Compositions, methods and uses comprising antibody-tlr agonist conjugates

Abstract

Disclosed herein are trastuzumab-linked TLR agonist derivative analogs comprising at least one non-natural amino acid, as well as methods of making such non-natural amino acids and polypeptides. The trastuzumab-linked TLR agonist derivative analogs may comprise a variety of possible functionalities, but typically have at least one oxime, carbonyl, dicarbonyl, and / or hydroxylamine group. Also disclosed herein are further post-translationally modified non-natural amino acid trastuzumab linked TLR agonist derivative analogs, methods of effecting such modifications, and methods for purifying such trastuzumab linked TLR agonist derivative analogs. In general, the modified trastuzumab linked TLR agonist derivative analogs comprise at least one oxime, carbonyl, dicarbonyl, and / or hydroxylamine group. Further disclosed are methods of using such non-natural amino acid trastuzumab linked TLR agonist derivative analogs, as well as modified non-natural amino acid trastuzumab linked TLR agonist derivative analogs, including therapeutic, diagnostic, and other biotechnological uses.

Description

[0001] Citation of Related Applications

[0002] This application is a divisional application of the Chinese invention patent application with application number 202080028048.7 (the application date of which is February 12, 2020, and the name of the invention is “Compositions Containing, Methods And Uses Of Antibody-TLR Agonist Conjugates”). The original application is a Chinese national phase application with international application number PCT / US2020 / 018015, which claims the benefit of U.S. Provisional Application No. 62 / 804,742, each of which is titled “Compositions Containing, Methods And Uses Of Antibody-TLR Agonist Conjugates” and filed on February 12, 2019. The contents are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted in ASCII format via EFS-Web and 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. Field of the Invention

[0005] The present disclosure relates to TLR agonist compounds and TLR agonist conjugates (TC) and their uses. The present invention further relates to pharmaceutical compositions containing (TC) as therapeutic or prophylactic agents. Background of the Invention

[0007] Using non-naturally encoded amino acids, targeting molecules or polypeptides such as antibodies and their fragments can be conjugated to TLR agonist compounds through site-specific conjugation to generate novel TLR agonist conjugates (TCs). Novel TCs can be constructed in such a way that, during systemic treatment, circulating TCs can target TLR agonists to tumor sites and locally stimulate beneficial immune responses, thereby minimizing systemic cytokine release syndrome. Summary of the Invention

[0008] The present invention relates to targeting polypeptides conjugated to agonist compounds of TLRs using one or more non-naturally encoded amino acids, 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 comprise a targeting biomolecule or polypeptide and a TLR agonist compound, which are conjugated together using non-naturally encoded amino acids through site-specific conjugation to produce a novel biomolecule TLR agonist conjugate (BTC). The targeting biomolecule or polypeptide can be a tumor-targeting biomolecule or polypeptide.

[0009] In another embodiment, the present invention also relates to TC further conjugated with a water-soluble polymer to form a stable dimer or multimer.

[0010] The present invention provides a method for inhibiting or reducing tumor or cancer growth, comprising contacting the tumor with an effective amount of TC of the present invention to stimulate the immune system near the patient's tumor. The present invention provides a method for inhibiting or reducing tumor or cancer growth, comprising contacting the tumor with an effective amount of PEGylated TC of the present invention or a stable dimer or multimer of TC. In one embodiment, the TC is non-PEGylated or mono-PEGylated. In one embodiment, the TC is di-PEGylated. In one embodiment, the TC has more than one and / or different TLR agonist molecules attached thereto. In one embodiment, the TC has more than one 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 modulate the 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 present invention, TC is co-administered with at least one chemotherapeutic agent and / or at least one immunotherapeutic agent.

[0011] 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 the variable region of an antibody light chain. In some embodiments, the ABP comprises the 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')2. In some embodiments, the ABP comprises two or more (Fab')2. In some embodiments, the ABP comprises an scFv. In some embodiments, the ABP comprises two or more scFv. In some embodiments, the ABP comprises a minibody. In some embodiments, the ABP comprises two or more minibodies. In some embodiments, the ABP comprises a diabody. In some embodiments, the ABP comprises two or more diabodies. In some embodiments, the ABP comprises a variable region of a light chain and a variable region of a heavy chain. In some embodiments, the ABP comprises a complete light chain and a complete heavy chain. In some embodiments, the ABP comprises one or more Fc domains or portions thereof. In some embodiments, the ABP comprises a combination of any 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, wherein the binding partner comprises an antigen, a polypeptide, a nucleic acid molecule, a polymer, or other molecule or substance. In some embodiments, the ABP associates with a non-antibody scaffold molecule or substance. In some embodiments, the antigen is a tumor antigen.

[0012] Toll-like receptors (TLRs) detect a wide range of conserved pathogen-associated molecular patterns (PAMPs). They play an important role in sensing invading pathogens and subsequently initiating innate immune responses. There are 10 known TLR family members in humans, which are type I transmembrane proteins characterized by an extracellular leucine-rich domain and a cytoplasmic tail containing a conserved Toll / interleukin (IL)-1 receptor (TIR) ​​domain. In this family, TLR3, TLR7, TLR8, and TLR9 are located in endosomes. By combining with specific small molecule ligands (i.e., TLR7 agonists or TLR8 agonists) or their natural ligands (i.e., single-stranded RNA, ssRNA), TLR7 and TLR8 can be activated. After the agonist binds to TLR7 or TLR8, the receptor in its dimerized form is considered to have undergone structural changes, resulting in the subsequent recruitment of adapter proteins in its cytoplasmic domain, including myeloid differentiation primary response gene 88 (MyD88). After the receptor signaling cascade is initiated through 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 interferon-α and other antiviral cytokine genes. TLR7 is mainly expressed on plasmacytoid cells and B cells. Changes in immune cell responsiveness may lead to a decrease in the innate immune response of cancer patients. Therefore, agonist-induced activation of TLR7 and / or TLR8 conjugated to a targeting moiety (such as an antibody or its fragment) may represent a new cancer treatment method. Treatment of TC with a TLR7 or TLR8 agonist represents a promising solution, thereby providing better effects and better tolerability. Suitable TLR7 and / or TLR8 agonists for use in preparing TCs in the present invention 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; U.S. Patent No. 8,673,932.

[0013] In some embodiments, the targeting polypeptide comprised by the TC 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) compared to the compatibility of the corresponding wild-type TC without the substitution, addition, or deletion. This increased compatibility enables the preparation of pharmaceutical formulations that maintain the physicochemical properties and biological activity of the protein during storage.

[0014] In some embodiments, one or more engineered bonds are generated using one or more non-natural amino acids. Intramolecular bonds can be generated in a variety of ways, including but not limited to, the reaction between two amino acids in a protein under suitable conditions (one or both amino acids can be non-natural amino acids); the reaction of two amino acids (each of which can be naturally encoded or non-naturally encoded) with a linker, polymer, or other molecule under suitable conditions.

[0015] In some embodiments, one or more amino acid substitutions in a TC polypeptide may be with one or more naturally occurring or non-naturally occurring amino acids. In some embodiments, amino acid substitutions in a TC polypeptide may 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 may be with one or more naturally occurring amino acids, and at least one substitution is with a non-naturally encoded amino acid. In some embodiments, a TC polypeptide may be an antibody or antibody fragment. In some embodiments, a TC polypeptide may be a tumor-targeting polypeptide.

[0016] 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.

[0017] In some embodiments, the non-naturally encoded amino acid comprises a carbonyl group. In some embodiments, the non-naturally encoded amino acid has the following structure:

[0018]

[0019] wherein 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 carboxyl terminus modification group.

[0020] 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.

[0021] 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 following structure:

[0022]

[0023] wherein n is 0-10; R1 is alkyl, aryl, substituted alkyl, substituted aryl, or absent; 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 carboxyl terminus modification group.

[0024] In some embodiments, the non-naturally encoded amino acid comprises an alkynyl group. In some embodiments, the non-naturally encoded amino acid has the following structure:

[0025]

[0026] wherein 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 carboxyl terminus modification group.

[0027] 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.

[0028] The present invention also provides isolated nucleic acids comprising a polynucleotide encoding a targeting polypeptide of TC, and the present invention provides isolated nucleic acids comprising a polynucleotide that hybridizes to the polynucleotide under stringent conditions. The present invention also provides isolated nucleic acids comprising a polynucleotide encoding a targeting polypeptide, wherein the polynucleotide comprises at least one selector codon. It will be apparent to one of ordinary skill in the art that many different polynucleotides can encode any polypeptide of the present invention.

[0029] 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.

[0030] The present invention also provides methods for preparing TC polypeptides linked to water-soluble polymers or to one or more TC polypeptides to form homodimers or homomultimers. In some embodiments, the methods comprise contacting an isolated TC polypeptide comprising a non-naturally encoded amino acid with a water-soluble polymer or a 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 toward a water-soluble polymer or linker that is unreactive toward any of the 20 common amino acids. In some embodiments, the non-naturally encoded amino acid incorporated into the TC polypeptide is reactive toward a linker, polymer, or bioactive molecule that is unreactive toward any of the 20 common amino acids.

[0031] In some embodiments, the TC polypeptide linked to a water-soluble polymer or linker is prepared by reacting a TC polypeptide comprising a carbonyl-containing amino acid with a poly(ethylene glycol) molecule or a linker comprising an aminooxy, hydrazine, hydrazide, or semicarbazide group. In some embodiments, the aminooxy, hydrazine, hydrazide, or semicarbazide group is linked to the poly(ethylene glycol) molecule or linker through an amide bond. In some embodiments, the aminooxy, hydrazine, hydrazide, or semicarbazide group is linked to the poly(ethylene glycol) molecule or linker through a carbamate bond.

[0032] In some embodiments, the TC polypeptide linked to the water soluble polymer is prepared by reacting a poly(ethylene glycol) molecule or a linker comprising a carbonyl group with a polypeptide comprising a non-naturally encoded amino acid comprising an aminooxy group, a hydrazine group, a hydrazide group, or a semicarbazide group.

[0033] In some embodiments, the TC polypeptide linked to a water soluble polymer or linker is prepared by reacting a TC comprising an alkyne-containing amino acid with a poly(ethylene glycol) molecule comprising an azide moiety. In some embodiments, the azide or alkyne group is linked to the poly(ethylene glycol) molecule or linker through an amide bond.

[0034] In some embodiments, the TC polypeptide linked to a water soluble polymer or linker is prepared by reacting a TC polypeptide comprising an azide-containing amino acid with a poly(ethylene glycol) molecule comprising an alkyne moiety. In some embodiments, the azide or alkyne group is linked to the poly(ethylene glycol) molecule or linker through an amide bond.

[0035] In some embodiments, the poly(ethylene glycol) molecule or linker has a molecular weight between about 0.1 kDa and about 100 kDa. In some embodiments, the poly(ethylene glycol) molecule or linker has a molecular weight between 0.1 kDa and 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 between 1 kDa and 100 kDa or between 1 kDa and 50 kDa.

[0036] 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 group, a hydrazide group, a hydrazine group, or a semicarbazide group. 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.

[0037] The present invention also provides cells comprising a polynucleotide encoding a TC-targeting polypeptide comprising a selector codon. In some embodiments, the cells comprise an orthogonal RNA synthetase and / or an orthogonal tRNA for substituting a non-naturally encoded amino acid into the TC-targeting polypeptide.

[0038] The present invention also provides methods for preparing a TC-targeting polypeptide comprising a non-naturally encoded amino acid. In some embodiments, the method comprises culturing cells comprising one or more polynucleotides encoding a TC-targeting polypeptide, an orthogonal RNA synthetase, and / or an orthogonal tRNA under conditions permissive for expression of the TC-targeting polypeptide or a variant thereof; and purifying the TC polypeptide from the cells and / or culture medium.

[0039] The present invention also provides methods for extending the therapeutic half-life, serum half-life, or circulation time of TC. The present invention also provides methods for modulating the immunogenicity of TC. In some embodiments, the methods comprise substituting any one or more amino acids in a naturally occurring TC targeting polypeptide with a non-naturally encoded amino acid, and / or linking the targeting polypeptide to a linker, a polymer, a water-soluble polymer, or a biologically active molecule.

[0040] The present invention also provides methods for 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 therapeutically effective amount of a pharmaceutical composition comprising TC containing 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.

[0041] 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 linked to the water-soluble polymer or linker is a non-naturally encoded amino acid present in the targeting polypeptide of the TC.

[0042] The present invention provides TC polypeptides comprising at least one linker, polymer, or bioactive molecule, wherein the linker, polymer, or bioactive 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 another water-soluble polymer, another TC, polypeptide, or bioactive molecule can be directly conjugated to the TC via a linker. In one embodiment, the linker is sufficiently long to allow flexibility and dimer formation. In one embodiment, the linker is at least 3 amino acids or 18 atoms in length, thereby allowing dimer formation. In some embodiments, the polypeptide is connected to the linker to allow multimer formation. In some embodiments, the linker is a bifunctional linker. In some embodiments, the compositions and / or TC of the present invention may comprise multiple linkers. In other embodiments, each linker may comprise one or more attached compounds. Linkers may also comprise alkylene, alkenylene, alkynylene, polyether, polyester, polyamide groups, as well as polyamino acids, polypeptides, cleavable peptides, or aminobenzyl carbamates. In some embodiments, the linkers may be the same or different linkers. Suitable linkers include, for example, cleavable and non-cleavable linkers. Suitable cleavable linkers include, for example, peptide linkers that can be cleaved by intracellular proteases such as lysosomal proteases 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. Linkers containing valine-citrulline or valine-alanine can contain maleimide or succinimide groups. Linkers containing valine-citrulline or valine-alanine can contain p-aminobenzyl alcohol (PABA) groups or p-aminobenzylcarbamate (PABC). Other suitable linkers include linkers that can be hydrolyzed at a pH below 5.5, such as hydrazone linkers. Other suitable cleavable linkers include disulfide linkers. In some embodiments, cleavable linkers can include linkers that are cleaved in the tumor microenvironment, such as tumor-infiltrating T cells. In some embodiments, non-cleavable linkers include, but are not limited to, maleimidocaproyl linkers. The maleimidocaproyl linker may comprise N-maleimidomethylcyclohexane-1-carboxylate, a succinimide group, a pentafluorophenyl group and / or one or more PEG molecules, but is not limited thereto. In some embodiments, any one of the compositions of the present invention, compounds, or salts thereof may be connected to a polypeptide via a linker. In some embodiments, any one of the compounds disclosed in Tables 3, 4, 5, 6, and 7 herein, or salts thereof, may be connected to a polypeptide via 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.

[0043] In some embodiments, the TC polypeptide is monoPEGylated. The present invention also provides a TC comprising a linker, polymer, or biologically active molecule attached to one or more non-naturally encoded amino acids, wherein the non-naturally encoded amino acids are ribosomally incorporated into the polypeptide at a preselected site.

[0044] 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 bonded to the non-natural amino acid of the polypeptide.

[0045] In another embodiment, the present invention provides a composition wherein the one or more targeting polypeptides are the same or different targeting polypeptides. In another embodiment, the present invention provides a composition wherein the one or more targeting polypeptides bind to a cell surface target, 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.

[0046] In other embodiments, the monospecific, bispecific or multispecific targeting polypeptide comprises a drug conjugate or a checkpoint inhibitor. It is contemplated that any suitable immune checkpoint inhibitor will be used together with the composition or TC 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-induced TNFR-related protein (GITR), inducible T cell co-stimulator (ICOS), CD96, poliovirus receptor-related 2 (PVRL2), PD-1, PD-L1, PD-L2, LAG-3, B7-H4, killer cell immunoglobulin receptor (KIR), OX40, OX40-L, indoleamine-2,3-dioxygenase-1 (IDO-1), indoleamine-2,3-dioxygenase-2 (IDO-2), CEACAM1, CD272, TEVI3, adenosine A2A receptor and VISTA protein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA4, PD-1 or PD-L1.

[0047] In another embodiment, the targeting polypeptide comprises an antibody or antibody fragment. In other embodiments, the targeting polypeptide is an antibody or antibody fragment that binds to a cellular antigen. In another embodiment, the targeting 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, E-cadherin 6, In some embodiments, the targeting polypeptide comprises an antibody or antibody fragment that binds to HER2. In another embodiment, the targeting polypeptide is trastuzumab.

[0048] In another embodiment, the antibody or antibody fragment comprises IgG, Fab, (Fab')2, Fv or single-chain Fv (scFv). In some embodiments, the antibody or antibody fragment comprises one or more Fab, (Fab')2, Fv or single-chain Fv (scFv) mutations. In some embodiments, the antibody or antibody piece 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.

[0049] 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 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 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 chain and the light chain, 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 chain and the light chain, 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 chain and the light chain, 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 chain and the light chain, 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 chain and the light chain, 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 chain and the light chain, 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 chain and the light chain, and the antibody or antibody fragment further comprises at least three Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy chain and the light chain, 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 chain and the light chain, and the antibody or antibody fragment further comprises at least four Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy chain and the light chain, 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 chain and the light chain, 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 chain and the light chain, 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, said antibody or antibody fragment further comprising at least six Fc mutations.

[0050] In another embodiment, the targeting polypeptide comprises one or more non-naturally encoded amino acids selected from the group consisting of p-acetylphenylalanine, p-nitrophenylalanine, p-sulfotyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-boronocarbonylphenylalanine, o-boronocarbonylphenylalanine, m-boronocarbonylphenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-OM e-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-amino Tyrosine, O-sulfotyrosine, 2-sulfophenylalanine, 3-sulfophenylalanine, 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 (L- In some embodiments, the non-natural amino acid is selected from the group consisting of p-acetyl-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine, and p-azidomethyl-phenylalanine. In some embodiments, the non-natural amino acid is selected from the group consisting of p-acetyl-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine, and p-azidomethyl-phenylalanine. In other embodiments, the non-naturally encoded amino acid is site-specifically incorporated into one or more targeting polypeptides.

[0051] In another embodiment, the TLR agonist is a TLR7 agonist, a TLR8 agonist, or a TLR7 / TLR8 dual agonist. In other embodiments, the TLR agonist is a TLR comprising a Figure 1 In another embodiment, the TLR agonist is any one of the TLR agonists selected from the group of structures according to Tables 3, 4, 5, 6, and 7 of the present invention.

[0052] In other embodiments, the targeting polypeptide is conjugated to one or more linkers, polymers, or bioactive molecules. In some embodiments, the targeting polypeptide is conjugated directly or indirectly to one or more linkers, polymers, or bioactive molecules. In some embodiments, the one or more linkers are cleavable or non-cleavable linkers.

[0053] In some embodiments, one or more linkers are 0.1 kDa to 50 kDa. In other embodiments, one or more linkers are 0.1 kDa to 10 kDa. In other embodiments, one or more linkers or polymers are linear, branched, multimeric or dendrimeric. In another embodiment, one or more linkers or polymers are bifunctional or multifunctional linkers or bifunctional or multifunctional polymers.

[0054] 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, PEG has a molecular weight between 0.1kDa and 100kDa. In other embodiments, PEG has a molecular weight between 0.1kDa and 50kDa. In other embodiments, PEG has a molecular weight between 0.1kDa and 40kDa. In other embodiments, PEG has a molecular weight between 0.1kDa and 30kDa. In other embodiments, PEG has a molecular weight between 0.1kDa and 20kDa. In other embodiments, PEG has a molecular weight between 0.1kDa and 10kDa. In some embodiments, poly (ethylene glycol) molecules have a molecular weight between about 0.1kDa and about 100kDa. In some embodiments, poly (ethylene glycol) molecules have a molecular weight between 0.1kDa and 50kDa. In some embodiments, the poly(ethylene glycol) has a molecular weight between 1 kDa and 25 kDa, or between 2 kDa and 22 kDa, or between 5 kDa and 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.

[0055] In another embodiment, at least one linker, polymer or bioactive molecule is connected to at least one non-naturally encoded amino acid. In some embodiments, the linker is PEG. In other embodiments, the linker is a PEG with a molecular weight between 0.1kDa and 50kDa. In other embodiments, the linker is a PEG with a molecular weight between 0.1kDa and 40kDa. In other embodiments, the linker is a PEG with a molecular weight between 0.1kDa and 30kDa. In other embodiments, the linker is a PEG with a molecular weight between 0.1kDa and 20kDa. In other embodiments, the linker is a PEG with a molecular weight between 0.1kDa and 10kDa. In other embodiments, the linker is a PEG with a molecular weight between 0.1kDa and 5kDa.

[0056] 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 enhance 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 in vitro synthesis.

[0057] In another embodiment, the non-naturally encoded amino acid is reactive toward a linker, polymer, or biologically active molecule that is not reactive toward 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 alkynyl group. In other embodiments, the non-naturally encoded amino acid comprises a carbonyl group.

[0058] In another embodiment, the targeting polypeptide is linked to a cytotoxic agent or an immunostimulatory agent. In another embodiment, the TC or BTC of the present invention is linked to a cytotoxic agent or an immunostimulatory agent. In another embodiment, the targeting polypeptide comprises a cytotoxic agent or an immunostimulatory agent. In another embodiment, the TC or BTC of the present invention comprises a cytotoxic agent or an immunostimulatory agent.

[0059] In another embodiment, the present invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist comprising a TLR agonist according to Figure 1In another embodiment, the TLR agonist is a TLR7 agonist, a TLR8 agonist, or a TLR7 / TLR8 dual agonist. In another embodiment, the TLR agonist comprises a TLR according to Figure 1AXC-621、AXC-62 2、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-725 XC-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-7 41、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-758 C-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-79 9、AXC-800、AXC-801、AXC-802、AXC-803、AXC-804、AXC-805、AXC-806、AXC-807、AXC-808、AXC-809、AXC-810、AXC-831、AXC-910 huaàhéwù.In another embodiment, the present invention provides any of the following TLR agonists: 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 In another embodiment, the TLR agonist comprises a structure according to Structure 1 further comprising a linker.

[0060] In other embodiments, the TLR agonist comprising a structure according to Structure 1 is selected from the group consisting of AXC-625, AXC-626, AXC-638, AXC-639, AXC-640, AXC-642, AXC-662, AXC-667, AXC-668, AXC-669, AXC-670, AXC-671, AXC-672, AXC-675, AXC-681, AXC-687, AXC-688, AXC-689, AXC-690 , AXC-691, AXC-697, AXC-699, AXC-700, AXC-701, AXC-702, AXC-709, AXC-710, AXC-711, AXC-713, AXC-714, AX C-717, AXC-719, AXC-722, AXC-723, AXC-724, AXC-725, AXC-726, AXC-727, AXC-731, AXC-732, AXC-733, AXC-73 4. AXC-735, AXC-736, AXC-737, AXC-738, AXC-739, AXC-740, AXC-741, AXC-743, AXC-742, AXC-747, AXC-748, A XC-750, AXC-751, AXC-752, AXC-754, AXC-755, AXC-756, AXC-757, AXC-758, AXC-759, AXC-760, AXC-761, AXC- In other embodiments, the TLR agonist comprising a structure according to Structure 1 is selected from the group consisting of AXC-801, AXC-802, AXC-831, and AXC-910 compounds. In other embodiments, the TLR agonist comprising a structure according to Structure 1 is selected from the group consisting of AXC-801, AXC-802, AXC-831, and AXC-910 compounds, further comprising a linker.

[0061] 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 chain and the light chain. In another embodiment, the one or more non-naturally encoded amino acids are selected from the group consisting of p-acetylphenylalanine, p-nitrophenylalanine, p-sulfotyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-boronocarbonylphenylalanine, o-boronocarbonylphenylalanine, m-boronocarbonylphenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-OMephenylalanine, Alanine, 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-sulfophenylalanine, 3-sulfophenylalanine, 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-poly(1,2-naphthyl)alanine, In some embodiments, the unnatural amino acid is p-acetyl-phenylalanine, p-acyl-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, p-azidoethoxyphenylalanine, and p-azidomethyl-phenylalanine. In other embodiments, the unnatural amino acid is p-acetyl-phenylalanine, 4-azido-L-phenylalanine, p-azidomethyl-phenylalanine, or p-azidoethoxyphenylalanine.

[0062] 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.

[0063] 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.

[0064] In another embodiment, the TLR agonist comprises a Figure 1 In another embodiment, the TLR agonist comprises a structure according to Structure 2 selected from the group of AXC-745, AXC-746, and AXC-753 compounds. In another embodiment, the TLR agonist comprises a structure according to any one of: AXC-745, AXC-746, and AXC-753 compounds, further comprising a linker. In another embodiment, the TLR agonist comprises a structure according to Structure 2 further comprising a linker.

[0065] In another embodiment, the TLR agonist comprises a Figure 1 In another embodiment, the TLR agonist comprising the structure according to Structure 3 is an AXC-837 or AXC-847 compound. In another embodiment, the TLR agonist comprises the structure according to an AXC-837 or AXC-847 compound, further comprising a linker. In another embodiment, the TLR agonist comprises the structure according to an AXC-847 compound, further comprising a linker. In another embodiment, the TLR agonist comprises the structure according to Structure 3 further comprising a linker.

[0066] In another embodiment, the TLR agonist comprises a Figure 1In another embodiment, the TLR agonist comprises a structure according to Structure 4 selected from the group consisting of AXC-844, AXC-842, AXC-843, AXC-845, AXC-846, AXC-836, or AXC-841 compounds. In another embodiment, the TLR agonist comprises a structure according to Structure 4 of any one of AXC-844, AXC-842, AXC-843, AXC-845, AXC-846, AXC-836, or AXC-841 compounds, further comprising a linker. In another embodiment, the TLR agonist comprises a structure according to Structure 4 further comprising a linker.

[0067] In another embodiment, the TLR agonist comprises a Figure 1In another embodiment, the TLR agonist comprising the structure according to 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 88. AXC-889, AXC-890, AXC-891, AXC-892, AXC-893, AXC-895, AXC-896, AXC-897, AXC-898, AXC-901, AXC-903, AXC- 904, AXC-905, AXC-906, AXC-907, AXC-908, AXC-909, AXC-911, AXC-912, AXC-913, AXC-914, AXC-915 or AXC-916 compounds. In other embodiments, the TLR agonist comprising a structure according to Structure 5 is selected from the group consisting 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, the TLR agonist comprises a structure according to any 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, In another embodiment, the TLR agonist comprises a structure according to Structure 5 further comprising a linker.

[0068] In another embodiment, the anti-HER2 antibody or antibody fragment comprises the amino acid sequence of at least one of SEQ ID NOs: 1-13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises the amino acid sequence of at least two 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) a light chain of any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises a) SEQ ID NO: 1; and b) any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises a) SEQ ID NO: 2; and b) any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 3. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 4. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 5. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 6. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 7. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 8. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 9. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 10. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 11. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 12. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 13.In another embodiment, the invention provides an anti-HER2 antibody or antibody fragment wherein a non-naturally encoded amino acid is incorporated in a site-specific manner at position 114 according to Kabat numbering.

[0069] In another embodiment, the present invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist comprising a TLR agonist according to Figure 1 In another embodiment, the invention provides a TLR agonist conjugate (TC), comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist of any one of the compounds selected from Tables 3-7, wherein the TLR agonist is conjugated to an antibody or antibody fragment by a linker covalently bonded to one or more non-naturally encoded amino acids incorporated into an 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 of any one of the compounds selected from Tables 3-7, wherein the TLR agonist is conjugated to an antibody or antibody fragment by a linker covalently bonded to one or more non-naturally encoded amino acids incorporated into an antibody or antibody fragment, and the TC further comprises a chemotherapeutic or immunotherapeutic agent.

[0070] In another embodiment, the present invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist comprising a TLR agonist according to Figure 1The invention provides a TLR agonist conjugate (TC), comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist of any one of the compounds selected from Tables 3-7, wherein the TLR agonist is conjugated to an antibody or antibody fragment by a linker covalently bonded to one or more non-naturally encoded amino acids incorporated into an 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 of any one of the compounds selected from Tables 3-7, wherein the TLR agonist is conjugated to an antibody or antibody fragment by a linker covalently bonded to one or more non-naturally encoded amino acids incorporated into an antibody or antibody fragment, and the TC further comprises a drug conjugate. In other embodiments, the drug conjugate is an antibody drug conjugate. In other embodiments, the TC further comprises a cytokine or a cytotoxin.

[0071] In another embodiment, the present invention provides a method for treating a subject or patient suffering from a cancer, disease, condition, indication, or illness, 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, illness, or illness is a HER2-positive tumor, cancer, indication, disease, illness, or illness. 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.

[0072] In another embodiment, the present invention provides a method for treating a subject or patient suffering from cancer, a disease, or a condition, comprising administering to the subject or patient a therapeutically effective amount of a composition of the present invention or TC further comprising a chemotherapeutic agent or an immunotherapeutic agent. In certain embodiments, 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, a fluoropyrimidine, a taxane, an anthracycline, lapatinib, capecitabine, letrozole, pertuzumab, docetaxel, and interferon-α. In another embodiment of the present invention, TC is co-administered with at least one chemotherapeutic agent.

[0073] In another embodiment, the present invention provides a method of treating a subject or patient suffering from 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 an antibody drug conjugate, a cytotoxic agent, or a checkpoint inhibitor.

[0074] In another embodiment, the present invention provides a method for killing cells, comprising contacting the cells with a TC of the present invention. In other embodiments, the cells are tumor cells or cancer cells. In certain embodiments, the tumor cells or cancer cells are cancer cells 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, or leukemia. 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 to be treated is a HER2-positive tumor, cancer, indication, disease, disorder, or condition.

[0075] The present invention provides a method for inhibiting or reducing tumor or cancer growth, comprising contacting the tumor with an effective amount of TC of the present invention to stimulate the immune system in the vicinity of the patient's tumor. The present invention provides a method for inhibiting or reducing tumor or cancer growth, comprising contacting the tumor with an effective amount of PEGylated TC of the present invention or a stable dimer or multimer of TC. In one embodiment, the TC is non-PEGylated or mono-PEGylated. In one embodiment, the TC is di-PEGylated. In one embodiment, the TC has more than one and / or different TLR agonist molecules attached thereto. Another embodiment of the present invention provides a method for using the TC of the present invention to modulate the immune response to tumor cells.

[0076] In some embodiments, the present invention provides methods for treating cancer using TC. In some embodiments, the TC of the present invention can be used to treat or prevent cancer-related diseases, disorders, and conditions, including conditions directly or indirectly related to cancer, such as angiogenesis and precancerous conditions, such as dysplasia. In some embodiments, the tumor is a liquid tumor or a solid tumor. In some embodiments, the condition to be 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 (leukemia), or a cancer or disease or condition related to any of these cancers. Cancer is a cancer that begins in epithelial cells, which are cells that cover the surface of the body, produce hormones, and make up glands. By way of non-limiting example, cancers include breast cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, rectal cancer, kidney cancer, bladder cancer, stomach cancer, prostate cancer, liver cancer, ovarian cancer, brain cancer, vaginal cancer, vulvar cancer, uterine cancer, oral cancer, penile cancer, testicular cancer, esophageal cancer, skin cancer, fallopian tube cancer, head and neck cancer, gastrointestinal stromal cancer, adenocarcinoma, cutaneous or intraocular melanoma, cancer of the anal region, cancer of the small intestine, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, urethra cancer, renal pelvis cancer, ureter cancer, endometrial cancer, cervical cancer, pituitary cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, brain stem glioma, and spinal axis tumors. In some cases, the cancer is skin cancer, such as basal cell carcinoma, squamous cell carcinoma, melanoma, non-melanoma, or actinic (solar) keratosis. In some embodiments, the present invention also relates to a method for treating acute leukemia in a mammal, comprising administering to the mammal a therapeutically effective amount of the TC of the present invention. The present invention also provides a method for inhibiting the proliferation of acute leukemic blast cells, comprising administering to a mammal suffering from acute leukemia a therapeutically effective dose of the TC of the present invention.

[0077] In another embodiment, the TC disclosed herein can be used to modulate an immune response. Modulation of an immune response can include stimulating, activating, enhancing, increasing, or upregulating an immune response. Modulation of an immune response can include suppressing, inhibiting, preventing, reducing, or downregulating an immune response.

[0078] In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the composition or TC of the present invention and a pharmaceutically acceptable carrier or excipient.

[0079] In another embodiment, the present invention provides use of the composition of the present invention in the manufacture of a medicament.

[0080] In another embodiment, the present invention provides a method comprising Figure 1In another embodiment, the present invention provides an immunostimulatory antibody conjugate (ISAC) of a TLR agonist comprising any one of the compounds according to 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 consisting 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.

[0081] In another embodiment, the present invention provides a Figure 1 In another embodiment, the present invention provides a salt of any compound of Table 3, 4, 5, 6, or 7. In another embodiment, the present invention provides a pharmaceutical composition or salt thereof of the composition, compound, and TC disclosed herein. In other embodiments, the pharmaceutical composition or salt further comprises a pharmaceutically acceptable excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 The general structure of a TLR agonist suitable for use in the invention is depicted.

[0083] Figure 2 The structures of various TC conjugates are depicted.

[0084] Figure 3 The structures of additional TC conjugates are depicted.

[0085] Figure 4 The biological activities of selected TC conjugates in cell proliferation assays are depicted.

[0086] Figure 5A and 5B TLR7 activity of various TLR7 agonists is depicted.

[0087] Figure 6 TLR7 activity of various TLR7 agonists attached to the linker is depicted.

[0088] Figure 7 TLR7 activity of additional TLR7 agonists and TLR7 agonists attached to a linker is depicted.

[0089] Figure 8 TLR7 activity of additional TLR7 agonists and TLR7 agonists attached to a linker is depicted.

[0090] Figure 9 Depicted are the TLR7 activities of different TLR7 agonists attached to linkers (Drug Linker or DL) compared to the unnatural amino acid pAF (DL-pAF).

[0091] Figures 10A-10C Depicted is the HPLC chromatogram of an unconjugated anti-HER2 antibody with an unnatural amino acid at amino acid position HA114 ( Figure 10A ), and at amino acid position HA114 with the TLR agonist AXC-875 ( Figure 10B ) and AXC-880( Figure 10C ) HPLC chromatogram of the conjugated anti-HER2 antibody.

[0092] Figures 11A-11C The SKOV3 HER2 high-expressing tumor cell line ( Figure 11A ); JIMT-1HER2 medium / low expression tumor cell line ( Figure 11B ); and A431 HER2-low expressing tumor cell line ( Figure 11C ) Tumor-dependent ISAC activity of various payload linkers conjugated to anti-HER2 antibodies.

[0093] Figure 12A and 12B The SKBR3 HER2 high expression tumor cell lines ( Figure 12A ) and HCC1806 HER2 very low expression tumor cell line ( Figure 12B ) Tumor-dependent ISAC activity of additional payload linkers conjugated to anti-HER2 antibodies.

[0094] Figure 13A and 13B The SKBR3 HER2 high expression tumor cell lines ( Figure 13A ) and HCC1806 HER2 very low expression tumor cell line ( Figure 13B ) Tumor-dependent ISAC activity of additional payload linkers conjugated to anti-HER2 antibodies.

[0095] Figure 14A and 14B The SKBR3 HER2 high expression tumor cell lines ( Figure 14A ) and HCC1806 HER2 very low expression tumor cell line ( Figure 14B ) Tumor-dependent ISAC activity of additional payload linkers conjugated to anti-HER2 antibodies.

[0096] Figure 15A and 15BThe SKBR3 HER2 high expression tumor cell lines ( Figure 15A ) and HCC1806 HER2 very low expression tumor cell line ( Figure 15B ) were used to evaluate the tumor-dependent ISAC activity of three (3) payload linkers conjugated to anti-HER2 antibodies, showing that HER2-AXC-879 had the best ISAC activity. DETAILED DESCRIPTION

[0097] 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 linkers. The TCs of the present invention may comprise a TLR agonist linked to an unnatural amino acid in the targeting moiety. Also included are methods for preparing such TCs comprising an unnatural amino acid incorporated into a targeting moiety polypeptide.

[0098] In certain embodiments, a pharmaceutical composition comprising any of the described compounds and a pharmaceutically acceptable carrier, excipient, or binder is provided.

[0099] In a further or alternative embodiment is a method of 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, and wherein the resulting heterocycle-containing non-natural amino acid polypeptide modulates the immunogenicity of the polypeptide relative to the homologous naturally occurring amino acid polypeptide.

[0100] It should be understood that the methods and compositions described herein are not limited to the specific methods, protocols, cell lines, constructs and reagents described herein, and as such, they may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the methods and compositions described herein, which will be limited only by the appended claims.

[0101] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0102] 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.

[0103] All publications and patents mentioned herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, constructs and methodologies described in the publications and that may be used in conjunction 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.

[0104] The term "aldol-based linkage" or "mixed aldol-based linkage" refers to the acid-catalyzed or base-catalyzed condensation of a carbonyl compound with an enol ester / enol of another carbonyl compound, which may be the same or different, to form a β-hydroxycarbonyl compound, an aldol.

[0105] As used herein, the term "affinity tag" refers to a tag that reversibly or irreversibly binds to another molecule, modifies it, destroys it, or forms a compound with it. For example, affinity tags include enzymes and their substrates or antibodies and their antigens.

[0106] The terms "alkoxy," "alkylamino," and "alkylthio" (or thioalkoxy) are used in their conventional sense and refer to those alkyl groups attached to the molecule through an oxygen atom, an amino group, or a sulfur atom, respectively.

[0107] Unless otherwise indicated, the term "alkyl" by itself or as part of another molecule means a straight or branched chain or cyclic hydrocarbon radical or combinations thereof, which may be fully saturated, mono- or polyunsaturated, and may include divalent and polyvalent radicals having the specified number of carbon atoms (i.e., C1-C 10 Represents one to ten carbons). The example of saturated hydrocarbon radical includes but is not limited to groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, for example, homologues and isomers of n-pentyl, n-hexyl, n-heptyl, n-octyl etc. Unsaturated alkyl is an alkyl with one or more double bonds or triple bonds. The example of unsaturated alkyl includes but is 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 homologues and isomers. Unless otherwise indicated, the term "alkyl" is also meant to include those alkyl derivatives defined in more detail herein, such as "heteroalkyl", "haloalkyl" and "homoalkyl".

[0108] The term "alkylene" by itself or as part of another molecule means a divalent radical derived from an alkane, for example (-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-. "Lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, typically having 8 or fewer carbon atoms. Unless otherwise indicated, the term "alkylene" is also intended to include those groups described herein as "heteroalkylene."

[0109] The term "amino acid" refers to naturally occurring and non-natural 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, such as, by way of example only, an α-carbon bound to a hydrogen, carboxyl, amino, and R groups. Such analogs can have modified R groups (for example, norleucine), or can have modified peptide backbones while still retaining the same basic chemical structure as naturally occurring amino acids. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium.

[0110] Amino acids may be referred to herein by their name, their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Additionally, nucleotides may be referred to by their generally accepted single-letter codes.

[0111] An "amino terminal modification group" refers to any molecule that can be attached to a terminal amine group. For example, such a terminal amine group can be at the end of a polymeric molecule, where such polymeric molecules include, but are 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 properties of polymeric molecules, including, but not limited to, extending the serum half-life of peptides.

[0112] "Antibody" herein refers to a protein consisting of one or more polypeptides that are substantially encoded by all or part of an antibody gene. Immunoglobulin genes include, but are not limited to, kappa, lambda, alpha, gamma (IgG1, IgG2, IgG3, and IgG4), delta, epsilon, and mu constant region genes, as well as many immunoglobulin variable region genes. Antibodies herein are intended to include full-length antibodies and antibody fragments, and include antibodies naturally occurring in any organism or engineered antibodies (e.g., variants).

[0113] By "antibody fragment," it is meant any antibody form other than the full-length form. Antibody fragments herein include antibodies that are smaller components present in a full-length antibody, as well as antibodies that have been engineered. 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, and the like (Maynard and Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9:395-402). Another functional substructure is a single-chain Fv (scFv), which consists of the variable regions of the immunoglobulin heavy and light chains, covalently linked by a peptide linker (S-zHu et al., 1996, Cancer Research, 56, 3055-3061). These small (Mr 25,000) proteins generally retain the specificity and affinity for the antigen in a single polypeptide and can provide a convenient structural unit (building block) for larger antigen-specific molecules. Unless otherwise specifically stated, statements and claims using the term "antibody" or "antibodies" specifically include "antibody fragments" and "antibody fragments."

[0114] As used herein, "antibody-drug conjugate" or "ADC" refers to an antibody molecule or fragment thereof covalently bonded to one or more biologically active molecules. The biologically active molecule can be conjugated to the antibody through a linker, a polymer or other covalent bond.

[0115] As used herein, the term "aromatic" or "aryl" refers to a closed ring structure having at least one ring with a conjugated π electron system, and includes carbocyclic aryl and heterocyclic aryl (or "heteroaryl" or "heteroaromatic") groups. A carbocyclic or heterocyclic aromatic group may contain 5 to 20 ring atoms. The term includes covalently linked monocyclic or fused-ring polycyclic (i.e., rings that share adjacent pairs of carbon atoms) groups. Aromatic groups may 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-mentioned aryl and heteroaryl ring systems are selected from the group of acceptable substituents described herein.

[0116] For the sake of brevity, when the term "aromatic" or "aryl" is used in combination with other terms (including but not limited to aryloxy, arylthioxy, arylalkyl), it includes aryl rings and heteroaryl rings as defined above. Thus, the term "aralkyl" or "alkaryl" is intended to include those groups in which an aryl group is attached to an alkyl group (including but not limited to benzyl, phenethyl, pyridylmethyl, etc.), including those alkyl groups in which a carbon atom (including but not limited to methylene) has been replaced by, for example, a heteroatom (for example only, an oxygen atom). Examples of such aryl groups include but are not limited to phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, etc.

[0117] As used herein, the term "arylene" refers to a divalent aromatic radical. Non-limiting examples of "arylene" include phenylene, pyridylene, pyrimidylene, and thienylene. Substituents for the arylene group are selected from the group of acceptable substituents described herein.

[0118] A "bifunctional polymer," also known as a "bifunctional linker," refers to a polymer comprising two functional groups that are capable of reacting specifically with other moieties to form a covalent or non-covalent linkage. Such moieties may include, but are not limited to, side groups on natural or unnatural amino acids or peptides containing such natural or unnatural amino acids. The other moieties that may be attached to the bifunctional linker or bifunctional polymer may be the same or different moieties. By way of example only, a bifunctional linker may have a functional group that is reactive with a group on a first peptide and another functional group that is 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, e.g., 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 known as a "multifunctional linker," refers to a polymer comprising two or more functional groups capable of reacting with other moieties. Such moieties may include, but are not limited to, side groups (including, but not limited to, amino acid side groups) on natural or non-natural amino acids or peptides containing such natural or non-natural amino acids to form covalent or non-covalent linkages. The bifunctional polymer or multifunctional polymer may have any desired length or molecular weight and may be selected to provide a particular desired spacing or conformation between the one or more molecules attached to the compound and the molecule or compound to which it is bound.

[0119] As used herein, the term "bioavailability" refers to the rate and extent to which a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the systemic circulation. Increased bioavailability refers to an increase in the rate and extent to which a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the systemic circulation. For example, increased bioavailability can be indicated as an increase in the concentration of a substance or its active moiety in the blood when compared to other substances or active moieties. Methods for evaluating increased bioavailability are known in the art and can be used to evaluate the bioavailability of any polypeptide.

[0120] 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 associated with an organism, including, but not limited to, viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans. In particular, as used herein, biologically active molecules include, but are not limited to, any substance intended for use in diagnosing, curing, alleviating, treating, or preventing disease in humans or other animals, or otherwise enhancing the physical or mental well-being of humans or animals. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, prodrugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles, and micelles. Classes of bioactive agents suitable for use with the methods and compositions described herein include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, antifungals, antivirals, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anxiolytics, hormones, growth factors, steroidal agents, toxins derived from microorganisms, and the like.

[0121] By "modulating a biological activity," it is meant increasing or decreasing the reactivity of a polypeptide, altering the selectivity of a polypeptide, or enhancing or decreasing the substrate selectivity of a polypeptide. Analysis of the modified biological activity can be performed by comparing the biological activity of a non-natural polypeptide with the biological activity of a natural polypeptide.

[0122] As used herein, the term "biological material" refers to material of biological origin, including but not limited to material obtained from bioreactors and / or recombinant methods and techniques.

[0123] As used herein, the term "biophysical probe" refers to a probe that can detect or monitor structural changes in molecules. Such molecules include, but are not limited to, proteins, and "biophysical probes" can be used to detect or monitor the interaction of proteins with other macromolecules. Examples of biophysical probes include, but are not limited to, spin labels, fluorophores, and photoactivatable groups.

[0124] As used herein, the term "biosynthesis" refers to any method that utilizes a translation system (cellular or non-cellular), including the use of at least one of the following components: polynucleotides, codons, tRNAs, and ribosomes. For example, non-natural amino acids can be "biosynthetically incorporated" into non-natural amino acid polypeptides using the methods and techniques described in WO 2002 / 085923, which is incorporated herein by reference in its entirety. In addition, methods for selecting useful non-natural amino acids that can be "biosynthetically incorporated" into non-natural amino acid polypeptides are described in WO 2002 / 085923, which is incorporated herein by reference in its entirety.

[0125] As used herein, the term "biotin analogue" or also referred to as "biotin mimetic" is any molecule other than biotin that binds with high affinity to avidin and / or streptavidin.

[0126] As used herein, the term "carbonyl" refers to a group containing a moiety selected from -C(O)-, -S(O)-, -S(O)2-, and -C(S)-, including but not limited to groups containing at least one ketone group, and / or at least one aldehyde group, and / or at least one ester group, and / or at least one carboxylic acid group, and / or at least one thioester group. Such carbonyl groups include ketones, aldehydes, carboxylic acids, esters, and thioesters. In addition, such groups can be part of linear, branched, or cyclic molecules.

[0127] The term "carboxyl terminal modification group" refers to any molecule that can be attached to a terminal carboxyl group. For example, such a terminal carboxyl group can be at the end of a polymeric molecule, where such polymeric molecules include, but are 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 properties of polymeric molecules, including, but not limited to, extending the serum half-life of peptides.

[0128] As used herein, the term "chemically cleavable group," also referred to as "chemically labile," refers to a group that breaks or cleaves upon exposure to an acid, base, oxidizing agent, reducing agent, chemical initiator, or free radical initiator.

[0129] As used herein, "cofolding" refers to a refolding process, reaction, or method that employs at least two molecules that interact with each other and result in the conversion of an unfolded or improperly folded molecule to a properly folded molecule. By way of example only, "cofolding" employs at least two polypeptides that interact with each other and result in the conversion of an unfolded or improperly folded polypeptide to a native, properly folded polypeptide. Such polypeptides can contain natural amino acids and / or at least one unnatural amino acid.

[0130] As used herein, a "conjugate" refers to a polypeptide linked (e.g., covalently linked) directly or through a linker to a compound or compound-linker described herein, such as a Figure 1or any compound or salt of any structure of Tables 3-7. "Targeting moiety" refers to a structure that has selective affinity for a target molecule relative to other non-target molecules. The targeting moiety of the present invention binds to a target molecule. The targeting moiety may include, for example, an antibody, a peptide, a ligand, a receptor, or a binding portion thereof. The target biomolecule may be a biological receptor or other structure of a cell, such as a tumor antigen. As used herein, the term "conjugate of the present invention," "targeting moiety conjugate," "targeting conjugate," "targeting moiety-active molecule conjugate," or "TC" refers to a targeting polypeptide or portion, analog, or derivative thereof that binds to a target present on a cell or a subunit thereof, which is conjugated to a bioactive molecule, portion, or analog thereof (including but not limited to a TLR7 and / or TLR8 agonist). As used herein, the term "tumor targeting moiety conjugate," "tumor targeting moiety-bioactive molecule conjugate," or "BTC" refers to a tumor-targeting polypeptide or portion, analog, or derivative thereof that binds to a target present on a tumor cell or a subunit thereof, which is conjugated to a bioactive 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 as alternatives to the term "conjugate of the invention."

[0131] The term "conservatively modified variant" applies to natural and non-natural amino acids and natural and non-natural nucleic acid sequences and combinations thereof. With respect to a particular nucleic acid sequence, "conservatively modified variant" refers to those natural and non-natural nucleic acids that encode the same or substantially the same natural and non-natural amino acid sequences, or when the natural and non-natural nucleic acids do not encode natural and non-natural amino acid sequences, refers to substantially the same sequences. For example, due to the degeneracy of the genetic code, many 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 each position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are a type of conservatively modified variations. Thus, for example, each natural or non-natural nucleic acid sequence encoding a natural or non-natural polypeptide herein also describes each possible silent variation 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 ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, each silent variation of a natural or non-natural nucleic acid encoding a natural or non-natural polypeptide is implicit in each described sequence.

[0132] With respect to amino acid sequences, individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, or delete a single natural and unnatural amino acid or a small percentage of natural and unnatural amino acids in the coded sequence are "conservatively modified variants," wherein the alteration results in an amino acid deletion, an amino acid addition, or the replacement of a natural and unnatural amino acid with a chemically similar amino acid. Conservative substitution tables that provide 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.

[0133] Conservative substitution tables providing functionally similar amino acids are known to those of ordinary skill in the art. The following eight groups each contain amino acids that are conservative substitutions for each other: 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 (WH Freeman & Co.; 2nd ed. (December 1993)).

[0134] Unless otherwise indicated, the terms "cycloalkyl" and "heterocycloalkyl" by themselves or in combination with other terms represent cyclic forms of "alkyl" and "heteroalkyl", respectively. Thus, cycloalkyl or heterocycloalkyl include saturated, partially unsaturated, and fully unsaturated ring connections. Additionally, for heterocycloalkyl, heteroatoms may occupy the position where the heterocycle is attached to the rest of the molecule. Heteroatoms may 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, etc. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophene-2-yl, tetrahydrothiophene-3-yl, 1-piperazinyl, 2-piperazinyl, etc. Additionally, the term includes polycyclic structures, including but not limited to bicyclic and tricyclic ring structures. Similarly, the term "heterocycloalkylene" by itself or as part of another molecule refers to a divalent radical derived from a heterocycloalkyl group, and the term "cycloalkylene" by itself or as part of another molecule refers to a divalent radical derived from a cycloalkyl group.

[0135] As used herein, the term "cyclodextrin" refers to a cyclic carbohydrate consisting of at least six to eight glucose molecules in a ring. The exterior of the ring contains water-soluble groups; in the center of the ring is a relatively nonpolar cavity that can accommodate small molecules.

[0136] As used herein, the term "cytotoxic" refers to a compound that harms cells.

[0137] As used herein, a denaturing agent ("denaturing agent" or "denaturant") refers to any compound or substance that will cause the reversible unfolding of a polymer. By way of example only, a "denaturing agent" can cause the reversible unfolding of a protein. The strength of the denaturing agent will be determined by the nature and concentration of the specific denaturing agent. For example, denaturing agents include, but are not limited to, chaotropes, detergents, organic solvents, water-miscible solvents, phospholipids, or combinations thereof. Non-limiting examples of chaotropes include, but are not limited to, urea, guanidine, and sodium thiocyanate. Non-limiting examples of detergents can include, but are not limited to, strong detergents such as sodium lauryl sulfate, or polyoxyethylene ethers (e.g., Tween or Triton detergents), sodium lauryl sarcosine (Sarkosyl); mild nonionic detergents (e.g., digitonin); mild cationic detergents such as N->2,3-(dioleyl)-propyl-N,N,N-trimethylammonium; mild ionic detergents (e.g., sodium cholate or sodium deoxycholate); or zwitterionic detergents including, but not limited to, sulfobetaines (Zwittergent), 3-(3-cholamidopropyl)dimethylamino-1-propane sulfate (CHAPS), and 3-(3-cholamidopropyl)dimethylamino-2-hydroxy-1-propane sulfonate (CHAPSO). Non-limiting examples of organic, water-miscible solvents include, but are not limited to, acetonitrile, lower alkanols (particularly C2-C4 alkanols such as ethanol or isopropanol), or lower alkanediols (C2-C4 alkanediols such as ethylene glycol), which can be used as denaturants. Non-limiting examples of phospholipids include, but are not limited to, naturally occurring phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol; or synthetic phospholipid derivatives or variants such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

[0138] 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. In addition, such groups can be part of linear, branched, or cyclic molecules.

[0139] As used herein, the term "detectable label" refers to a label that can be observed using analytical techniques including, but not limited to, fluorescence, chemiluminescence, electron spin resonance, ultraviolet / visible absorption spectroscopy, mass spectrometry, nuclear magnetic resonance, magnetic resonance, and electrochemical methods.

[0140] 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; and a group 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 a linear, branched, or cyclic molecule. The two moieties in a dicarbonyl group can be the same or different and can include substituents that will produce, by way of example only, an ester, ketone, aldehyde, thioester, or amide at either of the two moieties.

[0141] As used herein, the term "drug" refers to any substance used to prevent, diagnose, alleviate, treat, or cure a disease or condition.

[0142] As used herein, the term "effective amount" refers to a sufficient amount of an administered agent or compound to alleviate one or more symptoms of the disease or condition being treated to a certain extent. The result can be a reduction and / or alleviation of signs, symptoms, or causes of disease, or any other desired change in a biological system. For example, the administered agent or compound includes but is not limited to natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-amino acid polypeptides. For preventive, enhancing, and / or therapeutic treatments, 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. The appropriate "effective" amount in any individual case can be determined using techniques such as dose escalation studies.

[0143] The terms "enhance" and "enhancing" mean to increase or prolong a desired effect in terms of potency or duration. For example, "enhancing" the effect of a therapeutic agent refers to being able to increase or prolong the effect of the therapeutic agent in terms of potency or duration during the treatment of a disease, disorder or condition. As used herein, an "enhancing-effective amount" refers to an amount sufficient to enhance the effect of a therapeutic agent in treating a disease, disorder or condition. When used in a patient, the effective amount for this purpose 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.

[0144] As used herein, the term "eukaryote" refers to organisms belonging to the phylogenetic domain Eucarya, 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.

[0145] As used herein, the term "fatty acid" refers to a carboxylic acid having a hydrocarbon side chain of about C6 or longer.

[0146] As used herein, the term "fluorophore" refers to a molecule that emits a photon upon excitation and thereby emits fluorescence.

[0147] As used herein, the terms "functional group," "active moiety," "activating group," "leaving group," "reactive site," "chemically reactive group," and "chemically reactive moiety" refer to a portion or unit of a molecule where a chemical reaction occurs. These terms are somewhat synonymous in the chemical arts and are used herein to indicate a portion of a molecule that performs a certain function or activity and reacts with other molecules.

[0148] The term "halogen" includes fluorine, chlorine, iodine and bromine.

[0149] As used herein, the term "haloacyl" refers to an acyl group containing a halogen moiety, including but not limited to -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like.

[0150] As used herein, the term "haloalkyl" refers to an alkyl group containing a halogen moiety, including but not limited to -CF3 and -CH2CF3, etc.

[0151] As used herein, the term "heteroalkyl" refers to a straight or branched or cyclic hydrocarbon group or a combination thereof consisting of an alkyl group and at least one heteroatom selected from the group consisting of O, N, Si and S, wherein nitrogen and sulfur atoms may be optionally oxidized, and nitrogen heteroatoms may be optionally quaternized. The heteroatoms O, N and S and Si may be located at any interior position of the heteroalkyl group or at the position where the alkyl group is attached to the rest 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.

[0152] The term "heterocyclic-based linkage" or "heterocyclic linkage" refers to a moiety formed by the reaction of a dicarbonyl group with a diamine group. The resulting reaction product is a heterocycle, including a heteroaryl group or a heterocycloalkyl group. The resulting heterocyclic group is used as a chemical link between a non-natural amino acid or non-natural amino acid polypeptide and another functional group. In one embodiment, the heterocyclic linkage includes a nitrogen-containing heterocyclic linkage, including, by way of example only, a pyrazole linkage, a pyrrole linkage, an indole linkage, a benzodiazepine linkage, a benzophenone ... (benzodiazepine) linkages and pyrazolone linkages.

[0153] Similarly, the term "heteroalkylene" refers to a divalent radical derived from a heteroalkyl group, such as, but not limited to, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, the same or different heteroatoms may also occupy either or both of the chain termini (including, but not limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, and the like). Further, for alkylene and heteroalkylene linking groups, the orientation of the linking group is not implied by the direction in which the chemical formula of the linking group is written. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-.

[0154] As used herein, the term "heteroaryl" or "heteroaromatic" refers to an aromatic group containing at least one heteroatom selected from N, O, and S; wherein the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen atom may be optionally quaternized. A heteroaryl group may be substituted or unsubstituted. A heteroaryl group may be attached to the rest 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-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.

[0155] As used herein, the term "homoalkyl" refers to an alkyl group that is a hydrocarbon group.

[0156] As used herein, the term "identical" refers to that two or more sequences or subsequences are identical. In addition, as used herein, the term "substantially identical" refers to that two or more sequences or subsequences are identical. In addition, as used herein, the term "substantially identical" refers to that two or more sequences or subsequences are identical, as measured using a comparison algorithm or by manual comparison and visual inspection, when compared over a comparison window or a specified region and aligned to reach maximum correspondence, two or more sequences have a certain percentage of identical continuous units. For example only, if a continuous unit is 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 in a specified region, then two or more sequences can be "substantially identical". Such a percentage describes the "identity percentage" of two or more sequences. The identity of a sequence can be present in a region of at least about 75-100 continuous unit lengths, in a region of about 50 continuous unit lengths, or in the whole sequence when not specified. This definition also relates to the complementary sequence of a test sequence. By way of example only, two or more polypeptide sequences are identical when the amino acid residues are identical over a specified region, and 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. The 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, when not specified, across the entire sequence of a polypeptide sequence. Furthermore, by way of example only, two or more polynucleotide sequences are identical when the nucleic acid residues are identical across a specified region, and 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. Identity can exist over a region of at least about 75 to about 100 nucleic acid lengths, over a region of about 50 nucleic acid lengths, or, when not specified, over the entire sequence.

[0157] For sequence comparison, a sequence is typically used as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified, if necessary, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. Then, based on the program parameters, the sequence comparison algorithm calculates the percent sequence identity of the test sequence relative to the reference sequence.

[0158] As used herein, the term "immunogenicity" refers to the antibody response to administration of a therapeutic drug. The immunogenicity of therapeutic non-natural amino acid polypeptides can be obtained using quantitative and qualitative assays for detecting 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), luminescent immunoassays (LIA), and fluorescent immunoassays (FIA). Analyzing the immunogenicity of therapeutic non-natural amino acid polypeptides involves comparing the antibody response after administration of the therapeutic non-natural amino acid polypeptide with the antibody response after administration of the therapeutic natural amino acid polypeptide.

[0159] As used herein, the term "isolate" refers to separating a target component from non-target components and removing the target component from the non-target components. The separated material can be in a dry or semi-dry state, or in the form of a solution, including but not limited to an aqueous solution. The separated component can be in a homogeneous state, or the separated component can be part of a pharmaceutical composition containing 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. In addition, when the component of interest is isolated and is the predominant species present in the formulation, the component is described herein as being substantially purified. As used herein, the term "purified" can mean that the target component is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or more pure. By way of example only, a nucleic acid or protein is "isolated" when it is free of at least some of the cellular components with which it is associated in its natural state, or when the nucleic acid or protein has been concentrated to a level greater than its in vivo or in vitro production concentration. Also, for example, a gene is isolated when it is separated from open reading frames that flank the gene and encode a protein other than the gene of interest.

[0160] 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.

[0161] As used herein, the term "linkage" or "linker" refers to a bond or chemical moiety formed by a chemical reaction between a functional group of a linker and another molecule. Such bonds may include, but are not limited to, covalent bonds and non-covalent bonds, while such chemical moieties may include, but are not limited to, esters, carbonates, imidophosphoesters, hydrazones, acetals, orthoesters, peptide linkages, and oligonucleotide linkages. A hydrolytically stable linkage means that the linkage is substantially stable in water and does not react with water at useful pH values, including but not limited to, persisting under physiological conditions for an extended period, perhaps even indefinitely. A hydrolytically unstable or degradable linkage means that the linkage is degradable in water or in aqueous solutions, including, for example, blood. An enzymatically unstable or degradable linkage means that the linkage is degradable by one or more enzymes. By way of example only, PEG and related polymers may include degradable linkages in the polymer backbone or in the linking group between the polymer backbone and one or more terminal functional groups of the polymer molecule. Such degradable linkages include, but are not limited to, ester linkages formed by the reaction of a PEG carboxylic acid or an activated PEG carboxylic acid with an alcohol group on a bioactive agent, wherein such ester groups typically hydrolyze under physiological conditions to release the bioactive agent. Other hydrolytically degradable linkages include, but are not limited to, carbonate linkages; imine linkages resulting from the reaction of an amine and an aldehyde; phosphate linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages, which are the reaction product of a hydrazide and an aldehyde; acetal linkages, which are the reaction product of an aldehyde and an alcohol; orthoester linkages, which are the reaction product of a formate and an alcohol; peptide linkages formed by, but are not limited to, an amine group at the terminus of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by, but are not limited to, a phosphoramidite group at the terminus of a polymer and the 5' hydroxyl group of an oligonucleotide. Linkers include, but are not limited to, short linear, branched, multi-armed, or dendrimer 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. Many different cleavable linkers are known to those skilled in the art. See U.S. Patent Nos. 4,618,492; 4,542,225; and 4,625,014. Mechanisms for releasing the agent from these linking groups include, for example, irradiation of a photolabile bond and acid-catalyzed hydrolysis. For example, U.S. Patent No. 4,671,958 includes a description of immunoconjugates comprising a linker that is cleaved at the target site in vivo by the proteolytic enzymes of the patient's complement system. The length of the linker can be predetermined or selected based on the desired spatial relationship between the polypeptide and the molecule to which it is attached. In view of the numerous methods reported for attaching various radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and other agents to antibodies, one skilled in the art will be able to determine a suitable method for attaching a given agent or molecule to a polypeptide.

[0162] As used herein, the term "modification" refers to the presence of changes to a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. Such changes or modifications 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 in question is optionally modified, i.e., the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide in question can be modified or unmodified.

[0163] As used herein, the term "modulated serum half-life" refers to a positive or negative change in the circulating half-life of a modified bioactive molecule relative to its non-modified form. For example, modified bioactive molecules include, but are not limited to, natural amino acids, non-natural amino acids, natural amino acid polypeptides, or non-natural amino acid polypeptides. For example, serum half-life is measured by collecting blood samples at different time points after administration of the bioactive molecule or modified bioactive molecule and determining the concentration of the molecule in each sample. The correlation of serum concentration with time allows the calculation of serum half-life. For example, the regulated serum half-life can be an extension of serum half-life, which enables the improvement of the dosing regimen or the avoidance of toxic effects. Such an extension of serum half-life can be at least about two-fold, at least about three-fold, at least about five-fold, or at least about ten-fold. Methods for evaluating the extension of the serum half-life of any polypeptide are well known to those skilled in the art.

[0164] As used herein, the term "modulated therapeutic half-life" refers to a positive or negative change in half-life of a modified biologically active molecule of a therapeutically effective amount relative to its non-modified form. For 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, the therapeutic half-life is measured by measuring the pharmacokinetic and / or pharmacodynamic properties of the molecule at various time points after administration. An increase in therapeutic half-life can enable a specific beneficial dosing regimen, a specific beneficial total dose, or avoid undesired effects. For example, an extension of the therapeutic half-life can be caused by an increase in potency, an increase or decrease in the binding of the modified molecule to its target, an increase or decrease in another parameter or mechanism of action of the non-modified molecule, or an increase or decrease in the decomposition of the molecule by an enzyme (such as, for example only, a protease). Methods for evaluating the extension of the therapeutic half-life of any polypeptide are well known to those skilled in the art.

[0165] "Non-natural amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. Other terms that can be used synonymously with the term "non-natural amino acid" are "non-naturally encoded amino acids," "non-natural amino acids," "non-naturally occurring amino acids," and various forms thereof that are hyphenated and not hyphenated. The term "non-natural amino acid" includes, but is not limited to, amino acids that occur naturally by modifying naturally encoded amino acids (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine), but that they themselves are not incorporated into the growing polypeptide chain by the translation complex. Examples of such amino acids include, but are not limited to, N-acetylglucosamine-L-serine, N-acetylglucosamine-L-threonine, and O-phosphotyrosine. In addition, the term "non-natural amino acid" includes, but is not limited to, amino acids that are non-naturally occurring and can be synthesized or can be obtained by modifying non-natural amino acids. In some embodiments, non-natural amino acids include lysine analogs, for example, N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyl tetrazine lysine or allyloxycarbonyl lysine. In some embodiments, non-natural amino acids include sugar moieties. Examples of such amino acids include N-acetyl-L-glucosamine-L-serine, N-acetyl-L-galactosamine-L-serine, N-acetyl-L-glucosamine-L-threonine, N-acetyl-L-glucosamine-L-asparagine and O-mannosamine-L-serine. Examples of the amino acids also include naturally occurring N-linkages or O-linkages between amino acids and sugars that are typically not found in nature, including but not limited to examples of replacements of olefins, oximes, thioethers, amides, etc. Examples of such amino acids also include sugars not typically found in naturally occurring proteins, such as 2-deoxy-glucose, 2-deoxygalactose, and the like. Specific examples of unnatural amino acids include, but are not limited to, p-acetyl-L-phenylalanine, p-propargyloxyphenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propargyloxy-L-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine, and p-azidomethyl-phenylalanine, and the like.In some embodiments, the unnatural amino acid is selected from the group consisting of p-acetyl-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine, or p-azidomethyl-phenylalanine.

[0166] As used herein, the term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in 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 PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.); (iii) conservatively modified variants thereof (including but not limited to degenerate codon substitutions) and complementary sequences as well as explicitly indicated sequences. For 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)).

[0167] As used herein, the term "oxidant" refers to a compound or substance that is capable of removing electrons from a compound being oxidized. For example, oxidants include, but are not limited to, oxidized glutathione, cysteine, cystamine, oxidized dithiothreitol, oxidized erythritol, and oxygen. A variety of oxidants are suitable for use in the methods and compositions described herein.

[0168] As used herein, the term "pharmaceutically acceptable" refers to substances, including but not limited to salts, carriers, or diluents, that do not abrogate the biological activity or properties of the compound and are relatively non-toxic, i.e., the substance can be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any component of the composition in which it is contained.

[0169] As used herein, the term "polyalkylene glycol" or "poly(olefin glycol)" refers to a linear or branched polymeric polyether glycol. Such polyalkylene glycols include, but are not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof. Other exemplary embodiments are listed in, for example, commercial supplier catalogs, such as Shearwater Corporation's catalog "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001). By way of example only, such polymeric polyether glycols have an average molecular weight between about 0.1 kDa and about 100 kDa. For example, such polymeric polyether glycols include, but are not limited to, between about 100 Da and about 100,000 Da. The molecular weight of the polymer may be between about 100 Da and about 100,000 Da, including but not limited to about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da. In some embodiments, the molecular weight of the polymer is between about 100Da and about 50,000Da. In some embodiments, the molecular weight of the polymer is between about 100Da and about 40,000Da. In some embodiments, the molecular weight of the polymer is between about 1,000Da and about 40,000Da. In some embodiments, the molecular weight of the polymer is between about 1,000Da and about 40,000Da. In some embodiments, the molecular weight of the polymer is between about 2,000 and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da. In some embodiments, the poly(ethylene glycol) molecule is a branched polymer.The molecular weight of the branched chain PEG can be between about 1,000 Da and about 100,000 Da, including but not limited to about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 50 ... In some embodiments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 50,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched-chain PEG is between about 5,000 Da and about 20,000 Da. In other embodiments, the molecular weight of the branched-chain PEG is between about 2,000 Da and about 50,000 Da.

[0170] As used herein, the term "polymer" refers to a molecule composed of repeating subunits. Such molecules include, but are not limited to, polypeptides, polynucleotides, or polysaccharides or polyalkylene glycols.

[0171] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description referring to 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 is a non-natural amino acid. Furthermore, the terms "polypeptide," "peptide," and "protein" include amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0172] The term "post-translational modification" refers to any modification of a natural or unnatural amino acid that occurs after such amino acid has been 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.

[0173] As used herein, the term "prodrug" or "pharmaceutically acceptable prodrug" refers to an agent that is converted into a parent drug in vivo or in vitro, wherein it does not eliminate the biological activity or properties of the drug and is relatively non-toxic, that is, the substance can be administered to an individual without causing undesirable biological effects or interacting in a harmful manner with any component of the composition containing it. A prodrug is generally a drug precursor that is converted into an active species or a more active species after administration to a subject and subsequent absorption by a certain process, such as conversion by a metabolic pathway. Some prodrugs have chemical groups present on the prodrug that render it less active and / or impart solubility or some other property to the drug. Once the chemical group has been cleaved and / or modified from the prodrug, the active drug is produced. The prodrug is converted into the active drug in the body by enzymatic or non-enzymatic reactions. Prodrugs can provide improved physiochemical properties, such as better solubility; enhanced delivery characteristics, such as specific targeting to specific cells, tissues, organs or ligands; and improved therapeutic value of the drug. The benefits of such prodrugs include, but are not limited to, (i) ease of administration compared to the parent drug; (ii) the prodrug may be bioavailable by oral administration, 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 derivatives of active drugs that are pharmacologically inactive or have reduced activity. Prodrugs can be designed to regulate the amount of a drug or bioactive molecule that reaches the desired site of action by manipulating the properties of the drug, such as physiochemical properties, biopharmaceutical properties, or pharmacokinetic properties. Without limitation, an example of a prodrug is a non-natural amino acid polypeptide that is administered in the form of an ester ("prodrug") to facilitate transport across the cell membrane (where water solubility is detrimental to mobility), but then, once inside the cell where water solubility is beneficial, it is metabolically hydrolyzed into a carboxylic acid, the active entity. Prodrugs can be designed as reversible drug derivatives to serve as regulators that enhance drug transport to site-specific tissues.

[0174] 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 that is applied prophylactically to a patient and that will alleviate, to some extent, one or more symptoms of the disease, condition, or disorder being treated. In such prophylactic applications, such an amount may depend on the patient's health status, weight, etc. Such a prophylactically effective amount is considered to be within the skill of the art through routine experimentation, including, but not limited to, dose escalation clinical trials.

[0175] As used herein, the term "protected" refers to the presence of a "protecting group" or moiety that prevents the reaction of a chemically reactive functional group under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group being protected. By way of example only, (i) if the chemically reactive group is an amine or hydrazide, the protecting group may be selected from tert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc); (ii) if the chemically reactive group is a thiol, the protecting group may be o-dithiopyridine; and (iii) if the chemically reactive group is a carboxylic acid, such as butyric acid or propionic acid, or a hydroxyl group, the protecting group may be a benzyl or alkyl group, such as methyl, ethyl, or tert-butyl.

[0176] By way of example only, blocking / protecting groups may be selected from:

[0177]

[0178] In addition, protecting groups include, but are not limited to, photolabile groups such as Nvoc and MeNvoc, as well as other protecting groups known in the art. Other protecting groups are described in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, New York, NY, 1999, which is incorporated herein by reference in its entirety.

[0179] The term "recombinant host cell," also referred to as a "host cell," refers to a cell that contains an exogenous polynucleotide, wherein the methods used to insert the exogenous polynucleotide into the cell include, but are not limited to, direct uptake, transduction, f-mating, or other methods known in the art for producing recombinant host cells. By way of example only, such an exogenous polynucleotide may be a non-integrating vector, including, but not limited to, a plasmid, or may be integrated into the host genome.

[0180] 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, but are not limited to, ferrocene, quinones, Ru 2+ / 3+ Complex, Co 2+ / 3+ Complex and Os 2+ / 3+ complex.

[0181] As used herein, the term "reducing agent" refers to a compound or substance that is capable of adding electrons to a reduced compound. For example, reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2-aminoethanethiol), and reduced glutathione. By way of example only, such reducing agents can be used to maintain sulfhydryl groups in a reduced state and reduce intramolecular or intermolecular disulfide bonds.

[0182] As used herein, "refolding" describes any process, reaction, or method that converts an improperly folded or unfolded state to a native or properly folded conformation. By way of example only, refolding converts a disulfide bond-containing polypeptide from an improperly folded or unfolded state to a native or properly folded conformation with respect to the disulfide bonds. Such disulfide bond-containing polypeptides can be natural amino acid polypeptides or non-natural amino acid polypeptides.

[0183] 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 has been administered. For example, a drug that has been administered multiple times and produces only mild or no side effects is considered to have an excellent safety profile. Methods for evaluating the safety profile of any polypeptide are known in the art.

[0184] As used herein, the phrases "selectively hybridize" or "specifically hybridize" refer to the binding, duplexing or hybridization of a molecule to a particular nucleotide sequence under stringent hybridization conditions when the sequence is present in a complex mixture (including but not limited to total cellular or library DNA or RNA).

[0185] The phrase "stringent hybridization conditions" refers to hybridization of sequences of DNA, RNA, PNA or other nucleic acid mimics or combinations thereof under conditions of low ionic strength and high temperature. For example, under stringent conditions, a probe will hybridize to its target subsequence in a complex mixture of nucleic acids (including but not limited to total cellular or library DNA or RNA), but not to other sequences in the complex mixture. Stringent conditions are sequence-dependent and will be different in different circumstances. For example, longer sequences hybridize specifically at higher temperatures. Stringent hybridization conditions include, but are not limited to: (i) about 5-10°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH; (ii) a salt concentration of about 0.01 M to about 1.0 M 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, greater than 50 nucleotides); (iii) the addition of a destabilizing agent, including, but not limited to, formamide; (iv) 50% formamide, 5X SSC, and 1% SDS, incubated at 42°C, or 5X SSC, about 1% SDS, incubated at 65°C, washed in 0.2X SSC, and about 0.1% SDS, incubated 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 twice background. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993).

[0186] 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 can be, but is not limited to, a mammal, including, but not limited to, a human.

[0187] As used herein, the term "substantially purified" means that the target component is substantially or essentially free of other components that normally accompany or interact with the target component prior to purification. By way of example only, a preparation of the target component is "substantially purified" when it contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating components. Thus, a "substantially purified" target component can have a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or more. By way of example only, a natural amino acid polypeptide or non-natural amino acid polypeptide can be purified from natural cells, or from host cells in the case of recombinantly produced natural amino acid polypeptides or non-natural amino acid polypeptides. For example, a preparation of a natural amino acid polypeptide or non-natural amino acid polypeptide can be "substantially purified" when 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 material. For example, when a natural amino acid polypeptide or non-natural amino acid polypeptide is recombinantly produced by a host cell, the natural amino acid polypeptide or non-natural amino acid polypeptide may be present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cells. For example, when the natural amino acid polypeptide or non-natural amino acid polypeptide is recombinantly produced by host cells, the natural amino acid polypeptide 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 dry cell weight. For example, a "substantially purified" natural amino acid polypeptide or non-natural amino acid polypeptide can have a purity level of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more, as determined by an appropriate method, including but not limited to SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0188] The term "substituent" is also called "non-interfering substituent" and refers to a group that can be used to replace another group on a molecule. Such groups include, but are not limited to, halo, C1-C 10 Alkyl, C2-C 10 Alkenyl, C2-C10 Alkynyl, C1-C 10 Alkoxy, C5-C 12 Aralkyl, C3-C 12 Cycloalkyl, C4-C 12 Cycloalkenyl, phenyl, substituted phenyl, toluolyl, xylyl, biphenyl, C2-C 12 Alkoxyalkyl, C5-C 12 Alkoxyaryl, C5-C 12 Aryloxyalkyl, C7-C 12 Oxyaryl, C1-C6 alkylsulfinyl, C1-C 10 Alkylsulfonyl, -(CH2) m -O-(C1-C 10 alkyl), wherein m is 1-8, aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic, substituted heterocyclic, nitroalkyl, -NO2, -CN, -NRC(O)-(C1-C 10 alkyl), -C(O)-(C1-C 10 Alkyl), C2-C 10 Alkylthioalkyl, -C(O)O-(C1-C 10 alkyl), -OH, -SO2, =S, -COOH, -NR2, carbonyl, -C(O)-(C1-C 10 Alkyl)-CF3, -C(O)-CF3, -C(O)NR2, -(C1-C 10 Aryl)-S-(C6-C 10 Aryl), -C(O)-(C6-C 10 Aryl), -(CH2) m -O-(CH2) m -O-(C1-C 10 alkyl), wherein each m is 1 to 8, -C(O)NR2, -C(S)NR2, -SO2NR2, -NRC(O)NR2, -NRC(S)NR2, 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, they are written from left to right, and they also encompass chemically identical substituents that would result from writing the structure from right to left, e.g., -CH2O- is equivalent to -OCH2-.

[0189] By way of example only, substituents for alkyl and heteroalkyl groups (including those groups designated as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) include, but are not limited to, -OR, =O, =NR, =N-OR, -NR2, -SR, -halogen, -SiR3, -OC(O)R, -C(O)R, -C02R, -CONR2, -OC(O)NR2, -NRC(O)R, -NRC(O)NR2, -NR(O)2R, -NR-C(NR2)=NR, -S(O)R, -S(O)2R, -S(O)2NR2, -NRSO2R, -CN, and -NO2. Each R group in the foregoing list includes, but is not limited to, hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, including, but not limited to, aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy, or aralkyl. When two R groups are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR2 is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl.

[0190] For 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, -C02R, -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 a number ranging from zero to the total number of open valences on the aromatic ring system; and wherein each R group in the foregoing list is, but is not limited to, hydrogen, alkyl, heteroalkyl, aryl, and heteroaryl.

[0191] 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, disorder, or condition that is sufficient to cure, at least partially arrest, or alleviate to some extent, one or more symptoms of the disease, disorder, or condition being treated. The effectiveness of the composition will depend on various circumstances, 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 medications; and the judgment of the treating physician. By way of example only, a therapeutically effective amount can be determined through routine experimentation, including, but not limited to, a dose-escalation clinical trial.

[0192] As used herein, the term "thioalkoxy" refers to a sulfur-containing alkyl group attached to the molecule through an oxygen atom.

[0193] As used herein, the term "toxic moiety" or "toxic group" refers to a compound that can cause harm, disorder, or death. Toxic moieties include, but are not limited to, auristatin, DNA minor groove binder, DNA minor groove alkylating agent, enediyne, lexitropsin, duocarmycin, taxane, puromycin, TLR agonist, maytansinoid, vinca alkaloid, AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholinodoxorubicin, rhizoxin, cyanomorpholinodoxorubicin, TLR agonist-10, echinomycin, combretatstatin, chalicheamicin, maytansine, DM-1, fusobactin, podophyllotoxin (e.g., etoposide, teniposide, etc.), baccatin and its derivatives, anti-tubulin agent), cryptophysin, combretastatin, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, epothilone A, epothilone B, nocodazole, colchicine, colcemid, estramustine, cemadotin, discodermolide, maytansine, eleutherobin, Mechlorethamine, cyclophosphamide, melphalan, carmustine, lomustine, semustine, streptozocin, chlorozolin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphate, busulfan, dacarbazine, temozolomide, ytarabine, cytosine arabinoside, fluorouracil, floxuridine, 6-thioguanine, 6-mercaptopurine, pentostatin, 5-fluorouracil, methotrexate, 10-propargyl-5,8-dideazafolate, 5,8-dideazatetrahydrofolic acidacid), 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, rocithromycin, furazolidone, amoxicillin, ampicillin, carbenicillin, flucloxacillin, methicillin, penicillin, ciprofloxacin, Moxifloxacin, ofloxacin, doxycycline, minocycline, oxytetracycline, tetracycline, streptomycin, rifabutin, ethambutol, rifaximin, etc.), antiviral drugs (such as abacavir, acyclovir, ampligen, cidofovir, delavirdine, didanosine, efavirenz, entecavir, fosfonet, ganciclovir, ibacitabine, imunovir, iodine, inosine, lopinavir, methisazone, nesavir ( nexavir), nevirapine, oseltamivir, penciclovir, stavudine, trifluridine, truvada, valacyclovir, zanamivir, etc.), daunorubicin hydrochloride, daunorubicin, rubidomycin, cerubidine, idarubicin, doxorubicin, epirubicin and morpholino derivatives, phenoxazinone bicyclic peptides (such as dactinomycin), basic glycopeptides (such as bleomycin), anthraquinone glycosides (such as plicamycin, mithramycin), anthracenediones (e.g., mitoxantrone), aziridine pyrroloindolediones (e.g., mitomycin), macrocyclic immunosuppressants (e.g., cyclosporine, FK-506, tacrolimus, prograf, rapamycin, etc.), navelbene, CPT-11, anastrazole, letrazole, capecitabine, raloxifene, cyclophosphamide, ifosfamide, droloxafine, allocolchicine, Halichondrin B, colchicine, colchicine derivatives, maytansine, rhizoxin, paclitaxel, paclitaxel derivatives, docetaxel, thiocolchicine, tritylcysteineCysterin, vinblastine sulfate, vincristine sulfate, cisplatin, carboplatin, hydroxyurea, N-methylhydrazine, epidophyllotoxin, procarbazine, mitoxantrone, leucovorin, and tegafur. "Taxane" includes paclitaxel and any active taxane derivative or prodrug.

[0194] As used herein, the terms "treat," "treating," or "treatment" include alleviating, alleviating, or ameliorating the symptoms of a disease or condition; preventing additional symptoms; ameliorating or preventing the underlying metabolic causes of symptoms; inhibiting the disease or condition, such as arresting the development of the disease or condition; relieving the disease or condition; causing regression of the disease or condition; alleviating the condition caused by the disease or condition; or terminating the symptoms of the disease or condition. The terms "treat," "treating," or "treatment" include, but are not limited to, prophylactic and / or therapeutic treatment.

[0195] As used herein, the term "water-soluble polymer" refers to any polymer that is soluble in an aqueous solvent. Such water-soluble polymers include, but are not limited to, polyethylene glycol, polyethylene glycol propionaldehyde, its mono-C1-C 105,252,714, which is incorporated herein by reference), monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran derivatives (including dextran sulfate), polypropylene glycol, polyoxypropylene / 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 glycols and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ether and α-β-poly[(2-hydroxyethyl)-DL-asparagine, and the like, or mixtures thereof. By way of example only, such water-soluble polymers coupled to natural amino acid polypeptides or non-natural polypeptides can result in changes including, but not limited to, increased water solubility; increased or regulated serum half-life; increased or regulated therapeutic half-life relative to the unmodified form; increased bioavailability; regulated biological activity; increased circulation time; regulated immunogenicity; regulated physical association characteristics, including, but not limited to, aggregation and multimer formation, altered receptor binding, binding of activity modulators, or other targeting polypeptides, 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 can be used 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.

[0196] 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.

[0197] The compounds provided herein (including but not limited to non-natural amino acids, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) include isotopically labeled compounds, which are identical to those compounds recited in the various formulas and structures provided herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds herein include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, such as, respectively. 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 (e.g., into which a radioactive isotope such as 3 H and 14 C) are useful in drug and / or substrate tissue distribution assays. In addition, deuterium (i.e. 2 Isotopic substitutions of H) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements.

[0198] Some compounds herein (including but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reagents for producing the aforementioned compounds) have asymmetric carbon atoms and can therefore exist as enantiomers or diastereomers. Based on their physicochemical differences, diastereomeric mixtures can be separated into their individual diastereomers by known methods, such as by chromatography and / or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers, and converting the individual diastereomers (e.g., hydrolyzing) into the corresponding pure enantiomers. All such isomers (including diastereomers, enantiomers, and mixtures thereof) are considered to be part of the compositions described herein.

[0199] In another or further embodiment, the compounds described herein (including but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) are used in the form of prodrugs. In another or further embodiment, the compounds described herein (including but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) are metabolized after administration to an organism in need thereof to produce metabolites, which are then used to produce a desired effect, including a desired therapeutic effect. In further or additional embodiments, they are active metabolites of non-natural amino acids and "modified or unmodified" non-natural amino acid polypeptides.

[0200] The methods and preparations described herein include N-oxides, crystalline forms (also referred to 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 provided 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 non-solvated and solvated forms with pharmaceutically acceptable solvents (such as water, ethanol, etc.). The solvated forms of the non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides provided herein are also considered to be disclosed in this article.

[0201] Some compounds herein (including but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reagents for producing the aforementioned compounds) can exist in several tautomeric forms. All such tautomeric forms are considered to be part of the compositions described herein. Also, for example, all enol-keto forms of any compound herein (including but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reagents for producing the aforementioned compounds) are considered to be part of the compositions described herein.

[0202] 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 and reagents for producing any of the foregoing compounds) are acidic and can form salts with pharmaceutically acceptable cations. Some of the compounds herein (including but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reagents for producing the foregoing compounds) can be basic and therefore can 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 entities in aqueous, non-aqueous or partially aqueous media. The salts are recovered by using at least one of the following techniques: filtration, precipitation with a non-solvent, followed by filtration, evaporation of the solvent, or, in the case of aqueous solutions, lyophilization.

[0203] Pharmaceutically acceptable salts of the non-natural amino acid polypeptides disclosed herein can be formed when the acidic protons present in the parent non-natural amino acid polypeptide are replaced by metal ions, such as alkali metal ions, alkaline earth metal ions, or aluminum ions, or coordinated with organic bases. In addition, salts of the starting materials or intermediates can be used to prepare salt forms of the disclosed non-natural amino acid polypeptides. The non-natural amino acid polypeptides described herein can be made into 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 acid or organic acid. Alternatively, the non-natural amino acid polypeptides described herein can be made into 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 base or organic base.

[0204] The types of pharmaceutically acceptable salts include, but are not limited to: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; 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- (2) salts formed when the acidic protons present in the parent compound are replaced by metal ions such as alkali metal ions, alkaline earth metal ions or aluminum ions or coordinated with organic bases. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, etc.

[0205] Use various methods, including but not limited to, ion exchange chromatography, ion chromatography, capillary electrophoresis, inductively coupled plasma, atomic absorption spectroscopy, mass spectrometry or any combination thereof, can analyze and identify the corresponding counterion of the pharmaceutically acceptable salt of non-natural amino acid polypeptide. In addition, use the techniques and methods described in the Examples to test the therapeutic activity of such pharmaceutically acceptable salts of non-natural amino acid polypeptide.

[0206] It should be understood that reference to a salt includes solvent addition forms or its crystal forms, particularly solvates or polymorphs. Solvates contain stoichiometric and non-stoichiometric amounts of solvent and are often formed during crystallization with pharmaceutically acceptable solvents (such as water, ethanol, etc.). Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Polymorphs include different crystal packing arrangements of the same elemental composition of a compound. Polymorphs typically have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. Various factors such as recrystallization solvent, crystallization rate, and storage temperature can cause a single crystalline form to predominate.

[0207] Screening and characterization of pharmaceutically acceptable salt polymorphs and / or solvates 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 degradation 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 thermogravimetric-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, UV-Vis, and NMR (liquid and solid). Various microscopic 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 gas or water vapor atmosphere), IR microscopy, and Raman microscopy.

[0208] Although preferred embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Without departing from the present invention, numerous changes, variations, and substitutions will now occur to those skilled in the art. It should be understood that the various alternatives of the embodiments of the present invention described herein can be used for implementing the present invention. It is contemplated that the following claims define the scope of the present invention, and thus encompass methods and structures within the scope of these claims and their equivalents.

[0209] TLR agonist linker derivatives

[0210] At one level, described herein are tools (methods, compositions, techniques) for producing and using targeting polypeptides comprising a TC or analog of at least one non-natural amino acid or a modified non-natural amino acid having a carbonyl, dicarbonyl, oxime, or hydroxylamine group. Such targeting polypeptides comprising a TC of a non-natural amino acid may contain other functionalities, including but not limited to polymers; water-soluble polymers; derivatives of polyethylene glycol; a second protein or polypeptide or polypeptide analog; antibodies or antibody fragments; and any combination thereof. It is noted that the various functionalities mentioned above are not intended to imply that members of one functionality cannot be classified as members of another functionality. In fact, depending on the particular case, there will be overlap. By way of example only, water-soluble polymers overlap in scope with polyethylene glycol derivatives; however, the overlap is not complete, and therefore both functionalities are referenced above.

[0211] In one aspect, methods for selecting and designing TLR agonist linker derivatives and targeting polypeptides, as well as methods for modifying the targeting polypeptides using the methods, compositions, and techniques described herein, are provided. New TLR agonist linker derivatives and targeting polypeptides can be designed de novo, including, by way of example only, as part of a high-throughput screening process (in which case, multiple polypeptides can be designed, synthesized, characterized, and / or tested), or based on the interests of the researcher. New TLR linker derivatives and targeting polypeptides can also be designed based on the structure of a known or partially characterized polypeptide. By way of example only, TLR agonists have been the subject of intensive scientific research; new compounds can be designed based on the structure of a TLR agonist. Principles for selecting which amino acids to substitute and / or modify are described separately herein. The choice of which modification to employ is also described herein and can be used to meet the needs of the experimenter or end user. Such needs may include, but are not limited to, manipulating the therapeutic effectiveness of a polypeptide, improving the safety profile of a polypeptide, adjusting the pharmacokinetics, pharmacology, and / or pharmacodynamics of a polypeptide, such as, by way of example only, increasing water solubility, bioavailability, extending serum half-life, extending therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending circulation time. Additionally, such modifications include, by way of example only, providing additional functionality to the polypeptide, incorporation into antibodies, and any combination of the foregoing modifications.

[0212] Also described herein are TLR agonist linker derivatives and targeting polypeptides that have or can be modified to contain an oxime, carbonyl, dicarbonyl, or hydroxylamine group. This aspect also includes methods of producing, purifying, characterizing, and using such TLR agonist linker derivatives and targeting polypeptides.

[0213] The TLR agonist linker derivatives or targeting polypeptides 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 carbonyl or dicarbonyl groups, oxime groups, hydroxylamine groups or protected forms thereof. The TLR agonist linker derivatives or targeting polypeptides can be the same or different, for example, in a derivative comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different sites can be present.

[0214] As described herein, the present disclosure provides a targeting polypeptide coupled to another molecule having the formula "Targeting Polypeptide-LM," wherein L is a linker 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.

[0215] The targeting polypeptide and M can be linked together via L using standard linkers and procedures known to those skilled in the art. In some aspects, the targeting polypeptide and M are directly fused, and L is a bond. In other aspects, the targeting polypeptide and M are fused via a linker L. For example, in some embodiments, the targeting polypeptide and M are linked together via a peptide bond, optionally via a peptide or amino acid spacer. In some embodiments, the targeting polypeptide and M are chemically conjugated, optionally via a linker (L). In some embodiments, L is directly conjugated to each of the targeting polypeptide and M.

[0216] Chemical conjugation can occur by reacting a nucleophilic reactive group of one compound with an electrophilic reactive group of another compound. In some embodiments, when L is a bond, the targeting polypeptide is conjugated to M by reacting a nucleophilic reactive moiety on the targeting polypeptide with an electrophilic reactive moiety on Y, or by reacting an electrophilic reactive moiety on the targeting polypeptide with a nucleophilic reactive moiety on M. In embodiments, when L is a group that connects the targeting polypeptide and M together, the targeting polypeptide and / or M can be conjugated to L by reacting a nucleophilic reactive moiety on the targeting polypeptide and / or M with an electrophilic reactive moiety on L, or by reacting an electrophilic reactive moiety on the targeting polypeptide and / or M with a nucleophilic reactive moiety on L. Non-limiting examples of nucleophilic reactive groups include amino, thiol, and hydroxyl groups. Non-limiting examples of electrophilic reactive groups include carboxyl, acyl chloride, anhydride, ester, succinimidyl ester, alkyl halide, sulfonate, maleimido, 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 may be used to form an activated ester of the carboxylic acid.

[0217] 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 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'-tetramethyluronium hexafluorophosphate (HBTU).

[0218] In some embodiments, the targeting polypeptide comprises a nucleophilic reactive group (e.g., an amino group, a sulfhydryl group, or a hydroxyl group of a lysine, cysteine, or serine side chain) 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 of an Asp or Glu side chain) that can be conjugated to a nucleophilic reactive group on M or L. In some embodiments, the targeting polypeptide is chemically modified to comprise a reactive group that can be directly conjugated to M or to L. In some embodiments, the targeting polypeptide is modified at the N-terminus or C-terminus to comprise a natural or non-natural amino acid with a nucleophilic side chain. In an exemplary embodiment, the N-terminal or C-terminal amino acid of the targeting polypeptide is selected from the group consisting of: lysine, ornithine, serine, cysteine, and homocysteine. For example, the N-terminal or C-terminal amino acid of the targeting polypeptide can be modified to comprise a lysine residue. In some embodiments, the N-terminal or C-terminal amino acid of the targeting polypeptide is modified to comprise a natural or non-natural amino acid with an electrophilic side chain, such as Asp and Glu. In some embodiments, the internal amino acids of the targeting polypeptide are substituted with natural or non-natural amino acids having nucleophilic side chains as described above. In an exemplary embodiment, the internal amino acids of the targeting polypeptide that are substituted are selected from the group consisting of: lysine, ornithine, serine, cysteine, and homocysteine. For example, the internal amino acids of the targeting polypeptide can be substituted with lysine residues. In some embodiments, the internal amino acids of the targeting polypeptide are substituted with natural or non-natural amino acids having electrophilic side chains, such as Asp and Glu.

[0219] In some embodiments, M comprises a reactive group capable of directly conjugating to a targeting polypeptide or to L. In some embodiments, M comprises a nucleophilic reactive group (e.g., an amine, a thiol, a hydroxyl group) capable of conjugating to an electrophilic reactive group on a 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 a targeting polypeptide or L. In some embodiments, M is chemically modified to comprise a nucleophilic reactive group capable of conjugating to an electrophilic reactive group on a targeting polypeptide or L. In some embodiments, M is chemically modified to comprise an electrophilic reactive group capable of conjugating to a nucleophilic reactive group on a targeting polypeptide or L.

[0220] In some embodiments, conjugation can be performed via organosilanes, such as aminosilanes treated with glutaraldehyde; carbonyldiimidazole (CDI) activation of silanol groups; or using dendrimers. A variety of dendrimers are known in the art, including poly(amidoamine) (PAMAM) dendrimers, which are synthesized by a divergent process starting from an ammonia or ethylenediamine initiator core reagent; a subclass of PAMAM dendrimers based on a triaminoethyleneimine core; radially layered poly(amidoamine-organosilicon) dendrimers (PAMAMOS), which are reversed unimolecular micelles composed of a hydrophilic, nucleophilic polyamidoamine (PAMAM) interior and a hydrophobic organosilicon (OS) exterior; poly(propyleneimine) (PPI) dendrimers, which are generally polyalkylamines with primary amines as end groups, while the dendrimer interior consists of numerous tertiary tripropyleneamines; polyacrylamine (POPAM) dendrimers; diaminobutane (DAB) dendrimers; amphiphilic dendrimers; micellar dendrimers, which are unimolecular micelles of water-soluble hyperbranched polyphenylenes; polylysine dendrimers; and dendrimers based on a polybenzyl ether hyperbranched backbone.

[0221] In some embodiments, conjugation can be performed by olefin metathesis. In some embodiments, M and the targeting polypeptide, M and L, or both the targeting polypeptide and L comprise an olefin or alkyne moiety capable of undergoing metathesis. In some embodiments, a suitable catalyst (e.g., copper, ruthenium) is used to accelerate the metathesis reaction. Suitable methods for performing olefin metathesis reactions are described in the art. See, for example, Schafmeister et al., J. Am. Chem. Soc. 122: 5891-5892 (2000), Walensky et al., Science 305: 1466-1470 (2004), and Blackwell et al., Angew, Chem., Int. Ed. 37: 3281-3284 (1998).

[0222] In some embodiments, click chemistry can be used for conjugation. The "click reaction" is broad and easy to perform, uses only readily available reagents, and is insensitive to oxygen and water. In some embodiments, the click reaction is a cycloaddition reaction between an alkynyl group and an azide group to form a triazole group. In some embodiments, the click reaction uses a copper or ruthenium catalyst. Suitable methods for performing click reactions are described in the art. See, for example, Kolb et al., Drug Discovery Today 8:1128 (2003); Kolb et al., Angew. Chem. Int. Ed. 40:2004 (2001); Rostovtsev et al., Angew. Chem. Int. Ed. 41:2596 (2002); Tornoe et al., J. Org. Chem. 67:3057 (2002); Manetsch et al., J. Am. Chem. Soc. 126:12809 (2004); Lewis et al., J. Am. Chem. Soc. 126:12809 (2004); s et al., Angew. Chem. Int. Ed. 41: 1053 (2002); Speers, J. Am. Chem. Soc. 125: 4686 (2003); Chan et al., Org. Lett. 6: 2853 (2004); Zhang et al., J. Am. Chem. Soc. 127: 15998 (2005); and Waser et al., J. Am. Chem. Soc. 127: 8294 (2005).

[0223] Indirect conjugation via high affinity specific binding partners such as streptavidin / biotin or avidin / biotin or lectin / carbohydrate is also contemplated.

[0224] In some embodiments, the targeting polypeptide and / or M are functionalized to include nucleophilic reactive groups or electrophilic reactive groups with an organic derivatizing agent. This derivatizing agent can react with the selected side chain or N-terminal or C-terminal residue of the targeting amino acid on the targeting polypeptide and the functional group on M. The reactive groups on the targeting polypeptide and / or M include, for example, aldehyde groups, amino groups, ester groups, thiol groups, α-haloacetyl groups, maleimido groups, or hydrazino groups. Derivatizing agents include, for example, maleimidobenzoylsulfosuccinimide ester (conjugated 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 connected to each other through an intermediate carrier such as a polysaccharide or polypeptide carrier. Examples of polysaccharide carriers include aminodextran. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, their copolymers, and mixed polymers of these amino acids and other amino acids such as serine, thereby giving the desired solubility to the resulting load carrier.

[0225] Cysteinyl residues are most commonly reacted with α-haloacetates (and corresponding amines) such as chloroacetic acid or chloroacetamide to give carboxymethyl or carboxamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl disulfide, p-chloromercurylbenzoate, 2-chloromercuryl-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0226] Histidyl residues are derivatized by reaction with diethyl pyrocarbonate at pH 5.5-7.0, as this agent is relatively specific for the histidine side chain. P-Bromophenacyl bromide is also useful; the reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0.

[0227] Lysyl and amino terminal residues are reacted with succinic acid or other carboxylic anhydrides. Derivatization with these reagents has the effect of reversing the charge of the lysyl residues. Other suitable reagents for derivatizing α-amino-containing residues include imide esters such as methyl pyridinium imide, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactions with glyoxylate.

[0228] Arginyl residues are modified by reaction with one or more common reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed under alkaline conditions due to the high pKa of the guanidine functional group. Furthermore, these reagents react with lysine groups as well as the arginine epsilon-amino group.

[0229] Tyrosyl residues can be specifically modified, and of particular interest is the introduction of 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.

[0230] 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. In addition, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0231] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino group of lysine, arginine and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)), deamidation of asparagine or glutamine, acetylation of the N-terminal amine and / or amidation or esterification of the C-terminal carboxylic acid group.

[0232] Another type of covalent modification involves chemical or enzymatic coupling of glycosides to peptides. Sugars can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as the sulfhydryl group of cysteine, (d) free hydroxyl groups, such as the hydroxyl group of serine, threonine, or hydroxyproline, (e) aromatic residues, such as the residues 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).

[0233] 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 exemplary embodiments, L is an amide bond formed when an amine on the targeting polypeptide (e.g., the ε-amine of a lysine residue) reacts with a carboxyl group on M. In alternative embodiments, the targeting polypeptide and / or M are derivatized with a derivatizing agent prior to conjugation.

[0234] In some embodiments, L is a linking group. In some embodiments, L is a bifunctional linker and only comprises two reactive groups before being conjugated to the targeting polypeptide and M. In the embodiment in which both the targeting polypeptide and M have electrophilic reactive groups, L comprises two identical or two different nucleophilic groups (e.g., amine, hydroxyl, thiol) before being conjugated to the targeting polypeptide and M. In the embodiment in which both the targeting polypeptide and M have nucleophilic reactive groups, L comprises two identical or two different electrophilic groups (e.g., carboxyl, activated form of carboxyl, compound with leaving group) before being conjugated to the targeting polypeptide and M. In the embodiment in which one of the targeting polypeptide or M has nucleophilic reactive groups and the other of the targeting polypeptide or M has electrophilic reactive groups, L comprises one nucleophilic reactive group and one electrophilic group before being conjugated to the targeting polypeptide and M.

[0235] L can be any molecule having at least two reactive groups (before conjugation to the targeting polypeptide and M) that are capable of reacting with each of the targeting polypeptide and M. In some embodiments, L has only two reactive groups and is bifunctional. L (before conjugation to the peptide) can be represented by Formula VI:

[0236]

[0237] Wherein A and B are independently nucleophilic or electrophilic reactive groups. In some embodiments, both A and B are nucleophilic groups, or both are 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.

[0238] Table 1: Non-limiting combinations of nucleophilic and electrophilic groups

[0239]

[0240]

[0241]

[0242]

[0243] In some embodiments, A and B may include olefins and / or alkyne functional groups suitable for olefin metathesis reactions. In some embodiments, A and B include 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.

[0244] In some embodiments, L is hydrophobic. Hydrophobic linkers are known in the art. See, for example, Bioconjugate Techniques, G.T. Hermanson (Academic Press, San Diego, CA, 1996), which is incorporated herein by reference in its entirety. Suitable hydrophobic linking groups known in the art include, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoic acid. Prior to conjugation to the peptide of the composition, the hydrophobic linking group includes at least two reactive groups (A and B), as described herein and as follows:

[0245]

[0246] In some embodiments, the hydrophobic linking group comprises a maleimido or iodoacetyl and a carboxylic acid or activated carboxylic acid (e.g., NHS ester) as a reactive group. In these embodiments, with or without a coupling agent, the maleimido or iodoacetyl can be coupled to the thiol moiety on the targeting polypeptide or M, and the carboxylic acid or activated carboxylic acid can be coupled to the amine on the targeting polypeptide or M. Any coupling agent known to those skilled in the art, for example, DCC, DIC, HATU, HBTU, TBTU, and other activators described herein, can be used to couple the carboxylic acid to the free amine. In a specific embodiment, the hydrophilic linking group comprises a fatty chain with 2 to 100 methylene groups, wherein A and B are carboxyl groups or derivatives thereof (e.g., succinic acid). In other specific embodiments, L is iodoacetic acid.

[0247]

[0248] In some embodiments, the linking group is hydrophilic, such as, for example, a polyalkylene glycol. Prior to conjugation to the peptide of the composition, the hydrophilic linking group comprises at least two reactive groups (A and B), as described herein and as follows:

[0249]

[0250] In some embodiments, the linker is polyethylene glycol (PEG). In certain embodiments, PEG has a molecular weight of about 100 daltons to about 10,000 daltons, for example, 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.

[0251] In some embodiments, the hydrophilic linking group includes maleimido or iodoacetyl and carboxylic acid or activated carboxylic acid (e.g., NHS ester) as a reactive group. In these embodiments, with or without a coupling agent, maleimido or iodoacetyl can be coupled to the thiol moiety on the targeting polypeptide or M, and carboxylic acid or activated carboxylic acid can be coupled to the amine on the targeting polypeptide or M. Any suitable coupling agent known to those skilled in the art, for example, DCC, DIC, HATU, HBTU, TBTU, and other activators described herein, can be used to couple carboxylic acid to amine. In some embodiments, the linking group is maleimido-polymer (20-40kDa)-COOH, iodoacetyl-polymer (20-40kDa)-COOH, maleimido-polymer (20-40kDa)-NHS, or iodoacetyl-polymer (20-40kDa)-NHS.

[0252] In some embodiments, the linking group consists of an amino acid, a dipeptide, a tripeptide, or a polypeptide, wherein the amino acid, dipeptide, tripeptide, or polypeptide comprises at least two activating groups as described herein. In some embodiments, the linking group (L) comprises a moiety selected from the group consisting of amino, ether, thioether, maleimido, disulfide, amide, ester, thioester, olefin, cycloolefin, alkyne, trizoyl, carbamate, carbonate, cathepsin B-cleavable, and hydrazone.

[0253] In some embodiments, L comprises a chain of 1 to about 60 atoms, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms. In some embodiments, the chain atoms are all carbon atoms. In some embodiments, the chain atoms in the linker backbone are selected from C, O, N, and S. Chain atoms and linkers can be selected according to their expected solubility (hydrophilicity) to provide a more soluble conjugate. In some embodiments, L provides a functional group that is cleaved by an enzyme or other catalyst or by the hydrolysis conditions found in the target tissue, organ, or cell. In some embodiments, the length of L is long enough to reduce the possibility of steric hindrance.

[0254] 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, for example, when incubated in serum for 5 minutes, less than 25%, 20%, 15%, 10% or 5% of the conjugate is cleaved. In other embodiments, L is stable in serum for at least 10 minutes, or 20 minutes, or 25 minutes, or 30 minutes, or 60 minutes, or 90 minutes, or 120 minutes, or 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours or 24 hours. In these embodiments, L does not include a functional group that can undergo in vivo hydrolysis. In some exemplary embodiments, L is stable in serum for at least about 72 hours. Non-limiting examples of functional groups that can not undergo significant in vivo hydrolysis include amides, ethers and thioethers. For example, the following compounds do not undergo significant in vivo hydrolysis:

[0255]

[0256] In some embodiments, L is hydrolyzable in vivo. In these embodiments, L comprises a functional group capable of undergoing in vivo hydrolysis. Non-limiting examples of functional groups capable of undergoing in vivo hydrolysis include esters, anhydrides, and thioesters. For example, the following compound is capable of undergoing in vivo hydrolysis because it comprises an ester group:

[0257]

[0258] In some exemplary embodiments, L is unstable and undergoes extensive hydrolysis in plasma at 37° C. within 3 hours and complete hydrolysis within 6 hours. In some exemplary embodiments, L is not unstable.

[0259] In some embodiments, L is metastable in vivo. In these embodiments, L comprises a functional group that can be chemically or enzymatically cleaved in vivo, optionally over a period of time (e.g., an acid-labile, reduction-labile, or enzyme-labile functional group). In these embodiments, L may comprise, for example, a hydrazone moiety, a disulfide moiety, or a cathepsin-cleavable moiety. When L is metastable, and without wishing to be bound by any particular theory, the targeting polypeptide-LM conjugate is stable in the extracellular environment, e.g., in serum, for the aforementioned time period, but is unstable in the intracellular environment or under conditions simulating the intracellular environment, such that it cleaves upon entry into the cell. In some embodiments, when L is metastable, L is stable in serum for at least about 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 42 hours, or 48 hours, such as at least about 48 hours, 54 hours, 60 hours, 66 hours, or 72 hours, or about 24-48 hours, 48-72 hours, 24-60 hours, 36-48 hours, 36-72 hours, or 48-72 hours.

[0260] In another embodiment, the polymer derivative of the present invention comprises a polymer backbone having the structure:

[0261] X—CH2CH2O--(CH2CH2O) n --CH2CH2-O-(CH2) m -WN=N=N, where:

[0263] W is an aliphatic or aromatic linker moiety containing 1 to 10 carbon atoms;

[0264] n is 1 to about 4000; and X is a functional group as described above; and m is between 1 and 10.

[0265] 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 shown below, a water-soluble polymer backbone having an average molecular weight of about 800 Da to about 100,000 Da is reacted with an azide anion (which can be paired with any of a number of suitable counterions, including sodium, potassium, tert-butylammonium, etc.), wherein the polymer backbone has a first end bonded to a first functional group and a second end bonded to a suitable leaving group. The leaving group undergoes nucleophilic displacement and is replaced by the azide moiety, thereby providing the desired azide-containing polymer polymer;

[0266] X-polymer-LY+N3 - →X-Polymer-L N3

[0267] As shown, the polymer backbone suitable for the present invention has formula X-polymer-LY, wherein polymer is poly (ethylene glycol), X is the functional group that does not react with azide group, and Y is the leaving group suitable for.The example of suitable functional group includes but is 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.The example of suitable leaving group includes but is not limited to chloride, bromide, iodide, mesylate, trifluoroethylsulfonate (tresylate) and toluenesulfonate.

[0268] In another method of preparing the azide-containing polymer derivatives of the present invention, a linker carrying an azide functionality is contacted with a water-soluble polymer backbone having an average molecular weight of about 800 Da to about 100,000 Da, wherein the linker carries a chemical functionality that will selectively react with the chemical functionality on the polymer to form an azide-containing polymer derivative product, wherein the azide is separated from the polymer backbone by the linking group.

[0269] An exemplary reaction scheme is shown below:

[0270] X-polymer-Y+N-linker-N=N=N→PG-X-polymer-linker-N=N=N wherein:

[0271] The polymer is poly(ethylene glycol), and X is a capping group such as an alkoxy group or a functional group as described above; and Y is a functional group that does not react with the azide functionality but will react efficiently and selectively with the N functionality.

[0272] Examples of suitable functional groups include, but are not limited to, if N is an amine, Y is a carboxylic acid, carbonate, or active ester; if N is a hydrazide or aminooxy moiety, Y is a ketone; if N is a nucleophile, Y is a leaving group. If desired, purification of the crude product can be accomplished by known methods, including but not limited to precipitation of the product followed by chromatography.

[0273] In the case of polymeric diamines, a more specific example is shown below, where 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.

[0274] In this case, a variety of activating agents such as thionyl chloride or carbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazole can be used to couple the amine group to the carboxylic acid group, thereby generating an amide bond between the monoamine polymer derivative and the azide-bearing linker moiety. After the amide bond is successfully formed, the resulting N-tert-butyl-Boc-protected azide-containing derivative can be used directly to modify biologically active molecules or can be further processed to install other useful functional groups. For example, the Nt-Boc group can be hydrolyzed by treatment with a strong acid to generate an ω-amino-polymer-azide. The resulting amine can be used as a synthetic handle to install other useful functionalities, such as maleimide groups, activated disulfides, activated esters, etc., to generate valuable heterobifunctional reagents.

[0275] Heterobifunctional derivatives are particularly useful when it is desired to attach a different molecule to each end of the polymer. For example, an ω-N-amino-N-azide polymer will allow a molecule with an activated electrophilic group such as an aldehyde, ketone, activated ester, activated carbonate, etc. to be attached to one end of the polymer, and a molecule with an acetylene group to be attached to the other end of the polymer.

[0276] In another embodiment of the present invention, A is an aliphatic linker having 1 to 10 carbon atoms or a substituted aryl ring having 6 to 14 carbon atoms. X is a functional group that does not react with an azide group, and Y is a suitable leaving group.

[0277] Multiple targeting polypeptides can be connected by a linker polypeptide, wherein the linker polypeptide is optionally 6-14, 7-13, 8-12, 7-11, 9-11 or 9 amino acids in length. Other linkers include, but are not limited to, small polymers such as PEG, which can be multi-armed, thereby allowing multiple targeting polypeptide molecules to be linked together. By using such a linker or by direct chemical bonding between the respective N-termini of each polypeptide, multiple targeting polypeptides and modified targeting polypeptides can be connected to each other via their N-termini in a head-to-head configuration. For example, two targeting polypeptides can be connected to form a dimer by chemical bonding between their N-terminal amino groups or modified N-terminal amino groups. Moreover, a linker molecule designed to contain multiple chemical functional groups so as to bond to the N-terminus of each targeting polypeptide can be used to connect multiple targeting polypeptides at their corresponding N-termini. In addition, multiple targeting polypeptides can be connected by bonding between amino acids other than the N-terminal amino acid or the C-terminal amino acid. Examples of covalent bonds that can be used to form dimers and multimers of the targeting polypeptides described herein include, but are not limited to, disulfide bonds, sulfhydryl bonds, or thiol bonds. Additionally, certain enzymes (such as sortase) can be used to form a covalent bond between a targeting polypeptide and a linker (including at the N-terminus of the targeting polypeptide).

[0278] The linker can have a wide range of molecular weight or molecular length. Linkers of greater or lesser molecular weight can be used to provide desired spatial relationships or conformations between the targeting polypeptide and the connection entity or between the connection entity and its binding partner (if any). Linkers with longer or shorter molecular lengths can also be used to provide desired space or flexibility between the targeting polypeptide and the connection entity or between the connection entity and its binding partner.

[0279] 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 at at least a first end of a polymer backbone; and b) at least a second functional group at a second end of the polymer backbone. The second functional group may be the same as or different from the first functional group. In some embodiments, the second functional group does not react with the first functional group. In some embodiments, the present 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.

[0280] In an exemplary embodiment, the polymer is linked to the targeting polypeptide or modified targeting polypeptide via a linker. For example, the linker may comprise one or two amino acids that are bound to the polymer, such as an albumin-binding moiety, at one end and to any available position on the polypeptide backbone at the other end. Additional exemplary linkers include hydrophilic linkers, such as a chemical moiety comprising at least five non-hydrogen atoms, wherein 30%-50% of these non-hydrogen atoms are N or O. Additional exemplary linkers that can link the polymer to the targeting polypeptide or modified targeting polypeptide are disclosed in US 2012 / 0295847 and WO / 2012 / 168430, each of which is incorporated herein by reference in its entirety.

[0281] Optionally, multiple targeting polypeptides or modified targeting polypeptide molecules can be connected by a linker polypeptide, wherein the linker polypeptide is optionally 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12 amino acids in length, and longer, wherein optionally the N-terminus of one targeting polypeptide is fused to the C-terminus of a linker polypeptide, and the N-terminus of a linker polypeptide is fused to the N-terminus of another targeting polypeptide. Further exemplary linker polypeptides that can be utilized are disclosed in WO / 2013 / 004607, which is incorporated herein by reference in its entirety.

[0282] As used herein, the terms "electrophilic group", "electrophile", etc. 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, compounds containing halides, carbonyl groups, and epoxides. Common electrophilic reagents can be halides, such as thiophosgene, glycerol dichlorohydrin, phthaloyl chloride, succinyl chloride, chloroacetyl chloride, chlorosuccinyl chloride, etc.; ketones, such as chloroacetone, bromoacetone, etc.; aldehydes, such as glyoxal, etc.; isocyanates, such as hexamethylene diisocyanate, toluene diisocyanate, m-xylene diisocyanate, cyclohexylmethane-4,4-diisocyanate, etc., and derivatives of these compounds.

[0283] 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 may be an ionizable group that reacts as an anionic group. As used herein, "nucleophilic group" includes, but is not limited to, hydroxyl groups, primary amines, secondary amines, tertiary amines, and thiols.

[0284] Table 2 provides various starting electrophiles and nucleophiles that can be combined to produce the desired functional groups. The information provided is meant to be illustrative and not limiting of the synthetic techniques described herein.

[0285] Table 2: Examples of covalent linkages and their precursors

[0286]

[0287]

[0288] In general, carbon electrophiles are susceptible to attack by complementary nucleophiles, including carbon nucleophiles, where the attacking nucleophile donates an electron pair to the carbon electrophile, thereby forming a new bond between the nucleophile and the carbon electrophile.

[0289] Non-limiting examples of carbon nucleophiles include, but are not limited to, alkyl, alkenyl, aryl, and alkynyl Grignard reagents, organolithiums, organozincs, alkyl-, alkenyl, aryl-, and alkynyl-tin reagents (organostannanes), alkyl-, alkenyl-, aryl-, and alkynyl-borane reagents (organoboranes and organoborates); these carbon nucleophiles have the advantage of being kinetically stable in water or polar organic solvents. Other non-limiting examples of carbon nucleophiles include phosphorus ylides, enols, and enolate reagents; these carbon nucleophiles have the advantage of being relatively easy to generate from precursors well known to those skilled in the art of synthetic organic chemistry. When a carbon nucleophile is used in combination with a carbon electrophile, a new carbon-carbon bond is generated between the carbon nucleophile and the carbon electrophile.

[0290] Non-limiting examples of non-carbon nucleophiles suitable for coupling with 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 form heteroatom linkages (CXC), where X is a heteroatom including, but not limited to, oxygen, sulfur, or nitrogen.

[0291] In some cases, one end of the polymer used in the present invention terminates with a hydroxyl or methoxy group, that is, X is hydrogen or CH3 (" methoxy PEG "). Alternatively, polymers can terminate with reactive groups, thereby forming bifunctional polymers. Typical reactive groups can include those reactive groups that are commonly used for reacting with the functional group present in 20 kinds of common amino acids (including but not limited to maleimide groups, activated carbonates (including but not limited to p-nitrophenyl ester), activated esters (including but not limited to N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes), and 20 kinds of common amino acids that are inert but specifically react with complementary functional groups (including but not limited to azide, alkynyl). It is noted that the other end of the polymer represented by Y in the above formula will be attached directly or indirectly to the targeting polypeptide by naturally occurring or non-naturally encoded amino acids. For example, Y can be an amide, carbamate or urea connected to the amino group (including but not limited to the epsilon amine or N-terminal of lysine) of a polypeptide. Alternatively, Y can be a maleimide connected 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 often approached by 20 kinds of common amino acids. For example, the azide group on the polymer can react with the alkynyl group on the targeting polypeptide to form a Huisgen [3+2] cycloaddition product. Alternatively, the alkynyl group on the polymer can react with the azide group present in the targeting polypeptide to form a similar product. In some embodiments, where applicable, a strong nucleophile (including but not limited to hydrazine, hydrazide, hydroxylamine, semicarbazide) can react with the aldehyde group or ketone group present in the targeting polypeptide to form a hydrazone, oxime or semicarbazide (which can be further reduced by treating with an appropriate reducing agent in some cases). Alternatively, a strong nucleophile can be incorporated into the targeting polypeptide by non-naturally encoded amino acids and used to preferentially react with the ketone group or aldehyde group present in the water-soluble polymer.

[0292] Any molecular weight of the polymer can be used as needed, including but not limited to about 100 Daltons (Da) to 100,000 Da or more (including but not limited to sometimes 0.1-50 kDa or 10-40 kDa) as needed. The molecular weight of the polymer can have a wide range, including but not limited to between about 100 Da and about 100,000 Da or more. The polymer can be between about 100 Da and about 100,000 Da, including but not limited to 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 3 ... In some embodiments, the molecular weight of the polymer is about 100Da, about 200Da, about 300Da, about 400Da, about 500Da, about 600Da, about 700Da, about 800Da, about 900Da, about 1000Da, about 2000Da, about 3000Da, about 4000Da and about 100Da. In some embodiments, the molecular weight of the polymer is about 100Da, about 2500Da, about 2000Da, about 3000Da, about 4000Da and about 5000Da. In some embodiments, the molecular weight of the polymer is about 100Da, about 2500Da, about 2000Da, about 3000Da, about 4000Da and about 5000Da. In some embodiments, the molecular weight of the polymer is about 100Da, about 2500Da, about 2000Da, about 3000Da, about 4000Da and about 5000Da. In some embodiments, the molecular weight of the polymer is about 100Da, about 2500Da, about 2000Da, about 3000Da, about 4000Da and about 5000Da. The molecular weight of each chain of the branched polymer may be between about 1,000 Da and about 100,000 Da, including but not limited to 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, In some embodiments, the molecular weight of each chain of the branched polymer is between about 1,000 Da and about 50,000 Da. In some embodiments, the molecular weight of each chain of the branched polymer is between about 1,000 Da and about 40,000 Da.In some embodiments, the molecular weight of each chain of the branched polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of each chain of the branched polymer is between about 5,000 Da and about 20,000 Da. A wide range of polymer molecules are described in, including but not limited to, the Shearwater Polymers company catalog and the Nektar Therapeutics catalog, which are incorporated herein by reference.

[0293] In some embodiments, the invention provides polymer derivatives containing azide and acetylene, which comprise a water-soluble polymer backbone with a mean molecular weight of about 800Da to about 100,000Da. The polymer backbone of a water-soluble polymer can be polyethylene glycol. However, it should be understood that a variety of water-soluble polymers, including but not limited to polyethylene glycol and other related polymers, including poly (dextran) and poly (propylene glycol), are also suitable for 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 any form of polyethylene glycol, including difunctional PEG, multi-arm PEG, derivatization PEG, bifurcated PEG, branched PEG, pendant PEG (i.e., PEG or related polymers with one or more functional groups pendant to the polymer backbone) or PEG with degradable linkages therein.

[0294] 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 by subjecting ester linkages in the polymer backbone to hydrolysis. This hydrolysis results in the breakdown of the polymer into lower molecular weight fragments, as shown below: -polymer-CO2-polymer-+H2O → polymer-CO2H+HO-polymer-

[0295] Many polymers are also suitable for use in the present invention. In some embodiments, water-soluble polymer backbones with 2 to about 300 terminals 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, etc. Although the molecular weight of each chain of the polymer backbone can vary, it is typically within the range of about 800Da to about 100,000Da, typically about 6,000Da to about 80,000Da. The molecular weight of each chain of the polymer backbone can be between about 100 Da and about 100,000 Da, including but not limited to 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 40,000 Da, 5 In some embodiments, the molecular weight of each chain of the polymer backbone is between about 100Da and about 50,000Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 100Da and about 40,000Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 1,000Da and about 40,000Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 1,000Da and about 40,000Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 10,000 Da and about 40,000 Da.

[0296] In a feature of this embodiment of the invention, the intact polymer-conjugate undergoes minimal degradation following administration prior to hydrolysis, such that hydrolysis of the cleavable bond effectively controls the slow rate of release of the active targeting polypeptide into the bloodstream, as opposed to enzymatic degradation of the targeting polypeptide prior to its release into the systemic circulation.

[0297] 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 after storage and administration. For example, the targeting polypeptide or modified targeting polypeptide attached to the polymer should maintain its integrity after manufacturing the final pharmaceutical composition, after dissolution in an appropriate delivery vehicle (if employed), and after administration by any route.

[0298] The present invention also includes phosphate-based joints with adjustable stability for intracellular delivery of drug conjugates disclosed in US2017 / 0182181, which are incorporated herein by reference. Phosphate-based joints include monophosphates, diphosphates, triphosphates or tetraphosphate groups (phosphate groups) covalently linked to the distal end of the joint arm, and the joint arm comprises a tuning element, an optional spacer element and a reactive functional group from the distal end to the proximal end. The phosphate group of the phosphate-based joint 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 joint is: phosphate group-tuning element-optional spacer element-functional reactive group. Phosphate-based linkers conjugated to a payload have the following general structure: payload-phosphate group-tuning element-optional spacer element-functional reactive group, and when conjugated to a targeting ligand have the following general structure: payload-phosphate group-tuning element-optional spacer element-targeting ligand. These phosphate-based linkers have differentiated and tunable stabilities in blood relative to the intracellular environment (e.g., lysosomal compartment). The rate at which the phosphate group is cleaved in the intracellular environment to release the payload in its native or active form can be affected by the structure of the tuning element, and further effects are mediated by the substitution of the phosphate group and whether the phosphate group is a monophosphate, diphosphate, triphosphate, or tetraphosphate. In addition, these phosphate-based linkers provide the ability to construct conjugates, such as antibody-drug conjugates, in which the tendency of the conjugates to form aggregates is reduced compared to conjugates in which the same payload is conjugated to an antibody or targeting ligand using phosphate-based linkers not disclosed herein.

[0299] Structure and synthesis of TLR agonist linker derivatives: electrophilic and nucleophilic groups

[0300] TLR agonist derivatives with linkers containing hydroxylamine (also known as aminooxy) groups allow for reaction with a variety of electrophilic groups to form conjugates (including but not limited to with PEG or other water-soluble polymers). Like hydrazines, hydrazides, and semicarbazides, the enhanced nucleophilicity of aminooxy groups enables them 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). Although the result of the reaction with the hydrazine group is the corresponding hydrazone, the reaction of aminooxy groups with carbonyl or dicarbonyl-containing groups, such as, for example, ketones, aldehydes, or other functional groups with similar chemical reactivity, generally produces oximes. In some embodiments, TLR agonist derivatives with linkers comprising azide, alkyne, or cycloalkyne allow for the attachment of molecules via cycloaddition reactions, such as 1,3-dipolar cycloaddition, azide-alkyne Huisgen cycloaddition, etc. (described in U.S. Pat. No. 7,807,619, which is incorporated herein by reference to the extent relevant to the reaction).

[0301] Thus, in certain embodiments, described herein are TLR agonist derivatives having a linker comprising a hydroxylamine, an aldehyde, a protected aldehyde, a ketone, a protected ketone, a thioester, an ester, a dicarbonyl, a hydrazine, an amidine, an imine, a diamine, a keto-amine, a keto-alkyne, and an ene-dione hydroxylamine group, a hydroxylamine-like group (having similar reactivity to a hydroxylamine group and structurally similar to a hydroxylamine group), a masked hydroxylamine group (which can be readily converted to a hydroxylamine group), or a protected hydroxylamine group (having similar reactivity to a hydroxylamine group after deprotection). In some embodiments, the TLR agonist derivative having a linker comprises an azide, an alkyne, or a cycloalkyne.

[0302] 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 optionally undergo post-translational modification.

[0303] In certain embodiments, the compound of formula (I)-(VII) is stable in aqueous solution under weak acid conditions for at least 1 month. In certain embodiments, the compound of formula (I)-(VII) is stable under weak acid conditions for at least 2 weeks. In certain embodiments, the compound of formula (I)-(VII) is stable under weak acid conditions for at least 5 days. In certain embodiments, such acidic conditions are pH 2 to 8.

[0304] The methods and compositions provided and described herein include polypeptides comprising non-natural amino acids having at least one carbonyl or dicarbonyl group, oxime group, hydroxylamine group, or protected or masked forms thereof. Introducing at least one reactive group into a TLR agonist linker derivative or targeting polypeptide allows for the use of conjugation chemistries involving specific chemical reactions, including but not limited to reactions with one or more targeting polypeptides, rather than with ubiquitous amino acids. Once incorporated, the targeting polypeptide can also be modified by chemical methods utilizing specific functional groups or substituents described herein or suitable for use in TLR agonist linker derivatives or targeting polypeptides.

[0305] The TLR agonist linker derivatives and targeting polypeptide methods and compositions described herein provide conjugates of substances having a variety of functional groups, substituents or moieties with other substances, including but not limited to polymers; water-soluble polymers; derivatives of polyethylene glycol; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; and any combination thereof.

[0306] In certain embodiments, the TLR agonist linker derivatives, targeting polypeptides, TCs, linkers, and reagents described herein, including compounds of Formula (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 2 weeks. In other embodiments, such compounds are stable under weakly acidic conditions for at least 5 days.

[0307] 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. By way of example only, this aspect includes therapeutic, diagnostic, assay-based, industrial, cosmetic, plant biology, environmental, energy production, consumer products and / or military uses that would benefit from targeting polypeptides comprising "modified or unmodified" non-natural amino acid polypeptides or proteins.

[0308] The present invention provides TC molecules comprising at least one unnatural amino acid. In certain embodiments of the present invention, the TC having at least one unnatural amino acid comprises at least one post-translational modification. In one embodiment, the at least one post-translational modification comprises the attachment of a molecule comprising a second reactive group to at least one unnatural amino acid comprising a first reactive group using chemistry known to one of ordinary skill in the art suitable for specific reactive groups, including but not limited to 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 saccharide, a cyclodextrin, an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spin Labels, fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or non-covalently interact with other molecules, photocaged moieties, actinic radiation excitable moieties, photoisomerizable moieties, biotin, biotin derivatives, biotin analogs, moieties incorporating heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox-active agents, amino thioacids, 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, nanoemitters, radionuclides, radioactive emitters, neutron capture agents, or any combination of the above or any other desired compounds or substances. For example, the first reactive group is an alkynyl moiety (including but not limited to the unnatural amino acid p-propargyloxyphenylalanine, wherein the propargyl group is sometimes also referred to as an acetylene moiety), the second reactive group is an azido moiety, and a [3+2] cycloaddition chemistry approach is utilized. In another example, the first reactive group is an azido moiety (including but not limited to p-azido-L-phenylalanine or pAZ in the non-natural amino acid), and the second reactive group is an alkynyl moiety. In certain embodiments of the modified TC of the present invention, at least one non-natural amino acid (including but not limited to non-natural amino acids containing a ketone functional group) is used that comprises at least one post-translational modification, wherein the at least one post-translational modification comprises a sugar moiety. In certain embodiments, the post-translational modification is performed in vivo in eukaryotic cells or in non-eukaryotic cells. Linkers, polymers, water-soluble polymers, or other molecules can attach the molecule to the polypeptide. In another embodiment, the linker attached to the TC is long enough to allow dimer formation. The molecule can also be directly linked to the polypeptide.

[0309] In certain embodiments, the TC protein comprises at least one post-translational modification that is made in vivo by one host cell, where the post-translational modification is not normally made by another host cell type. In certain embodiments, the protein comprises at least one post-translational modification that is made in vivo by a eukaryotic cell, where the post-translational modification is not normally made by a non-eukaryotic cell. Examples of post-translational modifications include, but are not limited to, glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitate addition, phosphorylation, glycolipid linkage modification, and the like.

[0310] 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 linkage 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 linkage modification of the polypeptide. In some embodiments, the TC comprises one or more naturally encoded amino acids for glycosylation of the polypeptide.

[0311] In some embodiments, TC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation of a polypeptide. In some embodiments, TC comprises one or more deletions that enhance glycosylation of a polypeptide. In some embodiments, TC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation of a different amino acid in a polypeptide. In some embodiments, TC comprises one or more deletions that enhance glycosylation of a different amino acid in a polypeptide. In some embodiments, TC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation of a non-naturally encoded amino acid in a polypeptide. In some embodiments, TC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation of a naturally encoded amino acid in a polypeptide. In some embodiments, TC comprises one or more naturally encoded amino acid additions and / or substitutions that enhance glycosylation of a different amino acid in a polypeptide. In some embodiments, TC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation of a naturally encoded amino acid in a polypeptide. In some embodiments, TC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation of a non-naturally encoded amino acid in a polypeptide.

[0312] In one embodiment, the post-translational modification comprises the attachment of an oligosaccharide to an asparagine (including but not limited to, wherein the oligosaccharide comprises (GlcNAc-Man)2-Man-GlcNAc-GlcNAc, etc.) via a GlcNAc-asparagine linkage. In another embodiment, the post-translational modification comprises the attachment of an oligosaccharide (including but not limited to, Gal-GalNAc, Gal-GlcNAc, etc.) to a serine or threonine via a GalNAc-serine, GalNAc-threonine, GlcNAc-serine, or GlcNAc-threonine linkage. In certain embodiments, the protein or polypeptide of the invention may comprise a secretion or localization sequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, and / or the like. Examples of secretion signal sequences include but are not limited to prokaryotic secretion signal sequences, eukaryotic secretion signal sequences, eukaryotic secretion signal sequences optimized for bacterial expression 5', novel secretion signal sequences, pectate lyase secretion signal sequences, Omp A secretion signal sequences, and bacteriophage secretion signal sequences. Examples of secretory signal sequences include, but are not limited to, STII (prokaryotic), Fd GIII and M13 (phage), Bgl2 (yeast), and the signal sequence bla derived from a transposon. Any of these sequences may be modified to provide the desired result for the polypeptide, including but not limited to replacing a signal sequence with a different signal sequence, replacing a leader sequence with a different leader sequence, etc.

[0313] The 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 a 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 less than all, of the specific amino acids present in the naturally occurring form of the protein are substituted with an unnatural amino acid.

[0314] The present invention provides methods and compositions based on TCs comprising 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 chemistries involving specific chemical reactions, including but not limited to reactions with one or more non-naturally encoded amino acids, but not with the 20 commonly occurring amino acids. In some embodiments, the TC comprising a non-naturally encoded amino acid is 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 selectively modifying proteins with PEG derivatives or TLR-linker derivatives, which involves selectively incorporating non-genetically encoded amino acids into proteins in response to a selector codon, wherein the non-genetically encoded amino acids include but are not limited to those containing functional groups or substituents not found in the 20 naturally incorporated amino acids, including but not limited to ketone, azide, or acetylene moieties; and subsequently modifying these amino acids with appropriately reactive PEG derivatives. Once incorporated, the amino acid side chains can be modified to suit the specific functional groups or substituents present in the non-naturally encoded amino acid by utilizing chemical methods known to those of ordinary skill in the art. A variety of known chemical methods are suitable for use in the present invention for incorporating water-soluble polymers into proteins. Such methods include, but are not limited to, the Huisgen [3+2] cycloaddition reaction (see, for example, Padwa, A. Comprehensive Organic Synthesis, Vol. 4, (1991) edition Trost, BM, Pergamon, Oxford, pp. 1069-1109; and, Huisgen, R. 1,3-Dipolar Cycloaddition Chemistry, (1984) edition, Padwa, A., Wiley, New York, pp. 1-176), which have, respectively, including but not limited to, acetylene or azide derivatives.

[0315] Because the Huisgen [3+2] cycloaddition method involves a cycloaddition rather than a nucleophilic substitution reaction, proteins can be modified with extremely high selectivity. By adding a catalytic amount of Cu(I) salt to the reaction mixture, the reaction can be carried out with excellent regioselectivity (1,4>1,5) under aqueous conditions at room temperature. 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 the proteins of the present invention by [3+2] cycloaddition include almost any molecule with a suitable functional group or substituent, including but not limited to an azide or acetylene derivative. These molecules can be added to non-natural amino acids having an acetylene group, including but not limited to p-propargyloxyphenylalanine; or non-natural amino acids having an azide group, including but not limited to p-azido-phenylalanine.

[0316] The five-membered ring produced by the Huisgen [3+2] cycloaddition is generally irreversible in a reducing environment and is stable against hydrolysis in an aqueous environment for extended periods of time. Thus, the active PEG derivatives or TLR-linker derivatives of the present invention can be used to modify the physical and chemical properties of a variety of substances under harsh aqueous conditions. Even more importantly, because the azide and acetylene moieties are mutually specific (e.g., do not react with any of the 20 common genetically encoded amino acids), proteins can be modified with extremely high selectivity at one or more specific sites.

[0317] The present invention also provides water-soluble and hydrolytically stable derivatives of PEG derivatives or TLR linker derivatives and related hydrophilic polymers with one or more acetylene or azide moieties. PEG polymer derivatives containing acetylene moieties are highly selective for coupling with the azide moiety, and the azide moiety has been selectively introduced into the protein in response to a selection codon. Similarly, PEG polymer derivatives containing the azide moiety are highly selective for coupling with the acetylene moiety, and the acetylene moiety has been selectively introduced into the protein in response to a selection codon. More specifically, the azide moiety includes but is not limited to alkyl azides, aryl azides, and derivatives of these azides. As long as the acetylene-specific reactivity is maintained, the derivatives of alkyl and aryl azides can include other substituents. The acetylene moiety includes alkyl and aryl acetylene and respective derivatives. As long as the azide-specific reactivity is maintained, the derivatives of alkyl and aryl acetylene can include other substituents.

[0318] The present invention provides conjugates of substances having various functional groups, substituents or moieties with other substances, including but not limited to labels; dyes; polymers; water-soluble polymers; derivatives of polyethylene glycol; photocrosslinkers; radionuclides; cytotoxic compounds; drugs; affinity labels; photoaffinity labels; reactive compounds; resins; second proteins or polypeptides or polypeptide analogs; antibodies or antibody fragments; metal chelators; cofactors; fatty acids; carbohydrates; polynucleotides; DNA; RNA; antisense polynucleotides; sugars; water-soluble dendrimers; cyclodextrins; inhibitory ribonucleic acids; biomaterials; nanoparticles; spin labels; fluorophores; metal-containing moieties; radioactive moieties; novel functional groups; and

[0014] The present invention also includes conjugates of substances having an azide or acetylene moiety and a PEG polymer derivative having the corresponding acetylene or azide moiety. For example, a PEG polymer containing an azide moiety can be coupled to a biologically active molecule at a position in a protein containing a non-genetically encoded amino acid bearing an acetylene functional group. Linkages for conjugating PEG to biologically active molecules include, but are not limited to, Huisgen [3+2] cycloaddition products.

[0319] It is well established in the art that PEG can be used to modify the surface of biomaterials (see, for example, U.S. Patent 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 sites and one or more azide- or acetylene-containing polymers of the present invention coupled to the surface via a Huisgen [3+2] cycloaddition linkage. Biomaterials and other substances can also be coupled to azide- or acetylene-activated polymer derivatives via linkages other than azide or acetylene linkages, such as linkages comprising carboxylic acid, amine, alcohol, or thiol moieties, such that the azide or acetylene moiety is available for subsequent reactions.

[0320] The present invention includes methods for synthesizing azide-containing and acetylene-containing polymers of the present invention. In the case of azide-containing PEG derivatives, the azide can be directly bonded to the carbon atoms of the polymer. Alternatively, by attaching a linker having an azide moiety at one end to a conventional activated polymer, the resulting polymer has an azide moiety at its end, thereby preparing azide-containing PEG derivatives. In the case of acetylene-containing PEG derivatives, acetylene can be directly bonded to the carbon atoms of the polymer. Alternatively, by attaching a linker having an acetylene moiety at one end to a conventional activated polymer, the resulting polymer has an acetylene moiety at its end, thereby preparing acetylene-containing PEG derivatives.

[0321] More specifically, in the case of PEG derivatives containing azide, a water-soluble polymer with at least one active hydroxyl moiety undergoes such a reaction to produce a substituted polymer having a more reactive portion such as mesylate, trifluoroethylsulfonate (tresylate), toluenesulfonate or a halogen leaving group thereon. The preparation and use of PEG derivatives or TLR-linker derivatives containing sulfonyl halides, halogen atoms and other leaving groups are known to those of ordinary skill in the art. The resulting substituted polymer then undergoes such a reaction to replace the more reactive portion with an azide moiety at the end of the polymer. Alternatively, a water-soluble polymer with at least one active nucleophilic or electrophilic moiety undergoes a reaction with a linker having an azide at one end to form a covalent bond between the PEG polymer and the linker, and the azide moiety is positioned at the end of the polymer. Nucleophilic and electrophilic moieties, including amines, thiols, hydrazides, hydrazines, alcohols, carboxylates, aldehydes, ketones, thioesters, etc., are known to those of ordinary skill in the art.

[0322] More specifically, in the case of acetylene-containing PEG derivatives, a water-soluble polymer having at least one active hydroxyl moiety undergoes a reaction that displaces a halogen or other activated leaving group from a precursor containing the acetylene moiety. Alternatively, a water-soluble polymer having at least one active nucleophilic or electrophilic moiety undergoes a reaction with a linker having an acetylene at one terminus, such that a covalent bond is formed between the PEG polymer and the linker, 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 are well established by practitioners in the art.

[0323] The present invention also provides a method for selectively modifying proteins, thereby adding other substances to the modified proteins, including but not limited to water-soluble polymers containing azide or acetylene moieties such as PEG and PEG derivatives, or TLR-linker derivatives, linkers, or another TC polypeptide. Azide-containing and acetylene-containing PEG derivatives or TLR linker derivatives can be used to modify the properties of surfaces and molecules where biocompatibility, stability, solubility, and non-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.

[0324] General Recombinant Nucleic Acid Methods for Use with the Invention

[0325] In various embodiments of the present invention, nucleic acids encoding targeting polypeptides of interest are isolated, cloned, and often modified using recombinant methods. Such embodiments are useful, including but not limited to, for protein expression or in the generation of variants, derivatives, expression cassettes, or other sequences of targeting polypeptides derived from TCs. In some embodiments, sequences encoding polypeptides of the present invention are operably linked to a heterologous promoter.

[0326] The nucleotide sequence encoding the targeting polypeptide of the TC comprising the non-naturally encoded amino acid can be synthesized based on the amino acid sequence of the parent polypeptide, and then the nucleotide sequence is changed to achieve the introduction (i.e., incorporation or substitution) or removal (i.e., deletion or substitution) of the relevant amino acid residues. The nucleotide sequence can be conveniently modified by site-directed mutagenesis according to conventional methods. Alternatively, the nucleotide sequence can be prepared by chemical synthesis (including but not limited to by using an oligonucleotide synthesizer), wherein oligonucleotides are designed based on the amino acid sequence of the desired polypeptide, and preferably those codons that are favorable in the host cell in which the recombinant polypeptide will be produced are selected. For example, several small oligonucleotides encoding multiple parts of the desired polypeptide can be synthesized and assembled by PCR, ligation or ligation chain reaction. See, for example, Barany et al., Proc. Natl. Acad. Sci. 88: 189-193 (1991); U.S. Patent No. 6,521,427, which is incorporated herein by reference.

[0327] The present invention utilizes conventional techniques in the field of recombinant genetics. Basic texts disclosing the general methods used in the present invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd edition, 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994).

[0328] The present invention further relates to eukaryotic host cells, non-eukaryotic host cells and organisms for in vivo incorporation of non-natural amino acids by orthogonal tRNA / RS pairs. Host cells are genetically engineered (including but not limited to, conversion, transduction or transfection) using polynucleotides or constructs of the present invention, comprising polynucleotides of the present invention, including but not limited to vectors of the present invention, which can be, for example, cloning vectors or expression vectors.

[0329] Several well-known methods for introducing target nucleic acids into cells are available, any of which can be used in the present invention. These methods include: fusion of recipient cells with bacterial protoplasts containing the DNA, electroporation, microparticle bombardment, and infection with viral vectors (discussed further below). Bacterial cells can be used to amplify the number of plasmids containing the DNA constructs of the present invention. The bacteria are grown to logarithmic phase, and the plasmids within the bacteria can be isolated by various methods known in the art (see, e.g., Sambrook). In addition, kits for purifying plasmids from bacteria are commercially available (see, e.g., EasyPrep TM 、FlexiPrep TM , both from Pharmacia Biotech; StrataClean from Stratagene TM and QIAprep from Qiagen TM). The isolated and purified plasmid is then further manipulated to produce other plasmids for transfecting cells or incorporating related vectors to infect organisms. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters for regulating the expression of specific target nucleic acids. The vector optionally comprises a universal expression cassette containing at least one independent terminator sequence, a sequence allowing the cassette to replicate in eukaryotes or prokaryotes or both (including but not limited to a shuttle vector), and a selection marker for prokaryotic and eukaryotic systems. The vector is 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 ibid). In some embodiments, the present invention provides the bacterium that can be used for cloning and the bacteriophage of phage, for example, by the ATCC bacterium and bacteriophage catalogue (1992) of the people such as Gherna (editor) that ATCC announces.In the Recombinant DNA Second Edition Scientific American Books (NY) of the people such as Watson (1992), also found other basic procedures and basic theory considerations for other aspects of order-checking, cloning and molecular biology.In addition, basically any nucleic acid (and the nucleic acid of almost any mark, no matter standard nucleic acid or non-standard nucleic acid) all can be customized and ordered or standard order from multiple commercial sources, described source such as Midland Certified Reagent Company (Midland, TX, can obtain at world wide web mcrc.com), The Great American Gene Company (Ramona, CA, can obtain at world wide web genco.com), ExpressGen Inc. (Chicago, IL, can obtain at world wide web expressgen.com), Operon Technologies Inc. (Alameda, CA) and many other sources.

[0330] Selector codon

[0331] 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 codons (UAG), ochre codons, or opal codons (UGA); unnatural codons; codons of four or more bases; rare codons, and the like. It will be apparent to one of ordinary skill in the art that a wide range of selector codons can be introduced into a desired gene or polynucleotide, including, but not limited to, one or more, two or more, three or more, four, five, six, seven, eight, nine, ten, or more, in a single polynucleotide encoding at least a portion of a TC.

[0332] In one embodiment, the method relates to the use of a selector codon, which is a stop codon for incorporation into one or more non-natural amino acids in vivo. For example, an O-tRNA that recognizes a stop codon (including but not limited to UAG) is produced and aminoacylated by an O-RS with a desired non-natural amino acid. This O-tRNA is not recognized by the aminoacyl-tRNA synthetase of a naturally occurring host. Conventional site-directed mutagenesis can be used to introduce a stop codon at a site of interest in a polypeptide of interest, including but not limited to TAG. See, for example, Sayers, JR et al. (1988), 5'-3' Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res, 16:791-802. When O-RS, O-tRNA, and a nucleic acid encoding a polypeptide of interest are combined in vivo, a non-natural amino acid is incorporated in response to the UAG codon, thereby producing a polypeptide containing a non-natural amino acid at a specified position.

[0333] In vivo incorporation of unnatural amino acids can be performed without significantly perturbing the eukaryotic host cell. For example, because the suppression efficiency of the UAG codon depends on competition between O-tRNA (including but not limited to amber suppressor tRNA) and eukaryotic release factors (including but not limited to eRF) (which bind to the stop codon and initiate release of the growing peptide from the ribosome), the suppression efficiency can be modulated by (including but not limited to) increasing the expression level of O-tRNA and / or suppressor tRNA.

[0334] Unnatural amino acids can also be encoded using rare codons. For example, when the arginine concentration in an in vitro protein synthesis reaction is reduced, the rare arginine codon AGG has been shown to effectively insert Ala by a synthetic tRNA acylated with alanine. See, for example, Ma et al., Biochemistry, 32:7939 (1993). In this case, the synthetic tRNA competes with the naturally occurring tRNAArg, which 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 used to insert amino acids in in vitro transcription / translation extracts. See, for example, Kowal and Oliver, Nucl. Acid. Res., 25:4685 (1997). The components of the present invention can be generated to use these rare codons in vivo.

[0335] Selecting codons also includes extending codons, including but not limited to codons of four or more bases, such as codons of four, five, six or more bases. Examples of four-base codons include but are not limited to AGGA, CUAG, UAGA, CCCU, etc. Examples of five-base codons include but are not limited to AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC, etc. Features of the present invention include using extended codons based on frameshift suppression. Four or more base codons can be inserted into the same protein, including but not limited to one or more non-natural amino acids. For example, in the presence of a mutated O-tRNA (including but not limited to, a special frameshift suppressor tRNA with an anticodon loop, such as an anticodon loop with at least 8-10 nt), four or more base codons are read as single amino acids. In other embodiments, the anticodon loop can be decoded, including but not limited to, at least four-base codons, at least five-base codons, or at least six-base codons or more base codons. Since there are 256 possible four-base codons, codons of four or more bases can be used to encode multiple non-natural amino acids in the same cell. See, Anderson et al., (2002) Exploring theLimits of Codon and Anticodon Size, Chemistry and Biology, 9:237-244; Magliery, (2001) Expanding the Genetic Code: Selection of Efficient Suppressors ofFour-base Codons and Identification of “Shifty” Four-base Codons with a LibraryApproach in Escherichia coli, J. Mol. Biol. 307:755-769.

[0336] 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. 2-Naphthylalanine and an NBD derivative of lysine were simultaneously incorporated into streptavidin in vitro using two chemically acylated frameshift suppressor tRNAs, CGGG and AGGU. See, e.g., Hohsaka et al. (1999) J. Am. Chem. Soc., 121:12194. In an in vivo study, Moore et al. examined the ability of tRNALeu derivatives with NCUA anticodons to suppress UAGN codons (N can be U, A, G, or C) and found that the quadruple UAGA could be decoded by tRNALeu with UCUA anticodons with an efficiency of 13% to 26%, with almost no decoding in the 0 or -1 frames. 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 other unwanted sites.

[0337] 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 tRNAs that recognize natural three-base codons, and / or systems where three-base codons are rare codons.

[0338] The selector codon optionally includes an unnatural base pair. These unnatural base pairs further expand the existing genetic alphabet. An additional base pair increases the number of triplet codons from 64 to 125. The characteristics of the third base pair include: stable and selective base pairing, efficient enzymatic incorporation into DNA with high fidelity by polymerase, and efficient continued primer extension after the synthesis of the nascent unnatural base pair. Descriptions of unnatural base pairs applicable to methods and compositions include, for example, Hirao et al., (2002) An unnatural base pair for incorporating aminoacid analogues into protein, Nature Biotechnology, 20: 177-182. See also, for example, Wu, Y. et al., (2002) J. Am. Chem. Soc. 124: 14626-14630. Other relevant publications are listed below.

[0339] For in vivo use, non-natural nucleosides are membrane permeable and are phosphorylated to form the corresponding triphosphates. In addition, the added genetic information is stable and not destroyed by cellular enzymes. Previous efforts by Benner and others have utilized hydrogen bonding patterns that differ from those in canonical Watson-Crick pairs, with the most notable example being the iso-C:iso-G pair. See, for example, Switzer et al., (1989) J. Am. Chem. Soc., 111: 8322; and Piccirilli et al., (1990) Nature, 343: 33; Kool, (2000) Curr. Opin. Chem. Biol., 4: 602. These bases are typically mismatched with natural bases to some extent and cannot be enzymatically replicated. Kool and colleagues demonstrated that hydrophobic stacking interactions between bases can replace hydrogen bonds to 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. In order to develop non-natural base pairs that meet all of the above requirements, Schultz, Romesberg and their colleagues systematically synthesized and studied a series of non-natural hydrophobic bases. It was found that PICS:PICS self-pairs are more stable than natural base pairs and can be efficiently incorporated into DNA by the Klenow fragment (KF) of Escherichia coli DNA polymerase I. See, for example, McMinn et al., (1999) J. Am. Chem. Soc., 121: 11585-6; and Ogawa et al., (2000) J. Am. Chem. Soc., 122: 3274. KF can synthesize 3MN:3MN self-pairs with sufficient efficiency and selectivity for biological function. See, for example, Ogawa et al., (2000) J.Am.Chem.Soc., 122:8803. However, both bases serve as chain terminators for further replication. Recently, mutant DNA polymerases have evolved that can be used to replicate PICS pairs themselves. In addition, 7AI pairs themselves can be replicated. See, for example, Tae et al., (2001) J.Am.Chem.Soc., 123:7439. A new type of metallobase pair, Dipic:Py, has also been developed that forms a stable pair after binding to Cu(II). See, Meggers et al., (2000) J.Am.Chem.Soc., 122:10714. Because extended codons and unnatural codons are essentially orthogonal to natural codons, the method of the present invention can utilize this property to generate orthogonal tRNAs for them.

[0340] Unnatural amino acids can also be incorporated into desired polypeptides using a translational bypass system. In a translational bypass system, a large sequence is incorporated into a gene but is not translated into protein. The sequence contains a structure that acts as a cue, inducing the ribosome to skip over the sequence and resume translation downstream of the insertion.

[0341] Nucleic acid molecules encoding proteins of interest, such as TC targeting polypeptides, can be readily mutated to introduce cysteine ​​at any desired position in the polypeptide. Cysteine ​​is widely used to introduce active molecules, water-soluble polymers, proteins, or a variety of other molecules into proteins of interest. Methods suitable for incorporating cysteine ​​into desired positions in polypeptides are known to those of ordinary skill in the art, such as those described in U.S. Patent No. 6,608,183 (incorporated herein by reference), as well as standard mutagenesis techniques.

[0342] III. Non-naturally encoded amino acids

[0343] 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 TC. Generally speaking, the non-naturally encoded amino acids introduced are substantially chemically inert to the 20 common genetically encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). In some embodiments, the non-naturally encoded amino acids comprise side chain functional groups (including but not limited to azido, keto, aldehyde, and aminooxy groups) that react effectively and selectively with functional groups not found in the 20 common amino acids to form stable conjugates. For example, a targeting polypeptide comprising a TC containing a non-naturally encoded amino acid containing an azide functional group can be reacted with a polymer (including but not limited to poly(ethylene glycol) or, alternatively, a second polypeptide or linker containing an alkyne moiety) to form a Huisgen [3+2] cycloaddition product due to the selective reaction of the azide and alkyne functional groups, thereby forming a stable conjugate.

[0344] The general structure of an α-amino acid is shown below (Formula I):

[0345]

[0346]

[0347] Non-naturally encoded amino acids are generally any structure with the chemical formula listed above and can be applied to the present invention, wherein the R group is any substituent other than the substituent used in the twenty kinds of natural amino acids. Because the non-naturally encoded amino acids of the present invention are generally different from natural amino acids only in terms of side chain structure, 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 way they are formed 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-, ketone-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynyl, ether, thiol, seleno-, sulfonyl-, borate, borate ester, phosphate, phosphono, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino group etc. or any combination thereof. Other unnatural amino acids of interest that may be suitable for use in the present invention include, but are not limited to, amino acids comprising photoactivatable cross-linkers, spin-labeled amino acids, fluorescent amino acids, metal-binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or non-covalently interact with other molecules, photocaged and / or photoisomerizable amino acids, amino acids comprising biotin or a biotin analog, glycosylated amino acids such as sugar-substituted serine, 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 extended side chains compared to natural amino acids (including, but not limited to, polyethers or long-chain hydrocarbons of greater than about 5 or greater than about 10 carbon atoms), carbon-linked sugar-containing amino acids, redox-active amino acids, amino thioacid-containing amino acids, and amino acids comprising one or more toxic moieties.

[0348] Exemplary non-naturally encoded amino acids that can be applied to the present invention and can be used for reacting with water-soluble polymers include but are not limited to those with carbonyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactive groups. In some embodiments, non-naturally encoded amino acids include sugar moieties. Examples of such amino acids include N-acetyl-L-glucosamine-L-serine, N-acetyl-L-galactosamine-L-serine, N-acetyl-L-glucosamine-L-threonine, N-acetyl-L-glucosamine-L-asparagine and O-mannosamine-L-serine. Examples of such amino acids also include examples in which naturally occurring N-linkages or O-linkages between amino acids and sugars are replaced by covalent linkages (including but not limited to alkenes, oximes, thioethers, amides, etc.) that are not generally found in nature. Examples of such amino acids also include sugars that are generally not found in naturally occurring proteins, such as 2-deoxy-glucose, 2-deoxygalactose, etc.

[0349] Many 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 amino acids that are not commercially available are optionally synthesized as provided herein or using standard methods known to those of ordinary skill in the art. For organic synthesis techniques, see, for example, Fessendon and Fessendon's Organic Chemistry (1982, 2nd edition, Willard Grant Press, Boston Mass.); March's Advanced Organic Chemistry (3rd edition, 1985, Wiley and Sons, New York); and Carey and Sundberg's Advanced Organic Chemistry (3rd 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 non-natural amino acids containing novel side chains, non-natural amino acids suitable for use in the present invention optionally also contain modified backbone structures, including but not limited to, as shown in the structures of Formula II and III:

[0350] II

[0351]

[0352] III

[0353]

[0354] wherein Z typically comprises OH, NH2, SH, NH-R' or SR'; X and Y may be the same or different, typically comprising S or O, and R and R' (optionally the same or different) are typically selected from the same list of components of the R group described above for the non-natural amino acids of formula I, as well as hydrogen. For example, the non-natural amino acids of the present invention optionally comprise substitutions in the amino or carboxyl groups as shown in formula II and formula III. This type of non-natural amino acid includes, but is not limited to, α-hydroxy acids, α-thioacids, α-aminothiocarboxylates, including, but not limited to, side chains corresponding to the twenty common natural amino acids or non-natural side chains. In addition, substitutions at the α-carbon optionally include, but are not limited to, L, D, or α-α-disubstituted amino acids, such as D-glutamic acid, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Other structural alternatives include cyclic amino acids, such as proline analogs and 3-, 4-, 6-, 7-, 8-, and 9-membered ring proline analogs, β and γ amino acids, such as substituted β-alanine and γ-aminobutyric acid.

[0355] Many non-natural amino acids are based on natural amino acids, such as tyrosine, glutamine, phenylalanine, etc., and are suitable for use in the present invention. Tyrosine analogs include, but are not limited to, para-substituted tyrosine, ortho-substituted tyrosine, and meta-substituted tyrosine, wherein the substituted tyrosine comprises, but is not limited to, keto (including but not limited to acetyl), benzoyl, amino, hydrazine, hydroxylamine, thiol, carboxyl, isopropyl, methyl, C6-C 20Straight or branched hydrocarbons, saturated or unsaturated hydrocarbons, O-methyl, polyether, nitro, alkynyl, etc. In addition, polysubstituted aromatic rings are also considered. Glutamine analogs that can be used 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 can be used in the present invention include, but are not limited to, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine, wherein the substituents include (including but not limited to) hydroxyl, methoxy, methyl, allyl, aldehyde, azido, iodo, bromo, keto (including but not limited to acetyl), benzoyl, alkynyl, etc. 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-GlcNAcβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, and p-propargyloxy-phenylalanine. Examples of structures of various unnatural amino acids that may be suitable for use in the present invention are provided, for example, in WO 2002 / 085923, entitled "In vivo incorporation of unnatural amino acids." See also Kiick et al., (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudingerligation, PNAS 99:19-24, for additional methionine analogs, which is incorporated herein by reference. International Application No. PCT / US06 / 47822, entitled "Compositions Containing, Methods Involving, and Uses of Non-natural Amino Acids and Polypeptides," describes reductive alkylation and reductive amination of aromatic amine moieties, including but not limited to p-amino-phenylalanine, which is incorporated herein by reference.

[0356] In another embodiment of the present invention, a TC polypeptide is covalently modified with one or more non-naturally encoded amino acids. Selective chemical reactions that are orthogonal to the diverse functionalities of biological systems are considered important tools in chemical biology. As a relative novelty in synthetic chemical libraries, these bioorthogonal reactions have inspired new strategies for compound library synthesis, protein engineering, functional proteomics, and cell surface chemical remodeling. Azides have played a prominent role as unique chemical handles for bioconjugation. Staudinger ligation has been used with phosphines to tag azidosugars into metabolically introduced cellular glycoconjugates. Staudinger ligation can be performed in living animals without physiological damage; however, the Staudinger reaction is not without liability. The essential phosphines are susceptible to air oxidation, and their optimization to improve water solubility and increase reaction rate has proven to be a complex challenge.

[0357] An alternative mode of bioorthogonal reactivity for azide groups: the [3+2] cycloaddition reaction with alkynes described by Huisgen. In its classical form, the reaction has limited applicability in biological systems due to the high temperature (or pressure) requirement for reasonable reaction rates. Sharpless and co-workers overcame this obstacle by developing a copper(I)-catalyzed form, termed “click chemistry,” that readily proceeds at physiological temperatures and in functionally rich biological environments. This discovery enabled the selective modification of viral particles, nucleic acids, and proteins from complex tissue lysates. Unfortunately, the forced copper catalyst is toxic to both bacterial and mammalian cells, thus precluding applications where cells must remain viable. Catalyst-free Huisgen cycloadditions of alkynes activated by electron-withdrawing substituents have been reported to occur at ambient temperature. However, these compounds undergo Michael reactions with biological nucleophiles.

[0358] In one embodiment, compositions are provided that include a targeting polypeptide comprising a TC of an unnatural amino acid, such as p-(propargyloxy)-phenylalanine. Various compositions comprising p-(propargyloxy)-phenylalanine are also provided, including but not limited to proteins and / or cells. In one aspect, the composition comprising the p-(propargyloxy)-phenylalanine unnatural amino acid further comprises an orthogonal tRNA. The unnatural amino acid can be bonded (including but not limited to covalently) to the orthogonal tRNA, including but not limited to covalent bonding to the orthogonal tRNA via an amino-acyl bond, covalent bonding to the 3' OH or 2' OH of the terminal ribose sugar of the orthogonal tRNA, and the like.

[0359] Chemical moieties that can be incorporated into proteins via non-natural amino acids offer a variety of advantages and manipulations of proteins. For example, the unique reactivity of the keto functional group allows for selective modification of proteins in vivo and in vitro using any of a number of hydrazine- or hydroxylamine-containing reagents. For example, heavy atom non-natural amino acids can be used to phase X-ray structural data. Site-specific heavy atom introduction using non-natural amino acids also provides selectivity and flexibility in selecting the location of the heavy atom. For example, photoreactive non-natural 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 efficient in vivo and in vitro photocrosslinking of proteins. Examples of photoreactive non-natural amino acids include but are not limited to p-azido-phenylalanine and p-benzoyl-phenylalanine. Proteins with photoreactive non-natural amino acids can then be crosslinked by providing temporal control of the photoreactive groups through arbitrary excitation. In one example, (including but not limited to) using nuclear magnetic resonance and vibrational spectroscopy, the methyl groups of non-natural amino acids can be replaced with isotopically labeled groups (including but not limited to methyl groups) that serve as probes of local structure and dynamics. For example, alkyne or azido functional groups allow for the selective modification of proteins with molecules via [3+2] cycloaddition reactions.

[0360] The non-natural amino acid incorporated into a polypeptide at the amino terminus can consist of an R group, which is any substituent other than those used in the twenty natural amino acids, and a second reactive group, which is different from the NH2 group typically present in α-amino acids. Similar non-natural amino acids can be incorporated into the C-terminus, where the second reactive group is different from the COOH group typically present in α-amino acids.

[0361] The non-natural amino acids of the present invention can be selected or designed to provide additional characteristics not available in the twenty natural amino acids. For example, non-natural amino acids are optionally designed or selected to, for example, modify the biological properties of the protein into which they are incorporated. For example, by including non-natural amino acids in proteins, the following properties can be optionally modified: toxicity, biodistribution, solubility, stability (e.g., thermal, hydrolytic, oxidative, resistance to enzymatic degradation, etc.), ease of purification and processing, structural properties, spectral properties, chemical and / or photochemical properties, catalytic activity, redox potential, half-life, ability to react with other molecules (e.g., covalent or non-covalent), etc.

[0362] In some embodiments, the present invention provides TCs linked to water-soluble polymers, such as PEG, via an oxime bond. Many types of non-naturally encoded amino acids are suitable for forming oxime bonds. 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 herein by reference in their entireties. Non-naturally encoded amino acids are also described in U.S. Patent Nos. 7,083,970 and 7,045,337, which are incorporated herein by reference in their entireties.

[0363] Some embodiments of the present invention utilize TC polypeptides substituted with p-acetylphenylalanine amino acids at one or more positions. The synthesis of p-acetyl-(+ / -)-phenylalanine and m-acetyl-(+ / -)-phenylalanine is described in Zhang, Z. et al., Biochemistry 42:6735-6746 (2003), which is incorporated herein by reference. Other carbonyl- or dicarbonyl-containing amino acids can be similarly prepared by one of ordinary skill in the art. In addition, non-limiting exemplary syntheses of unnatural amino acids encompassed herein are set forth in U.S. Patent No. 7,083,970, which is incorporated herein by reference in its entirety.

[0364] Amino acids with electrophilic reactive groups allow for the attachment of molecules through a variety of reactions, including nucleophilic addition reactions. Such electrophilic reactive groups include carbonyl groups (including keto groups and dicarbonyl groups), carbonyl-like groups (which have similar reactivity to carbonyl groups (including keto groups and dicarbonyl groups) and are structurally similar to carbonyl groups), masked carbonyl groups (which can be easily converted to carbonyl groups (including keto groups and dicarbonyl groups)), or protected carbonyl groups (which have similar reactivity to carbonyl groups (including keto groups and dicarbonyl groups) after deprotection). Such amino acids include amino acids having the structure of formula (IV):

[0365]

[0366] in:

[0367] A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

[0368] B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2 or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;

[0369] J is

[0370] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0371] each R" is independently hydrogen, alkyl, substituted alkyl, or a protecting group, or when more than one R" group is present, two R"s optionally form a heterocycloalkyl;

[0372] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0373] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

[0374] R3 and R4 are each independently hydrogen, halogen, lower alkyl or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or heterocycloalkyl group;

[0375] Alternatively, the -ABJR groups taken together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl group containing at least one carbonyl (including dicarbonyl), protected carbonyl (including protected dicarbonyl), or masked carbonyl (including masked dicarbonyl);

[0376] Alternatively, the -JR groups taken together form a monocyclic or bicyclic cycloalkyl or heterocycloalkyl group containing at least one carbonyl (including dicarbonyl), protected carbonyl (including protected dicarbonyl), or masked carbonyl (including masked dicarbonyl);

[0377] provided that when A is phenylene and each R3 is H, B is present; and when A is -(CH2)4- and each R3 is H, B is not -NHC(O)(CH2CH2)-; and when A and B are not present and each R3 is H, R is not methyl.

[0378] Additionally, structures having formula (V) are included:

[0379]

[0380] in:

[0381] A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

[0382] B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2 or 3), -S(O) k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;

[0383] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0384] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0385] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

[0386] provided that when A is phenylene, B is present; and when A is -(CH2)4-, B is not -NHC(O)(CH2CH2)-; and when A and B are not present, R is not methyl.

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

[0388]

[0389] in:

[0390] B is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2 or 3), -S(O) k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;

[0391] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0392] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0393] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

[0394] Each R a independently selected from the group consisting of: H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (where k is 1, 2 or 3), -C(O)N(R')2, -OR' and -S(O) k R', wherein each R' is independently H, alkyl, or substituted alkyl.

[0395] Additionally, the following amino acids are included:

[0396]

[0397] Wherein such compound is optionally amino protecting group, carboxyl protecting or its salt.In addition, any of the following non-natural amino acids can be incorporated into the non-natural amino acid polypeptide.

[0398] Additionally, the following amino acids having the structure of formula (VII) are included:

[0399]

[0400] in

[0401] B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2 or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;

[0402] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0403] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0404] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

[0405] Each R a independently selected from the group consisting of: H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (where k is 1, 2 or 3), -C(O)N(R')2, -OR' and -S(O) k R', wherein each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8;

[0406] Provided that when A is -(CH2)4-, B is not -NHC(O)(CH2CH2)-.

[0407] Additionally, the following amino acids are included:

[0408]

[0409] Wherein this type of compound is optionally amino-protected, optionally carboxyl-protected, optionally amino-protected and carboxyl-protected, or its salt.In addition, these non-natural amino acids and any following non-natural amino acids can be incorporated into the non-natural amino acid polypeptide.

[0410] Additionally, the following amino acids having the structure of formula (VIII) are included:

[0411]

[0412] wherein A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

[0413] B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2 or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -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) kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;

[0414] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0415] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide.

[0416] Additionally, the following amino acids having the structure of formula (IX) are included:

[0417]

[0418] B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2 or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;

[0419] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0420] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0421] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

[0422] Each R a independently selected from the group consisting of: H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (where k is 1, 2 or 3), -C(O)N(R')2, -OR' and -S(O) k R', wherein each R' is independently H, alkyl, or substituted alkyl.

[0423] Additionally, the following amino acids are included:

[0424]

[0425] Wherein such compound is optionally amino-protected, optionally carboxyl-protected, optionally amino-protected and carboxyl-protected, or its salt.In addition, these non-natural amino acids and any following non-natural amino acids can be incorporated into the non-natural amino acid polypeptide.

[0426] Additionally, the following amino acids having the structure of formula (X) are included:

[0427]

[0428] wherein B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2 or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) kN(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;

[0429] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0430] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0431] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

[0432] Each R a independently selected from the group consisting of: H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (where k is 1, 2 or 3), -C(O)N(R')2, -OR' and -S(O) k R', wherein each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8.

[0433] Additionally, the following amino acids are included:

[0434]

[0435] Wherein such compound is optionally amino-protected, optionally carboxyl-protected, optionally amino-protected and carboxyl-protected, or its salt.In addition, these non-natural amino acids and any following non-natural amino acids can be incorporated into the non-natural amino acid polypeptide.

[0436] In addition to single carbonyl structures, the unnatural amino acids described herein can include groups such as dicarbonyls, dicarbonyl-like groups, masked dicarbonyls, and protected dicarbonyls.

[0437] For example, the following amino acids having the structure of formula (XI) are included:

[0438]

[0439] wherein A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

[0440] B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2 or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;

[0441] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0442] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0443] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide.

[0444] Additionally, the following amino acids having the structure of formula (XII) are included:

[0445]

[0446] B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2 or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;

[0447] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0448] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0449] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

[0450] Each R a independently selected from the group consisting of: H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (where k is 1, 2 or 3), -C(O)N(R')2, -OR' and -S(O) k R', wherein each R' is independently H, alkyl, or substituted alkyl.

[0451] Additionally, the following amino acids are included:

[0452] Wherein such compound is optionally amino-protected, optionally carboxyl-protected, optionally amino-protected and carboxyl-protected, or its salt.In addition, these non-natural amino acids and any following non-natural amino acids can be incorporated into the non-natural amino acid polypeptide.

[0453] Additionally, the following amino acids having the structure of formula (XIII) are included:

[0454]

[0455] wherein B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2 or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;

[0456] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0457] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0458] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

[0459] Each R aindependently selected from the group consisting of: H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (where k is 1, 2 or 3), -C(O)N(R')2, -OR' and -S(O) k R', wherein each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8.

[0460] Additionally, the following amino acids are included:

[0461]

[0462] Wherein this type of compound is optionally amino-protected, optionally carboxyl-protected, optionally amino-protected and carboxyl-protected, or its salt.In addition, these non-natural amino acids and any following non-natural amino acids can be incorporated into the non-natural amino acid polypeptide.

[0463] Additionally, the following amino acids having the structure of Formula (XIV) are included:

[0464]

[0465] in:

[0466] A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

[0467] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0468] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0469] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

[0470] X1 is C, S or S(O); and L is alkylene, substituted alkylene, N(R')(alkylene) or N(R')(substituted alkylene), wherein R' is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.

[0471] Additionally, the following amino acids having the structure of formula (XIV-A) are included:

[0472]

[0473] in:

[0474] A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

[0475] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0476] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0477] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

[0478] L is alkylene, substituted alkylene, N(R')(alkylene), or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0479] Additionally, the following amino acids having the structure of Formula (XIV-B) are included:

[0480]

[0481] in:

[0482] A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

[0483] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0484] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0485] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

[0486] L is alkylene, substituted alkylene, N(R')(alkylene), or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0487] Additionally, the following amino acids having the structure of Formula (XV) are included:

[0488]

[0489] in:

[0490] A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

[0491] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0492] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0493] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

[0494] X1 is C, S or S(O); n is 0, 1, 2, 3, 4 or 5; and in 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 may together form a cycloalkyl group.

[0495] Additionally, the following amino acids having the structure of Formula (XV-A) are included:

[0496]

[0497] in:

[0498] A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

[0499] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0500] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0501] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

[0502] n is 0, 1, 2, 3, 4 or 5; and in 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 may together form a cycloalkyl group.

[0503] Additionally, the following amino acids having the structure of Formula (XV-B) are included:

[0504]

[0505] in:

[0506] A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

[0507] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0508] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0509] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

[0510] n is 0, 1, 2, 3, 4 or 5; and in 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 may together form a cycloalkyl group.

[0511] Additionally, the following amino acids having the structure of Formula (XVI) are included:

[0512]

[0513] in:

[0514] A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

[0515] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0516] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0517] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

[0518] X1 is C, S or S(O); and L is alkylene, substituted alkylene, N(R')(alkylene) or N(R')(substituted alkylene), wherein R' is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.

[0519] Additionally, the following amino acids having the structure of Formula (XVI-A) are included:

[0520]

[0521] in:

[0522] A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

[0523] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0524] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0525] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

[0526] L is alkylene, substituted alkylene, N(R')(alkylene), or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0527] Additionally, the following amino acids having the structure of Formula (XVI-B) are included:

[0528]

[0529] in:

[0530] A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

[0531] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0532] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0533] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

[0534] L is alkylene, substituted alkylene, N(R')(alkylene), or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0535] Additionally, amino acids having the structure of Formula (XVII) are included:

[0536]

[0537] in:

[0538] A is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

[0539] m is -C(R3)-,

[0540] (a) , wherein (a) indicates bonding to the A group, and (b) indicates bonding to the corresponding carbonyl group, R3 and R4 are independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl, or R3 and R4 or two R3 groups or two R4 groups optionally form a cycloalkyl or heterocycloalkyl;

[0541] R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0542] T3 is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0543] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0544] R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide.

[0545] Additionally, amino acids having the structure of Formula (XVIII) are included:

[0546]

[0547] in:

[0548] m is -C(R3)-,

[0549] wherein (a) indicates bonding to an A group, and (b) indicates bonding to the corresponding carbonyl group, R3 and R4 are independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl, or R3 and R4 or two R3 groups or two R4 groups optionally form a cycloalkyl or heterocycloalkyl group;

[0550] R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0551] T3 is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0552] R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

[0553] R2 is optional, and when present, is OH,...

Claims

1. A composition 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 bonded to the non-natural amino acid of the targeting polypeptide.

2. The composition of claim 1, wherein the one or more targeting polypeptides are the same or different targeting polypeptides.

3. The composition of claim 1, wherein the one or more targeting polypeptides bind to a cell surface target or a tumor cell target.

4. The composition of claim 1, wherein the one or more targeting polypeptides are monospecific, bispecific or multispecific targeting polypeptides.

5. The composition of claim 4, wherein the monospecific, bispecific or multispecific targeting polypeptide comprises a drug conjugate or a checkpoint inhibitor.

6. The composition of claim 1, wherein the targeting polypeptide comprises an antibody or an antibody fragment.

7. The composition of claim 6, wherein the targeting polypeptide is an antibody or antibody fragment that binds to an antigen of a cell.

8. The composition of claim 6, wherein the targeting 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, calcium Mucin 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.

9. The composition of claim 8, wherein the targeting polypeptide comprises an antibody or antibody fragment that binds to HER2.

10. The composition according to claim 9, wherein the tumor targeting polypeptide is trastuzumab.

11. The composition of claim 6, wherein the antibody or antibody fragment comprises IgG, Fab, (Fab')2, Fv or single-chain Fv (scFv).

12. The composition of claim 6, wherein the antibody or antibody fragment comprises one or more Fab, (Fab')2, Fv or single chain Fv (scFv) mutations.

13. The composition of claim 6, wherein the antibody or antibody fragment comprises one or more Fc mutations.

14. The composition of claim 6, wherein the antibody or antibody fragment comprises one to six Fc mutations.

15. The composition of claim 6, wherein 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 chain and the light chain.

16. The composition of claim 6, wherein the antibody or antibody fragment further comprises one or more Fc mutations.

17. The composition of claim 1, wherein one or more of the targeting polypeptides comprises one or more non-naturally encoded amino acids selected from the group consisting of p-acetylphenylalanine, p-nitrophenylalanine, p-sulfotyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-borylcarbonylphenylalanine, o-borylcarbonylphenylalanine, m-borylcarbonylphenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, o-acylphenylalanine, Phenylalanine, m-acylphenylalanine, p-OMephenylalanine, o-OMephenylalanine, m-OMephenylalanine, 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-nitroTr p, 3-aminotyrosine, 2-aminotyrosine, O-sulfotyrosine, 2-sulfophenylalanine, 3-sulfophenylalanine, 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-filament 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, p-azidoethoxyphenylalanine, and p-azidomethyl-phenylalanine.

18. The composition of claim 17, wherein the unnatural amino acid is selected from the group consisting of p-acetyl-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine, or p-azidomethyl-phenylalanine.

19. The composition of claim 17, wherein the non-naturally encoded amino acid is site-specifically incorporated into the one or more targeting polypeptides.

20. The composition of claim 1, wherein the TLR agonist is a TLR7 agonist, a TLR8 agonist, or a TLR7 / TLR8 dual agonist.

21. The composition of claim 1, wherein the TLR agonist is a TLR agonist comprising a molecular structure according to any one of Structures 1, 2, 3, 4 or 5 of Figure 1.

22. The composition of claim 1, wherein the TLR agonist is any one of the TLR agonists selected from the group of structures according to Tables 3, 4, 5, 6, 7.

23. The composition of claim 1, wherein the targeting polypeptide is conjugated to one or more linkers, polymers, or biologically active molecules.

24. The composition of claim 23, wherein the one or more linkers are cleavable or non-cleavable linkers.

25. The composition of claim 23, wherein the one or more linkers are between 0.01 kDa and 50 kDa.

26. The composition of claim 25, wherein the one or more linkers are between 0.01 kDa and 10 kDa.

27. The composition of claim 23, wherein the one or more linkers or polymers are linear, branched, polymeric or dendritic.

28. The composition of claim 23, wherein the one or more linkers or polymers are bifunctional or multifunctional linkers or bifunctional or multifunctional polymers.

29. The composition of claim 23, wherein the one or more polymers are water soluble polymers.

30. The composition of claim 23, wherein the at least one linker, polymer, or biologically active molecule is linked to at least one non-naturally encoded amino acid.

31. The composition of claim 29, wherein the water soluble polymer is polyethylene glycol (PEG).

32. The composition of claim 31, wherein the PEG has a molecular weight between 0.1 kDa and 100 kDa.

33. The composition of claim 32, wherein the PEG has a molecular weight between 0.1 kDa and 50 kDa.

34. A composition according to embodiment 23, wherein the linker is PEG with a molecular weight between 0.1 kDa and 50 kDa.

35. The composition of claim 1, wherein the targeting polypeptide comprises one or more amino acid substitutions, additions or deletions that increase the stability or solubility of the composition.

36. The composition of claim 1, wherein 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 in vitro synthesis.

37. The composition of claim 1, wherein the non-naturally encoded amino acid is reactive toward a linker, polymer, or biologically active molecule that is unreactive toward any of the 20 common amino acids in polypeptides.

38. The composition of claim 37, wherein 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.

39. The composition of claim 38, wherein the non-naturally encoded amino acid comprises a carbonyl group.

40. The composition of claim 1, wherein the targeting polypeptide is linked to a cytotoxic agent or an immunostimulatory agent.

41. A TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist, wherein the TLR agonist comprises a structure according to any structure of Figure 1, wherein the TLR agonist is conjugated to the antibody or antibody fragment via a linker covalently bonded to one or more non-naturally encoded amino acids incorporated into the antibody or antibody fragment.

42. The TC according to claim 41, wherein the TLR agonist is a TLR7 agonist, a TLR8 agonist or a TLR7 / TLR8 dual agonist.

43. The TC of claim 41, wherein the TLR agonist comprises a structure according to Structure 1 of Figure 1.

44. The TC of claim 43, wherein the TLR agonist comprising a structure according to Structure 1 is selected from the group consisting of AXC-621, AXC-622, AXC-625, AXC-626, AXC-627, AXC-638, AXC-639, AXC-640, AXC-642, AXC-662, AXC-665, AXC-666, AXC-667, AXC-668, AXC-669, AXC-670, AXC-671, AXC-672, AXC-675, AXC-678, AXC-679, AXC-680, 1. 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- A XC-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-79 9. 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.

45. The TC of claim 41, wherein the anti-HER2 antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy chain and the light chain.

46. ​​The TC according to claim 45, wherein the anti-HER2 antibody or antibody fragment further comprises one or more mutations in the Fc region.

47. The TC according to claim 41, wherein the one or more non-naturally encoded amino acids are selected from the group of amino acids: p-acetylphenylalanine, p-nitrophenylalanine, p-sulfotyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-boronocarbonylphenylalanine, o-boronocarbonylphenylalanine, m-boronocarbonylphenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, o-acylphenylalanine, m-acylphenylalanine Acid, 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-aminotyr amino acid, 2-aminotyrosine, O-sulfotyrosine, 2-sulfoxyphenylalanine, 3-sulfophenylalanine, 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, Levodopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propargyloxy-L-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine, and p-azidomethyl-phenylalanine.

48. The TC according to claim 47, wherein the unnatural amino acid is p-acetyl-phenylalanine, 4-azido-L-phenylalanine, p-azidomethyl-phenylalanine or p-azidoethoxyphenylalanine.

49. The TC according to claim 41, wherein the linker is a cleavable or non-cleavable linker.

50. The TC according to claim 49, wherein the linker is a bifunctional linker.

51. The TC of claim 41, wherein the TLR agonist comprises a structure according to Structure 2 of Figure 1.

52. The TC according to claim 51, wherein the TLR agonist comprising a structure according to Structure 2 is selected from the group of AXC-745, AXC-746 and AXC-753 compounds.

53. The TC of claim 41, wherein the TLR agonist comprises a structure according to Structure 3 of Figure 1.

54. The TC according to claim 53, wherein the TLR agonist comprising a structure according to Structure 3 is an AXC-837 or AXC-847 compound.

55. The TC of claim 41, wherein the TLR agonist comprises a structure according to Structure 4 of Figure 1.

56. The TC of claim 55, wherein the TLR agonist comprising a structure according to Structure 4 is selected from the group consisting of AXC-844, AXC-842, AXC-843, AXC-845, AXC-846, AXC-836 or AXC-841 compounds.

57. The TC of claim 41, wherein the TLR agonist comprises a structure according to Structure 5 of Figure 1.

58. The TC of claim 57, wherein the TLR agonist comprising a structure according to 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-891,AXC-892,AXC-893,AXC-895,AXC-896,AXC-897,AXC-898,AXC-901,AXC-903,A compounds 59. The TC according to any one of claims 41-58, wherein the anti-HER2 antibody or antibody fragment comprises the amino acid sequence of at least one of SEQ ID NOs: 1-13.

60. The TC according to any one of claims 41-59, wherein the anti-HER2 antibody or antibody fragment comprises the amino acid sequence of at least two of SEQ ID NOs: 1-13.

61. The TC according to any one of claims 41-60, wherein the anti-HER2 antibody or antibody fragment comprises a) SEQ ID NO: 1 or 2; and b) any one of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13.

62. The TC according to claim 41, further comprising a chemotherapeutic agent or an immunotherapeutic agent.

63. The TC according to claim 41, further comprising an antibody drug conjugate.

64. A method of treating a subject or patient suffering from cancer or a disease or condition, the method comprising administering to the subject or patient a therapeutically effective amount of the composition or TC of any one of the preceding claims.

65. The method of claim 64, further comprising a chemotherapeutic agent or an immunotherapeutic agent.

66. The method of claim 64, further comprising an antibody drug conjugate, a cytotoxic agent, or a checkpoint inhibitor.

67. A pharmaceutical composition comprising a therapeutically effective amount of the composition or TC according to any one of the preceding claims and a pharmaceutically acceptable carrier or excipient.

68. Use of a composition according to any one of claims 1 to 39 in the manufacture of a medicament.

69. The TC of claim 44, wherein the TLR agonist comprising a structure according to Structure 1 is selected from the group consisting of AXC-625, AXC-626, AXC-638, AXC-639, AXC-640, AXC-642, AXC-662, AXC-667, AXC-668, AXC-669, AXC-670, AXC-671, AXC-672, AXC-675, AXC-681, AXC-687, AXC-688, AXC-689.

9. AXC-690, AXC-691, AXC-697, AXC-699, AXC-700, AXC-701, AXC-702, AXC-709, AXC-710, AXC-711, AXC-713, AXC -714, AXC-717, AXC-719, AXC-722, AXC-723, AXC-724, AXC-725, AXC-726, AXC-727, AXC-731, AXC-732, AXC-733, AXC-734, AXC-735, AXC-736, AXC-737, AXC-738, AXC-739, AXC-740, AXC-741, AXC-743, AXC-742, AXC-747, AXC- 748、AXC-750、AXC-751、AXC-752、AXC-754、AXC-755、AXC-756、AXC-757、AXC-758、AXC-759、AXC-760、AXC-761、A XC-762, AXC-764, AXC-771, AXC-772, AXC-773, AXC-777, AXC-778, AXC-779, AXC-789, AXC-793, AXC-800, AXC-80 1. AXC-802, AXC-803, AXC-804, AXC-805, AXC-806, AXC-807, AXC-808, AXC-809, AXC-810, AXC-831 and AXC-910 compounds.

70. The TC according to claim 44, wherein the TLR agonist comprising a structure according to Structure 1 is selected from the group consisting of AXC-801, AXC-802, AXC-831 and AXC-910 compounds.

71. The TC of claim 54, wherein the TLR agonist comprises the structure of the AXC-847 compound.

72. The TC of claim 58, wherein the TLR agonist comprising a structure according to Structure 5 is selected from the group consisting 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.

73. The TC according to any one of claims 43-44, 51-58 or 69-72, further comprising a linker.

74. An immunostimulatory antibody conjugate (ISAC) according to any one of claims 41-63.

75. The ISAC of claim 74, wherein the TLR agonist comprises a compound selected from the group consisting 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.

76. A salt of any of the compounds having a structure according to Figure 1.

77. A salt of any of the compounds of Tables 3, 4, 5, 6, and 7.

78. A method of killing cells, comprising contacting the cells with the TC according to claim 41.

79. The method of claim 78, wherein the cell is a tumor cell or a cancer cell.

80. A pharmaceutical composition according to claim 41 or a salt thereof.

81. The pharmaceutical composition or salt of claim 80, further comprising a pharmaceutically acceptable excipient.

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