A nucleotide sequence encoding a targeting polypeptide of a TC containing a non-naturally encoded amino acid can be synthesized based on the amino acid sequence of the parent polypeptide, and then the nucleotide sequence can be altered to result in 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 using conventional methods. Alternatively, the nucleotide sequence can be prepared by chemical synthesis, including, but not limited to, using an oligonucleotide synthesizer, where oligonucleotides are designed based on the amino acid sequence of the desired polypeptide, preferably selecting those codons that are preferred in the host cell in which the recombinant polypeptide will 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. Pat. No. 6,521,427 (incorporated herein by reference).

[0304] The present invention utilizes conventional techniques in the field of recombinant genetics. A basic text disclosing the general methods for use in the present invention is Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd Edition). 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, non-eukaryotic host cells, and organisms for the in vivo incorporation of unnatural amino acids via orthogonal tRNA / RS pairs. Host cells are genetically engineered (including, but not limited to, transformed, transduced, or transfected) with polynucleotides of the invention or constructs comprising polynucleotides of the invention, including, but not limited to, vectors of the invention, which can be, for example, cloning vectors or expression vectors.

[0306] Several well-known methods for introducing target nucleic acids into cells are available, any of which can be used in the present invention. These include fusion of recipient cells with bacterial protoplasts containing DNA, electroporation, projectile 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. Bacteria can be grown to logarithmic phase, and the plasmids within the bacteria can be isolated by various methods known in the art (see, e.g., Sambrook). In addition, kits are commercially available for purifying plasmids from bacteria (see, e.g., EasyPrep™, FlexiPrep™ (both Pharmacia Biotech), StrataClean™ (Stratagene), and QIAprep™ (Qiagen)). The isolated and purified plasmids can then be further manipulated to produce other plasmids and used to transfect cells, or incorporated into related vectors and used to infect organisms. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulating the expression of specific target nucleic acids. Vectors optionally contain at least one independent terminator sequence, a general expression cassette containing sequences that allow replication of the cassette in eukaryotes, prokaryotes, or both (including, but not limited to, shuttle vectors), and selectable markers for both prokaryotic and eukaryotic systems. Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or both. See Gillam & Smith, Gene 8:81 (1979); Roberts, et al., Nature, 328:731 (1987); Schneider, E., et al., Protein Expr. Purif. 6(1):10-14 (1995); Ausubel, Sambrook, Berger (all supra).Catalogs of bacteria and bacteriophages useful for cloning are provided, for example, by the ATCC, e.g., The ATCC Catalogue of Bacteria and Bacteriophage (1992) Gherna et al. (eds.). Additional basic procedures for sequencing, cloning, and other aspects of molecular biology, as well as underlying theoretical considerations, are provided in Watson et al. (1992) Recombinant DNA. Second Edition Scientific American Books, NY Additionally, essentially any nucleic acid (and virtually any labeled nucleic acid, whether standard or non-standard) can be custom or standard ordered from any of a variety of commercial sources, including 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 others.

[0307] Selector codon The selector codons of the present invention expand the genetic codon framework of the protein biosynthetic machinery. For example, selector codons include, but are not limited to, unique three-base codons, nonsense codons such as stop codons (including but not limited to, amber codon (UAG), ochre codon, or opal codon (UGA)), unnatural codons, four or more base codons, rare codons, etc. It will be readily apparent to one of skill in the art that the number of selector codons that can be introduced into a desired gene or polynucleotide can vary widely, 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 a TC.

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

[0309] Incorporation of unnatural amino acids in vivo can be achieved without significant perturbation of the eukaryotic host cell. For example, because the efficiency of suppression of the UAG codon depends on competition between an O-tRNA, including but not limited to, an amber suppressor tRNA, and a eukaryotic release factor (including but not limited to, an eRF), which binds to the stop codon and initiates release of the growing peptide from the ribosome, the efficiency of suppression can be modulated by, including but not limited to, increasing the expression level of the O-tRNA and / or the suppressor tRNA.

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

[0311] Selector codons also include, but are not limited to, extended codons containing four or more base codons, for example, four, five, six, or more base codons. Examples of four-base codons include, but are not limited to, AGGA, CUAG, UAGA, and CCCU. Examples of five-base codons include, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU, and UAGGC. A feature of the present invention involves the use of extended codons based on frameshift suppression. Four or more base codons, including, but not limited to, one or more unnatural amino acids, can be inserted into the same protein. For example, in the presence of a mutant O-tRNA with an anticodon loop, including, but not limited to, a special frameshift suppressor tRNA with an anticodon loop of at least 8-10 nt, the four or more base codon is read as a single amino acid. In other embodiments, anticodon loops containing, but not limited to, at least four-base codons, at least five-base codons, or at least six-base codons or more can be decoded. Because there are 256 possible four-base codons, multiple unnatural 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, e.g., Ma et al., (1993) Biochemistry, 32:7939, and Hohsaka et al., (1999) J. Am. Chem. Soc., 121:34. CGGG and AGGU were used to simultaneously incorporate 2-naphthylalanine and an NBD derivative of lysine into streptavidin in vitro using two chemically acylated frameshift suppressor tRNAs. See, e.g., Hohsaka et al., (1999) J. Am. Chem. Soc., 121:12194. In an in vivo study, Moore et al. investigated the ability of tRNALeu derivatives with an NCUA anticodon to suppress the UAGN codon (where N can be U, A, G, or C) and found that the quadruple UAGA could be decoded by tRNALeu with a UCUA anticodon with 13-26% efficiency, with little decoding in the 0 or -1 frame. See Moore et al., (2000) J. Mol. Biol., 298:195. In one embodiment, extended codons based on rare or nonsense codons can be used in the present invention, which can reduce missense readthrough and frameshift suppression at otherwise undesirable sites.

[0313] For a given system, the selector codon can also include one of the natural three base codons, where the endogenous system does not use (or rarely uses) the natural base codon, for example, this includes systems that lack a tRNA that recognizes the natural three base codon and / or systems where the three base codon is a rare codon.

[0314] The selector codon optionally contains an unnatural base pair. These unnatural base pairs further expand the existing genetic alphabet. One extra base pair increases the number of triplet codons from 64 to 125. Properties of the third base pair include stable selective base pairing, efficient enzymatic incorporation into DNA with high fidelity by polymerases, and efficient continued primer extension after synthesis of the nascent unnatural base pair. Descriptions of unnatural base pairs that can be adapted for 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 relevant publications are listed below.

[0315] For in vivo use, unnatural nucleosides are membrane-permeable and phosphorylated to form the corresponding triphosphate. Additionally, the amplified genetic information is stable and not destroyed by cellular enzymes. Previous attempts by Benner et al. utilized hydrogen-bonding patterns different from those of canonical Watson-Crick pairs, the most notable example of which is 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 degree with natural bases and cannot be enzymatically replicated. Kool and colleagues showed that hydrophobic packing interactions between bases can replace hydrogen bonds and drive base pair formation. See Kool, (2000) Curr. Opin. Chem. Biol., 4:602 and Guckian and Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. With the aim 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. The PICS:PICS self-pair was found to be more stable than natural base pairs and can be efficiently incorporated into DNA by the Klenow fragment of Escherichia coli DNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem. Soc., 121:11585-6 and Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274. The 3MN:3MN self-pair can be synthesized by KF with sufficient efficiency and selectivity for biological function. See, for example, Ogawa et al., (2000) J. Am. Chem. Soc., 122:8803. However, both bases act as chain terminators for further replication. A mutant DNA polymerase has recently been devised that can be used to replicate the PICS self-pair. In addition, it can replicate the 7AI self-pair.See, e.g., Tae et al., (2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py, has also been developed, which forms a stable pair upon binding Cu(II). See Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Because extended codons and unnatural codons are inherently orthogonal to natural codons, the methods of the invention can take advantage of this property to generate orthogonal tRNAs for them.

[0316]

[0003] Translational bypassing systems can also be used to incorporate unnatural amino acids into a desired polypeptide. In translational bypassing systems, a large sequence is incorporated into a gene but is not translated into protein. This sequence contains a structure that acts as a cue to direct the ribosome to skip the sequence and resume translation downstream of the insertion.

[0317] Nucleic acid molecules encoding proteins of interest, such as targeting polypeptides of TC, can be easily mutated to introduce cysteine ​​into 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 into proteins of interest.Suitable methods for incorporating cysteine ​​into desired positions of polypeptides are known to those skilled in the art, such as those described in U.S. Patent No. 6,608,183, which is incorporated herein by reference, and standard mutagenesis techniques.

[0318] III. Non-Naturally Encoded Amino Acids A 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 a TC. Typically, the introduced non-naturally encoded amino acid is substantially chemically inert toward the 20 common genetically encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). In some embodiments, the non-naturally encoded amino acid contains a side chain functional group that reacts efficiently and selectively with functional groups not found on the 20 common amino acids (including, but not limited to, azide, ketone, aldehyde, and aminooxy groups) to form stable conjugates. For example, a targeting polypeptide of a TC comprising 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 to form a Huisgen [3+2] cycloaddition product.

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

[0320] [ka]

[0321] Non-naturally encoded amino acids are typically any structure having the formula listed above, where the R group is any substituent other than those used in the 20 naturally occurring amino acids, and may be suitable for use in the present invention. Non-naturally encoded amino acids of the present invention typically differ from natural amino acids only in the structure of their side chains; therefore, non-naturally encoded amino acids form amide bonds with other amino acids, including, but not limited to, natural or non-naturally encoded, in the same manner as they do in naturally occurring polypeptides. However, non-naturally encoded amino acids have side chain groups that distinguish them from natural amino acids. For example, R optionally includes alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether, thiol, seleno, sulfonyl, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino group, etc., or any combination thereof. Other non-naturally occurring amino acids of interest that may be suitable for use in the present invention include, but are not limited to, amino acids comprising photoactivatable crosslinkers, 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, photocaged and / or photoisomerizable amino acids, amino acids comprising biotin or biotin analogs, glycosylated amino acids such as sugar-substituted serines, other carbohydrate-modified amino acids, keto-containing amino acids, amino acids comprising polyethylene glycol or polyethers, heavy atom-substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids with elongated side chains compared to natural amino acids (including, but not limited to, polyethers or long-chain hydrocarbons containing, but not limited to, more than about 5 or more than about 10 carbons), carbon-linked sugar-containing amino acids, oxygen-reducing active amino acids, aminothioacid-containing amino acids, and amino acids comprising one or more toxin moieties.

[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 with carbonyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide, and alkyne reactive groups. In some embodiments, the non-naturally encoded amino acid comprises a sugar moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine. Examples of such amino acids also include those in which the naturally occurring N- or O-linkage between the amino acid and the sugar is replaced with a covalent bond not commonly found in nature, including, but not limited to, an alkene, oxime, thioether, amide, etc. Examples of such amino acids also include sugars not commonly found in naturally occurring proteins, such as 2-deoxy-glucose and 2-deoxygalactose.

[0323] Many of the non-naturally encoded amino acids provided herein are commercially available, for example, from Sigma-Aldrich (St. Louis, MO, USA), Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), or Peptech (Burlington, MA, USA). Those that are not commercially available are optionally synthesized as provided herein or using standard methods known to those of skill in the art. For organic synthesis techniques, see, for example, Organic Chemistry by Fessendon and Fessendon, (1982, Second Edition, Willard Grant Press, Boston Mass.), Advanced Organic Chemistry See, for example, "The Organic Chemistry of the Invention" 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] [ka]

[0325] [ka]

[0326] (wherein Z typically comprises OH, NH, SH, NH-R', or S-R'; X and Y can be the same or different and typically comprise S or O; and R and R' can optionally be the same or different and are typically selected from the same list of R group components described above for the unnatural amino acids with Formula I and hydrogen. For example, unnatural amino acids of the invention optionally comprise substitutions at the amino or carboxyl group, as shown by Formulas II and III. Unnatural amino acids of this type include, but are not limited to, α-hydroxy acids, α-thioacids, α-aminothiocarboxylates, having side chains including, but not limited to, those corresponding to the 20 common natural amino acids or unnatural side chains. Additionally, substitutions at the α carbon optionally include, but are not limited to, α-α-disubstituted amino acids such as L, D, or D-glutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, etc. 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, and are suitable for use in the present invention. Tyrosine analogs include, but are not limited to, para-substituted tyrosines, ortho-substituted tyrosines, and meta-substituted tyrosines, where the substituted tyrosines may have a keto group (including, but not limited to, an acetyl group), a benzoyl group, an amino group, a hydrazine, a hydroxylamine, a thiol group, a carboxy group, an isopropyl group, a methyl group, a C6-C 20Examples of aryl groups include, but are not limited to, straight-chain or branched hydrocarbons, saturated or unsaturated hydrocarbons, O-methyl groups, polyether groups, nitro groups, alkynyl groups, and the like. 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, aldehyde, azido, iodo, bromo, keto groups (including, but not limited to, acetyl groups), benzoyl, alkynyl groups, and the like. Specific examples of unnatural amino acids that may be suitable for use in the present invention include, but are not limited to, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAc β-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, and p-propargyloxy-phenylalanine. Examples of structures of various unnatural amino acids that may be suitable for use in the present 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, incorporated herein by reference. International Application No. PCT / US06 / 47822, entitled "Compositions Containing, Methods Involving, and Uses of Non-natural Amino Acids and Polypeptides," incorporated herein by reference, describes reductive alkylation of aromatic amine moieties, including, but not limited to, p-amino-phenylalanine and reductive amination.

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

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

[0330] In one embodiment, a composition of a TC targeting polypeptide is provided that includes an unnatural amino acid (such as p-(propargyloxy)-phenylalanine). Various compositions that include p-(propargyloxy)-phenylalanine are also provided, including, but not limited to, proteins and / or cells. In one aspect, the composition that includes the p-(propargyloxy)-phenylalanine unnatural amino acid further includes an orthogonal tRNA. The unnatural amino acid can be attached (including but not limited to, covalently) to the orthogonal tRNA, including, but not limited to, covalently attached to the orthogonal tRNA through an amino-acyl bond, covalently attached to the 3' OH or 2' OH of the terminal ribose sugar of the orthogonal tRNA, etc.

[0331] Chemical moieties that can be incorporated into proteins via unnatural amino acids offer a variety of advantages and manipulations of proteins. For example, the inherent reactivity of the keto functional group allows for the selective modification of proteins in vitro and in vivo with any of several hydrazine- or hydroxylamine-containing reagents. Heavy atom unnatural amino acids can be useful, for example, for phasing X-ray structural data. Site-specific introduction of heavy atoms using unnatural amino acids also offers selectivity and flexibility in choosing the location of the heavy atoms. Photoreactive unnatural amino acids (including, but not limited to, amino acids with benzophenone and aryl azide (including, but not limited to, phenyl azide) side chains) allow, for example, efficient photocrosslinking of proteins in vivo and in vitro. Examples of photoreactive unnatural amino acids include, but are not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine. Proteins bearing photoreactive unnatural amino acids can then be optionally crosslinked by excitation of the photoreactive group, providing temporal control. In one example, the methyl group of the unnatural amino can be replaced with isotopically labeled, including but not limited to, methyl groups as probes of local structure and dynamics, including but not limited to, the use of nuclear magnetic resonance and vibrational spectroscopy. For example, alkynyl or azido functional groups allow for the selective modification of proteins with molecules through a [3+2] cycloaddition reaction.

[0332] Unnatural amino acids incorporated into polypeptides at the amino terminus may be comprised of an R group that is any substituent other than those used for the 20 natural amino acids, and a second reactive group that is different from the NH group normally present in alpha-amino acids. Similar unnatural amino acids can be incorporated at the C-terminus with a second reactive group that is different from the COOH group normally present in alpha-amino acids.

[0333]

[00130] The unnatural amino acids of the invention can be selected or designed to provide additional characteristics not available with the 20 natural amino acids. For example, unnatural amino acids can be optionally designed or selected to, e.g., modify the biological properties of the protein into which they are incorporated. For example, the following properties can be optionally modified by including an unnatural amino acid in a protein: toxicity, biodistribution, solubility, stability (e.g., resistance to heat, hydrolysis, oxidation, enzymatic degradation, etc.), ease of purification and processing, structural properties, spectroscopic properties, chemical and / or photochemical properties, catalytic activity, redox potential, half-life, ability to react with other molecules (e.g., covalently or non-covalently, etc.).

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

[0335] Some embodiments of the present invention utilize TC polypeptides 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), which is incorporated by reference. Other carbonyl- or dicarbonyl-containing amino acids can be similarly prepared by one of skill in the art. Additionally, non-limiting exemplary syntheses of unnatural amino acids included herein are provided in U.S. Patent No. 7,083,970, which is incorporated by reference in its entirety.

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

[0337] [ka]

[0338] During the ceremony, A is optional and, if present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, if present, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(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 is a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')- (each R' is independently H, alkyl, or substituted alkyl); J is

[0339] [ka]

[0340] and R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; each R" is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R" group is present, two R" optionally form a heterocycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4, or two R3 groups, optionally form a cycloalkyl or heterocycloalkyl, or or -ABJR groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl containing at least one carbonyl group including a dicarbonyl group, a protected carbonyl group including a protected dicarbonyl group, or a masked carbonyl group including a masked dicarbonyl group; or -JR groups together form a monocyclic or bicyclic cycloalkyl or heterocycloalkyl containing at least one carbonyl group including a dicarbonyl group, a protected carbonyl group including a protected dicarbonyl group, or a masked carbonyl group including a masked dicarbonyl group; provided that when A is phenylene and each R is H, then B is present; when A is —(CH)— and each R is H, then B is not —NHC(O)(CHCH)—; and when A and B are absent and each R is H, then R is not methyl.

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

[0342] [ka]

[0343] During the ceremony, A is optional and, if present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, if present, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(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 is a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')- (each R' is independently H, alkyl, or substituted alkyl); R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if 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] Additionally, amino acids having the structure of formula (VI) are included:

[0345] [ka]

[0346] During the ceremony, 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) kis a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')- (each R' is independently H, alkyl, or substituted alkyl); R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each R a are independently H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) k R', where each R' is independently H, alkyl, or substituted alkyl.

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

[0348] [ka]

[0349] (wherein such compounds are optionally amino-protected, carboxyl-protected, or salts thereof.) Additionally, any of the following unnatural amino acids can be incorporated into the unnatural amino acid polypeptides:

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

[0351] [ka]

[0352] During the ceremony, B is optional and, if present, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(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 is a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')- (each R' is independently H, alkyl, or substituted alkyl); R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each R a are independently H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) kR' (each R' is independently H, alkyl, or substituted alkyl), and n is 0 to 8; provided that when A is -(CH2)4-, then B is not -NHC(O)(CH2CH2)-.

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

[0354] [ka]

[0355] (wherein such compounds are optionally amino-protected, optionally carboxyl-protected, optionally amino-protected and carboxyl-protected, or salts thereof.) In addition, these unnatural amino acids, as well as any of the following unnatural amino acids, can be incorporated into unnatural amino acid polypeptides.

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

[0357] [ka]

[0358] wherein A is optional and, if present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, if present, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(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 is a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')- (each R' is independently H, alkyl, or substituted alkyl); R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

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

[0360] [ka]

[0361] B is optional and, if present, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(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 is a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')- (each R' is independently H, alkyl, or substituted alkyl); R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each R a are independently H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) kR', where each R' is independently H, alkyl, or substituted alkyl.

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

[0363] [ka]

[0364] (wherein such compounds are optionally amino-protected, optionally carboxyl-protected, optionally amino-protected and carboxyl-protected, or salts thereof.) In addition, these unnatural amino acids, as well as any of the following unnatural amino acids, can be incorporated into unnatural amino acid polypeptides.

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

[0366] [ka]

[0367] In the formula, B is optional and, if present, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(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 is a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')- (each R' is independently H, alkyl, or substituted alkyl); R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each R a are independently H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) k R' (each R' is independently H, alkyl, or substituted alkyl), and n is 0-8.

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

[0369] [ka]

[0370] (wherein such compounds are optionally amino-protected, optionally carboxyl-protected, optionally amino-protected and carboxyl-protected, or salts thereof.) In addition, these unnatural amino acids, as well as any of the following unnatural amino acids, can be incorporated into unnatural amino acid polypeptides.

[0371] In addition to monocarbonyl structures, the unnatural amino acids described herein can contain 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] [ka]

[0374] wherein A is optional and, if present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, if present, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(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 is a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')- (each R' is independently H, alkyl, or substituted alkyl); R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

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

[0376] [ka]

[0377] B is optional and, if present, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k-(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 is a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')- (each R' is independently H, alkyl, or substituted alkyl); R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each R a are independently H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) k R', where each R' is independently H, alkyl, or substituted alkyl.

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

[0379] [ka]

[0380] (wherein such compounds are optionally amino-protected, optionally carboxyl-protected, optionally amino-protected and carboxyl-protected, or salts thereof.) In addition, these unnatural amino acids, as well as any of the following unnatural amino acids, can be incorporated into unnatural amino acid polypeptides.

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

[0382] [ka]

[0383] In the formula, B is optional and, if present, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(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) kis a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')- (each R' is independently H, alkyl, or substituted alkyl); R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each R a are independently H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) k R' (each R' is independently H, alkyl, or substituted alkyl), and n is 0-8.

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

[0385] [ka]

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

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

[0388] [ka]

[0389] During the ceremony, A is optional and, if present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if 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.

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

[0391] [ka]

[0392] During the ceremony, A is optional and, if present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if 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] Additionally, the following amino acids having the structure of formula (XIV-B) are included:

[0394] [ka]

[0395] During the ceremony, A is optional and, if present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if 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] Additionally, the following amino acids having the structure of formula (XV) are included:

[0397] [ka]

[0398] During the ceremony, A is optional and, if present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; X1 is C, S, or S(O), n is 0, 1, 2, 3, 4, or 5, and each CR 8 R 9 Each R on the group 8 and R 9are independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 and R 9 can be taken together to form =O or cycloalkyl, or any adjacent R 8 The groups can be taken together to form a cycloalkyl.

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

[0400] [ka]

[0401] During the ceremony, A is optional and, if present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; n is 0, 1, 2, 3, 4, or 5, and each CR 8 R 9 Each R on the group 8 and R 9 are independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 and R 9can together form =O or cycloalkyl, or any adjacent R 8 The groups can be taken together to form a cycloalkyl.

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

[0403] [ka]

[0404] During the ceremony, A is optional and, if present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; n is 0, 1, 2, 3, 4, or 5, and each CR 8 R 9 Each R on the group 8 and R 9 are independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 and R 9 can together form =O or cycloalkyl, or any adjacent R 8 The groups can be taken together to form a cycloalkyl.

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

[0406] [ka]

[0407] During the ceremony, A is optional and, if present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if 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] Additionally, the following amino acids having the structure of formula (XVI-A) are included:

[0409] [ka]

[0410] During the ceremony, A is optional and, if present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if 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.

[0411] Additionally, the following amino acids having the structure of formula (XVI-B) are included:

[0412] [ka]

[0413] During the ceremony, A is optional and, if present, is lower alkylene, substituted lower alkylene, l...

Claims

1. 1. A composition comprising one or more targeting polypeptides having one or more unnatural amino acids incorporated therein, at least one of the polypeptides is linked to a TLR agonist molecule via a linker that is covalently attached to the unnatural amino acid of the targeting polypeptide; The TLR agonist is a TLR agonist having the molecular structure shown in Core 5 below, or a salt thereof; 【Chemistry 1】 where: X is O, S, NH, or H; R １ is C １ ~C １２ Alkyl, substituted C １ ~C １２ Alkyl, oxygen-containing C １ ~C １２ Alkyl, heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, -N ３ , terminal C １ ~C １２ Alkyl, terminally substituted C １ ~C １２ alkyl, or absent, R ２ or R ３ are respectively, C ４ ~C ８ cycloalkyl, or independently —H, C １ ~C １２ linked to form an alkyl, nitro-containing alkyl, oxygen-containing alkyl, or aromatic ring; R ４ HA, -ONH ２ , terminal C １ ~C １２ Alkyl, C １ ~C １２ Alkyl, C １ ~C １２ Substituted alkyl, C ４ ~C ８ is cycloalkyl, an aromatic ring, a substituted aromatic ring, a heteroaromatic ring, a substituted heteroaromatic ring, or absent; Z １ is C １ ~C ６ Alkyl, C ３ ~C １０ cycloalkyl, or C ３ ~C １０ is a nitro-containing heterocycle, Z ２ is an aromatic ring, an aromatic heterocycle, C ２ ~C ８ cycloalkyl or absent, and Z ３ is C １ ~C １２ Alkyl, substituted C １ ~C １２ Alkyl, C ３ ~C ８ Cycloalkyl, substituted C ３ ~C ８ Cycloalkyl, substituted C ３ ~C ８ Nitro-containing heterocycles, C ３ ~C ８ a nitro-containing heterocycle or is absent, where: The term "alkyl" is also meant to include those derivatives of alkyl defined in more detail herein, such as "heteroalkyl," "haloalkyl," and "homoalkyl," where: The term "heteroalkyl" refers to a straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of an alkyl group and at least one heteroatom selected from the group consisting of O, N, Si, and S, wherein the nitrogen and sulfur atoms can be optionally oxidized, and the nitrogen heteroatom can be optionally quaternized; where: The term "cycloalkyl," by itself or in combination with other terms, represents a cyclic version of "alkyl."

2. 1. A composition comprising an anti-HER2 antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain and the light chain are: (a) SEQ ID NOs: 1 and 7; (b) SEQ ID NOs: 2 and 3; (c) SEQ ID NOs: 2 and 4; (d) SEQ ID NOs: 2 and 5; (e) SEQ ID NOs: 2 and 7; and (f) SEQ ID NOs: 2 and 8 and comprising an amino acid sequence selected from the group consisting of: one or more of SEQ ID NOs: 2, 4, 5, 7, and 8 comprises a para-acetyl-phenylalanine covalently attached to a Toll-like receptor (TLR) agonist by an oxime bond; and The TLR agonist has the following structural formula: 【Chemistry 2】 or a salt thereof.

3. The composition of claim 2 , wherein the anti-HER2 antibody comprises an IgG.

4. 4. The composition of claim 2 or 3, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 1 and the light chain comprises the amino acid sequence of SEQ ID NO: 7, wherein SEQ ID NO: 7 has a single para-acetyl-phenylalanine at position 121 according to Kabat numbering.

5. 4. The composition of claim 2 or 3, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 2 and the light chain comprises the amino acid sequence of SEQ ID NO: 3, wherein SEQ ID NO: 2 has a para-acetyl-phenylalanine at position 114 according to Kabat numbering.

6. 4. The composition of claim 2 or 3, wherein the heavy chain and the light chain comprise the amino acid sequences of SEQ ID NOs: 2 and 4, wherein SEQ ID NO: 2 has a single para-acetyl-phenylalanine at position 114 according to Kabat numbering and SEQ ID NO: 4 has a single para-acetyl-phenylalanine at position 110 according to Kabat numbering.

7. 4. The composition of claim 2 or 3, wherein the heavy chain and the light chain comprise the amino acid sequences of SEQ ID NOs: 2 and 5, wherein SEQ ID NO: 2 has a single para-acetyl-phenylalanine at position 114 according to Kabat numbering and SEQ ID NO: 5 has a single para-acetyl-phenylalanine at position 112 according to Kabat numbering.

8. 4. The composition of claim 2 or 3, wherein the heavy chain and the light chain comprise the amino acid sequences of SEQ ID NOs: 2 and 7, wherein SEQ ID NO: 2 has a single para-acetyl-phenylalanine at position 114 according to Kabat numbering and SEQ ID NO: 7 has a single para-acetyl-phenylalanine at position 121 according to Kabat numbering.

9. 4. The composition of claim 2 or 3, wherein the heavy chain and the light chain comprise the amino acid sequences of SEQ ID NOs: 2 and 8, wherein SEQ ID NO: 2 has a single para-acetyl-phenylalanine at position 114 according to Kabat numbering and SEQ ID NO: 8 has a single para-acetyl-phenylalanine at position 127 according to Kabat numbering.

10. The anti-HER2 antibody fragment comprises: (i) Fab, (Fab')2, Fv, or single-chain Fv (scFv); (ii) one or more Fab, (Fab')2, Fv, or single-chain Fv (scFv) variants; or (iii) one or more Fc mutations The composition according to any one of claims 2 to 9, comprising:

11. 11. The composition of claim 2, wherein the TLR agonist is: 【Transformation 3】 The composition.

12. 11. The composition of claim 2, wherein the TLR agonist is: 【Chemistry 4】 The composition is a salt of

13. A composition described in any one of claims 2 to 12, wherein the drug-to-antibody ratio (DAR) is about 1 to about 2, or 1 or 2.

14. The composition described in claim 13, wherein the DAR is 2.

15. The composition comprises an anti-HER2 antibody-drug conjugate (ADC) comprising an anti-HER2 antibody or antigen-binding fragment thereof, wherein the heavy chain and the light chain comprise the amino acid sequences of SEQ ID NOs: 1 and 7, where SEQ ID NO: 7 has a para-acetyl-phenylalanine at position 121 according to Kabat numbering, and the anti-HER2 ADC has the following structure: 【Transformation 5】 the drug-antibody ratio (DAR) is 2; The composition of claim 2.

16. The composition comprises an anti-HER2 antibody-drug conjugate (ADC) comprising an anti-HER2 antibody or antigen-binding fragment thereof, wherein the heavy chain and the light chain comprise the amino acid sequences of SEQ ID NOs: 2 and 3, where SEQ ID NO: 2 has a para-acetyl-phenylalanine at position 114 according to Kabat numbering, and the anti-HER2 ADC has the following structure: 【Transformation 6】 the drug-antibody ratio (DAR) is 2; The composition of claim 2.

17. A pharmaceutical composition comprising a therapeutically effective amount of the composition of any one of claims 1 to 16 and a pharmaceutically acceptable carrier or excipient.

18. A pharmaceutical composition for treating HER2-positive cancer, comprising the composition according to any one of claims 1 to 16.

19. A pharmaceutical composition for use in treating HER2-positive cancer, comprising the composition of any one of claims 1 to 16 and a pharmaceutically acceptable carrier or excipient.

20. Use of a composition according to any one of claims 1 to 16 in the manufacture of a medicament for the treatment of HER2-positive cancer.

21. A composition according to any one of claims 1 to 16 for use in the treatment of HER2-positive cancer.

22. 20. The pharmaceutical composition of claim 18, or the pharmaceutical composition for use of claim 19, wherein the HER2-positive cancer is breast cancer, ovarian cancer, bladder cancer, gastric cancer, head and neck cancer, esophageal cancer, brain cancer or lung cancer.

23. The use of claim 20, wherein the HER2-positive cancer is breast cancer, ovarian cancer, bladder cancer, gastric cancer, head and neck cancer, esophageal cancer, brain cancer or lung cancer.

24. The composition described in claim 21, wherein the HER2-positive cancer is breast cancer, ovarian cancer, bladder cancer, gastric cancer, head and neck cancer, esophageal cancer, brain cancer or lung cancer.

Images