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 relevant applications

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

[0003] sequence list

[0004] This application contains a sequence list that has been submitted in ASCII format via EFS-Web and is fully incorporated herein by reference. An ASCII copy created on February 7, 2020, is named AMBX_0230_PCT_SL.txt and is 30,527 bytes in size. Invention Field

[0005] This invention discloses TLR agonist compounds and TLR agonist conjugates (TCs) and their uses. The invention further relates to pharmaceutical compositions containing a (TC) as a therapeutic or preventative agent. Background of the Invention

[0007] Using non-naturally encoded amino acids, target molecules or peptides such as antibodies and their fragments can be conjugated to TLR agonist compounds through site-specific conjugation, thereby generating novel TLR agonist conjugates (TCs). These novel TCs can be constructed in such a way that, during systemic therapy, 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] This invention relates to targeting peptides conjugated with one or more non-naturally encoded amino acids to a TLR agonist compound, including but not limited to TLR7 and / or TLR8. Such conjugates are referred to herein as TLR agonist conjugates (TCs). The TCs of this invention comprise a targeting biomolecule or peptide and a TLR agonist compound, which are conjugated together via site-specific conjugation using non-naturally encoded amino acids to produce novel biological TLR agonist conjugates (BTCs). The targeting biomolecule or peptide may be a tumor-targeting biomolecule or peptide.

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

[0010] This invention provides a method for inhibiting or reducing tumor or cancer growth, comprising contacting the tumor with an effective amount of the present invention's TC to stimulate the immune system near the tumor in a patient. This invention provides a method for inhibiting or reducing tumor or cancer growth, comprising contacting the tumor with an effective amount of the present invention's polyethylene glycol-modified TC or a stable dimer or polymer of TC. In one embodiment, the TC is non-polyethylene glycol-modified or monopolyethylene glycol-modified. In one embodiment, the TC is dipolyethylene glycol-modified. 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 molecule attached thereto. Another embodiment of the invention provides a method for modulating an immune response against tumor cells using the present invention's TC. In some embodiments, the TC is co-administered with at least one chemotherapeutic agent and / or at least one immunotherapeutic agent. Chemotherapy agents can be selected from the group consisting of: temozolomide, gemcitabine, doxorubicin, cyclophosphamide, paclitaxel, cisplatin, fluoropyrimidine, taxane, anthracyclines, lapatinib, capecitabine, letrozole, pertuzumab, docetaxel, and IFN-α. In another embodiment of the invention, TC is administered co-administered with at least one chemotherapeutic agent and / or at least one immunotherapy agent.

[0011] In some embodiments, the TC comprises a targeting peptide, including but not limited to an antigen-binding peptide (ABP) comprising one or more non-naturally encoded amino acids. In some embodiments, the ABP comprises an intact antibody heavy chain. In some embodiments, the ABP comprises an intact antibody light chain. In some embodiments, the ABP comprises a variable region of the antibody light chain. In some embodiments, the ABP comprises a variable region of the antibody heavy chain. In some embodiments, the ABP comprises at least one CDR of the antibody light chain. In some embodiments, the ABP comprises at least one CDR of the antibody heavy chain. In some embodiments, the ABP comprises at least one CDR of the light chain and at least one CDR of the heavy chain. In some embodiments, the ABP comprises 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 scFv. In some embodiments, the ABP comprises two or more scFvs. In some embodiments, the ABP comprises a microantibody. In some embodiments, the ABP comprises two or more microantibodies. In some embodiments, the ABP comprises a biantibody. In some embodiments, the ABP comprises two or more biantibodies. 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 an intact light chain and an intact 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, homopolymer, or heteropolymer of any of the above embodiments. In some embodiments, the ABP comprises a polypeptide that binds to a binding partner, wherein the binding partner includes an antigen, polypeptide, nucleic acid molecule, polymer, or other molecule or substance. In some embodiments, the ABP associates with a non-antibody scaffold molecule or substance. In some embodiments, the antigen is a tumor antigen.

[0012] Toll-like receptors (TLRs) detect a wide range of conserved pathogen-associated molecular patterns (PAMPs). They play important roles in sensing invading pathogens and subsequently initiating innate immune responses. There are 10 known members of the TLR family in humans; these are type I transmembrane proteins characterized by an extracellular leucine-rich domain and a cytoplasmic tail containing a conserved Toll / interleukin (IL)-1 receptor (TIR) ​​domain. Within this family, TLR3, TLR7, TLR8, and TLR9 reside in endosomes. TLR7 and TLR8 can be activated by binding to specific small molecule ligands (i.e., TLR7 or TLR8 agonists) or their native ligands (i.e., single-stranded RNA, ssRNA). Upon agonist binding to TLR7 or TLR8, the receptor, in its dimerized form, is thought to undergo a structural change, leading to the subsequent recruitment of adaptor proteins, including myeloid differentiation primary response gene 88 (MyD88), into its cytoplasmic domain. Following the initiation of the receptor signaling cascade via the MyD88 pathway, cytoplasmic transcription factors such as interferon regulatory factor 7 (IRF-7) and nuclear factor κB (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 primarily expressed on plasmacytoid cells and B cells. Altered immune cellular responsiveness may lead to a reduction in the innate immune response in cancer patients. Therefore, agonist-induced activation of TLR7 and / or TLR8 conjugated to target moieties (such as antibodies or fragments thereof) represents a novel approach to cancer treatment. Treatment with TCs containing TLR7 or TLR8 agonists represents a promising solution, offering better efficacy and tolerability. Suitable TLR7 and / or TLR8 agonists for the preparation of TC in this 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; and U.S. Patent No. 8,673,932.

[0013] In some embodiments, the targeting peptide contained in TC further comprises amino acid substitutions, additions, or deletions. These amino acid substitutions, additions, or deletions increase the compatibility of the TC peptide with pharmaceutical preservatives (e.g., m-cresol, phenol, benzyl alcohol) compared to the compatibility of the corresponding wild-type TC without substitutions, additions, or deletions. This increased compatibility will enable the preparation of pharmaceutical formulations that preserve the physicochemical properties and biological activity of the protein during storage.

[0014] In some implementations, 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 reactions between two amino acids in a protein (one or both amino acids may be non-natural) under suitable conditions; and reactions between two amino acids (each amino acid may be naturally encoded or non-naturally encoded) and linkers, polymers, or other molecules under suitable conditions.

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

[0016] In some implementations, the non-naturally encoded amino acid comprises a carbonyl group, an acetyl group, an aminooxy group, a hydrazyl group, an acylhydrazyl group, an aminourea group, an azide group, or an alkynyl group.

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

[0018]

[0019] Where n is 0-10; R1 is alkyl, aryl, substituted alkyl or substituted aryl; R2 is H, alkyl, aryl, substituted alkyl or substituted aryl; R3 is H, amino acid, polypeptide or amino-terminal modifying group, and R4 is H, amino acid, polypeptide or carboxyl-terminal modifying group.

[0020] In some embodiments, the non-naturally encoded amino acid contains an aminooxy group. In some embodiments, the non-naturally encoded amino acid contains an acylhydrazine group. In some embodiments, the non-naturally encoded amino acid residue contains a hydrazine group. In some embodiments, the non-naturally encoded amino acid residue contains an aminourea group.

[0021] In some embodiments, the non-naturally encoded amino acid residues contain an azide group. In some embodiments, the non-naturally encoded amino acid has the following structure:

[0022]

[0023] Where 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, amino acid, polypeptide or amino-terminal modifying group, and R3 is H, amino acid, polypeptide or carboxyl-terminal modifying group.

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

[0025]

[0026] Where n is 0-10; R1 is alkyl, aryl, substituted alkyl or substituted aryl; X is O, N, S or absent; m is 0-10; R2 is H, amino acid, polypeptide or amino-terminal modifying group, and R3 is H, amino acid, polypeptide or carboxyl-terminal modifying 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 bioactive molecules.

[0028] The present invention also provides isolated nucleic acids comprising polynucleotides encoding a targeting polypeptide, and further provides isolated nucleic acids comprising polynucleotides hybridizing with said polynucleotides under stringent conditions. The present invention also provides isolated nucleic acids comprising polynucleotides encoding a targeting polypeptide, wherein said polynucleotides contain at least one selector codon. It will be apparent to those skilled in the art that many different polynucleotides can encode any of the polypeptides of the present invention.

[0029] In some implementations, the codons are selected from the following groups: amber codons, ochre codons, opal codons, unique codons, rare codons, pentabase codons, and tetrabase codons.

[0030] This invention also provides a method for preparing TC peptides linked to water-soluble polymers or to one or more TC peptides to form homodimers or homopolymers. In some embodiments, the method includes contacting an isolated TC peptide containing a non-naturally encoded amino acid with a water-soluble polymer or a linker containing a portion that reacts with the non-naturally encoded amino acid. In some embodiments, the non-naturally encoded amino acid incorporated into the TC peptide is reactive to the water-soluble polymer or linker, while the water-soluble polymer or linker is not reactive to any of the 20 common amino acids. In some embodiments, the non-naturally encoded amino acid incorporated into the TC peptide is reactive to the linker, polymer, or bioactive molecule, while the linker, polymer, or bioactive molecule is not reactive to any of the 20 common amino acids.

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

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

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

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

[0035] In some embodiments, the poly(ethylene glycol) molecule or connector has a molecular weight between about 0.1 kDa and about 100 kDa. In some embodiments, the poly(ethylene glycol) molecule or connector has a molecular weight between 0.1 kDa and 50 kDa. In some embodiments, the poly(ethylene glycol) molecule or connector is a branched polymer or connector. In some embodiments, each branch of the poly(ethylene glycol) branched polymer or connector 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 peptide comprises a polyalkylene glycol moiety. In some embodiments, the non-naturally encoded amino acid residues incorporated into the TC peptide comprise carbonyl, aminooxy, hydrazide, hydrazine, aminourea, azide, or alkynyl groups. In some embodiments, the non-naturally encoded amino acid residues incorporated into the TC peptide comprise a carbonyl moiety, and the water-soluble polymer comprises an aminooxy, hydrazide, hydrazine, or aminourea moiety. In some embodiments, the non-naturally encoded amino acid residues incorporated into the TC peptide comprise an alkynyl moiety, and the water-soluble polymer comprises an azide moiety. In some embodiments, the non-naturally encoded amino acid residues incorporated into the TC peptide comprise an azide moiety, and the water-soluble polymer comprises an alkynyl moiety. The present invention also provides compositions comprising a TC peptide 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.

[0037] The present invention also provides cells comprising polynucleotides encoding a targeting polypeptide containing a select codon (TC). In some embodiments, the cells comprise orthogonal RNA synthases and / or orthogonal tRNAs for replacing non-naturally encoded amino acids into the TC targeting polypeptide.

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

[0039] The present invention also provides methods for prolonging 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 include replacing any one or more amino acids of a naturally occurring TC-targeting polypeptide with a non-naturally encoded amino acid, and / or linking the targeting polypeptide to a linker, polymer, water-soluble polymer, or bioactive molecule.

[0040] The present invention also provides a method of treating a patient requiring such treatment with an effective amount of the TC molecule of the present invention. In some embodiments, the method includes 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 unglycosylated.

[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 site. 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] This invention provides TC peptides comprising at least one linker, polymer, or bioactive molecule, wherein the linker, polymer, or bioactive molecule is attached to the peptide via a functional group of a non-naturally encoded amino acid in the target peptide via ribosome incorporation. In TC conjugates, PEG or other water-soluble polymers, another TC, peptide, or bioactive molecule can be directly conjugated to the TC via the linker. In one embodiment, the linker is long enough 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 peptide is linked to the linker to allow polymer formation. In some embodiments, the linker is a bifunctional linker. In some embodiments, the compositions and / or TCs of this invention may comprise multiple linkers. In other embodiments, each linker may comprise one or more compounds to which it is attached. The linker may also comprise alkylene, alkenylene, alkyneene, polyether, polyester, polyamide groups, and also polyamino acids, peptides, cleavable peptides, or aminobenzylcarbamates. 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 may include valine-citrulline linkers or valine-alanine peptides. In some embodiments, the linker may be a dipeptide linker, such as a valine-citrulline or phenylalanine-lysine linker. Linkers containing valine-citrulline or valine-alanine may contain maleimide or succinimide groups. Linkers containing valine-citrulline or valine-alanine may contain a p-aminobenzyl alcohol (PABA) group or p-aminobenzyl carbamate (PABC). Other suitable linkers include linkers that are hydrolyzable at pH below 5.5, such as hydrazone linkers. Additional suitable cleavable linkers include disulfide linkers. In some embodiments, cleavable linkers may 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, maleimide-hexanoyl linkers. The maleimide hexanoyl linker may comprise, but is not limited to, N-maleimide methylcyclohexane-1-carboxylate, succinimide, pentafluorophenyl, and / or one or more PEG molecules. In some embodiments, any of the compositions, compounds, or salts thereof of the present invention may be linked to a peptide via the linker. In some embodiments, any of the compounds or salts thereof disclosed in Tables 3, 4, 5, 6, and 7 herein may be linked to a peptide via the linker. In some embodiments, the peptide is a targeting peptide, a biological targeting peptide, or a tumor-targeting peptide. In some embodiments, the targeting peptide is an antibody or an antibody fragment.

[0043] In some embodiments, the TC peptide is monopolyglycolate. The present invention also provides a TC comprising a linker, polymer, or bioactive molecule attached to one or more non-naturally encoded amino acids, wherein the non-naturally encoded amino acids are incorporated into the peptide via ribosomes at preselected sites.

[0044] In some embodiments, the present invention provides a composition comprising one or more targeting peptides having one or more incorporated non-naturally encoded amino acids, wherein at least one of the peptides is linked to a TLR agonist molecule via a linker covalently bonded to a non-natural amino acid of the peptide.

[0045] In another embodiment, the present invention provides a composition wherein the one or more targeting peptides are the same or different targeting peptides. In another embodiment, the present invention provides a composition wherein the one or more targeting peptides bind to a cell surface target, a tumor cell target, or a cancer cell target. In another embodiment, the one or more targeting peptides are single-specific, bispecific, or multispecific targeting peptides.

[0046] In other embodiments, the single-specific, bispecific, or multispecific targeting peptide comprises a drug conjugate or a checkpoint inhibitor. The use of any suitable immune checkpoint inhibitor in conjunction with the compositions of the present invention or TC is considered. 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 may also be used in the present invention. In some embodiments, immune checkpoint inhibitors are CTLA4, TIGIT, glucocorticoid-induced TNFR-related protein (GITR), inducible T cell co-stimulatory agents (ICOS), CD96, poliovirus receptor-associated 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, immune checkpoint inhibitors are inhibitors of CTLA4, PD-1, or PD-L1.

[0047] In another embodiment, the targeting peptide comprises an antibody or antibody fragment. In another embodiment, the targeting peptide is an antibody or antibody fragment that binds to a cellular antigen. In yet another embodiment, the targeting peptide is an antibody or antibody fragment that binds to a target selected from the group consisting of: HER2, HER3, PD-1, PDL-1, EGFR, TROP2, PSMA, VEGFR, CTLA-4, EpCAM, MUC1, MUC16, c-met, GPC3, ENPP3, TIM-1, FOLR1, STEAP1, mesothelin, 5T4, CEA, CA9, cadherin 6, ROR1, SLC34A2, SLC39A6, SLC44A4, LY6E, DLL3, ePhA2, GPNMB, SLITRK6, CD3, CD19, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD47, CD52, CD56, CD70, CD96, CD97, CD99, CD117, CD123, CD179, CD223, and CD276. In some embodiments, the targeting peptide comprises an antibody or antibody fragment that binds to HER2. In another embodiment, the targeting peptide is trastuzumab.

[0048] In another embodiment, the antibody or antibody fragment includes IgG, Fab, (Fab')2, Fv, or single-chain Fv (scFv). In some embodiments, the antibody or antibody fragment contains one or more Fab, (Fab')2, Fv, or single-chain Fv (scFv) mutations. In some embodiments, the antibody or antibody fragment contains one or more Fc mutations. In other embodiments, the antibody or antibody fragment contains one to six Fc mutations. In some embodiments, the antibody or antibody fragment contains two or more Fc mutations. In other embodiments, the antibody or antibody fragment contains three or more Fc mutations. In some embodiments, the antibody or antibody fragment contains four or more Fc mutations. In other embodiments, the antibody or antibody fragment contains five or more Fc mutations. In other embodiments, the antibody or antibody fragment contains 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, light chain, or both heavy and light chains. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain and light chain. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, light chain, or both heavy and light chains, and further comprises one or more Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into each of the heavy chain and light chain, and the antibody or antibody fragment further comprises one or more Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, light chain, or both heavy and light chains, and further comprises at least two Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into each of the heavy chain and light chain, and the antibody or antibody fragment further comprises at least two Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, light chain, or both heavy and light chains, and further comprises at least three Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into each of the heavy and light chains, and the antibody or antibody fragment further comprises at least three Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains, and further comprises at least four Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into each of the heavy and light chains, and the antibody or antibody fragment further comprises at least four Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains, and further comprises at least five Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into each of the heavy and light chains, and the antibody or antibody fragment further comprises at least five Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains, and further comprises at least six Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into each of the heavy and light chains, and the antibody or antibody fragment further comprises at least six Fc mutations.

[0050] In another embodiment, the targeting peptide comprises one or more non-naturally encoded amino acids selected from the group consisting of: p-acetylphenylalanine, p-nitrophenylalanine, p-sulfonyltyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-boroncarbonylphenylalanine, o-boroncarbonylphenylalanine, m-boroncarbonylphenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-OM e-phenylalanine, o-OMe phenylalanine, m-OMe phenylalanine, p-sulfonylphenylalanine, o-sulfonylphenylalanine, m-sulfonylphenylalanine, 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 O-Sulfotyrosine, 2-Sulfooxyphenylalanine, 3-Sulfooxyphenylalanine, o-Carboxyphenylalanine, m-Carboxyphenylalanine, p-Acetyl-L-Phenylanine, p-Propyl-Phenylanine, O-Methyl-L-Tyrosine, L-3-(2-Naphthyl)alanine, 3-Methyl-Phenylanine, O-4-Allyl-L-Tyrosine, 4-Propyl-L-Tyrosine, Tri-O-Acetyl-GlcNAcβ-Serine, L-DOPA (L- Dopa), fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propynyloxy-L-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine, and p-azidomethyl-phenylalanine. In another embodiment, the non-natural amino acid is selected from the group consisting of: p-acetyl-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine, or p-azidomethyl-phenylalanine. In other embodiments, the non-naturally encoded amino acid is site-specifically incorporated into one or more targeting peptides.

[0051] In another embodiment, the TLR agonist is a TLR7 agonist, a TLR8 agonist, or a dual TLR7 / TLR8 agonist. In other embodiments, the TLR agonist comprises, according to Figure 1 The TLR agonist has a molecular structure of any one of structures 1, 2, 3, 4, or 5. In another embodiment, the TLR agonist is any one of the TLR agonists selected from the group consisting of structures of Tables 3, 4, 5, 6, and 7 according to the present invention.

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

[0053] In some embodiments, one or more connectors are from 0.1 kDa to 50 kDa. In other embodiments, one or more connectors are from 0.1 kDa to 10 kDa. In other embodiments, one or more connectors or polymers are linear, branched, polymeric, or dendritic. In yet another embodiment, one or more connectors or polymers are bifunctional or multifunctional connectors or 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 0.1 kDa and 100 kDa. In other embodiments, PEG has a molecular weight between 0.1 kDa and 50 kDa. In other embodiments, PEG has a molecular weight between 0.1 kDa and 40 kDa. In other embodiments, PEG has a molecular weight between 0.1 kDa and 30 kDa. In other embodiments, PEG has a molecular weight between 0.1 kDa and 20 kDa. In other embodiments, PEG has a molecular weight between 0.1 kDa and 10 kDa. In some embodiments, the poly(ethylene glycol) molecule has a molecular weight between about 0.1 kDa and about 100 kDa. In some embodiments, the poly(ethylene glycol) molecule has a molecular weight between 0.1 kDa and 50 kDa. 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. 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 the average molecular weight. In some embodiments, the average molecular weight is the number-average molecular weight (Mn). The average molecular weight can be determined or measured using GPC or SEC, SDS / PAGE analysis, RP-HPLC, mass spectrometry, or capillary electrophoresis.

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

[0056] In another embodiment, the targeting peptide comprises one or more amino acid substitutions, additions, or deletions that increase the stability or solubility of the composition. In another embodiment, the targeting peptide comprises one or more amino acid substitutions, additions, or deletions that enhance / decrease ADCP or ADCC activity. In another embodiment, the targeting peptide 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 the expression of the targeting peptide in recombinant host cells or its in vitro synthesis.

[0057] In another embodiment, the non-naturally encoded amino acid is reactive to the linker, polymer, or bioactive molecule, which is not reactive to any of the 20 common amino acids in the polypeptide. In another embodiment, the non-naturally encoded amino acid comprises a carbonyl, aminooxy, hydrazine, acylhydrazine, aminourea, azide, or alkynyl group. In other embodiments, the non-naturally encoded amino acid comprises a carbonyl group.

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

[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, said TLR agonist comprising, according to Figure 1The structure of any TLR agonist, 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. In another embodiment, the TLR agonist is a TLR7 agonist, a TLR8 agonist, or a TLR7 / TLR8 dual agonist. In another embodiment, the TLR agonist comprises 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 Compounds AXC-759, AXC-760, AXC-761, AXC-762, AXC-764, AXC-771, AXC-772, AXC-773, AXC-777, AXC-778, AXC-779, AXC-789, AXC-793, AXC-799, AXC-800, AXC-801, AXC-802, AXC-803, AXC-804, AXC-805, AXC-806, AXC-807, AXC-808, AXC-809, AXC-810, AXC-831, or AXC-910. In another embodiment, the TLR agonist comprises a structure according to Structure 1 that further includes a linker.

[0060] In other embodiments, the TLR agonist comprising the 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, and 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- Compounds 762, AXC-764, AXC-771, AXC-772, AXC-773, AXC-777, AXC-778, AXC-779, AXC-789, AXC-793, AXC-800, AXC-801, AXC-802, AXC-803, AXC-804, AXC-805, AXC-806, AXC-807, AXC-808, AXC-809, AXC-810, AXC-831, and AXC-910. In other embodiments, the TLR agonist comprising the structure according to Structure 1 is selected from the group consisting of compounds AXC-801, AXC-802, AXC-831, and AXC-910. In other embodiments, the TLR agonist comprising the structure according to Structure 1 is selected from the group consisting of compounds AXC-801, AXC-802, AXC-831 and AXC-910, said compounds further comprising a connector.

[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, light chain, or both the heavy and light chains. In another embodiment, the one or more non-naturally encoded amino acids are selected from the group consisting of: p-acetylphenylalanine, p-nitrophenylalanine, p-sulfotyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-boroncarbonylphenylalanine, o-boroncarbonylphenylalanine, m-boroncarbonylphenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-OMephenylalanine. Alanine, o-OMe phenylalanine, m-OMe phenylalanine, p-sulfonylphenylalanine, o-sulfonylphenylalanine, m-sulfonylphenylalanine, 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-sulfonyltyrosine, 2-sulfonoxyphenylalanine, 3-sulfonoxyphenylalanine, o-carboxyphenylalanine, m-carboxyphenylalanine, p-acetyl-L-phenylalanine, p-propynyl-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-poly(hydroxyphenylalanine) The non-natural amino acids are p-acetyl-phenylalanine, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propynyloxy-L-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine, and p-azidomethyl-phenylalanine. In other embodiments, the non-natural 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 includes one or more mutations in the Fc region. In another embodiment, the anti-HER2 antibody or antibody fragment further includes two or more mutations in the Fc region. In another embodiment, the anti-HER2 antibody or antibody fragment further includes three or more mutations in the Fc region. In another embodiment, the anti-HER2 antibody or antibody fragment further includes four or more mutations in the Fc region. In another embodiment, the anti-HER2 antibody or antibody fragment further includes five or more mutations in the Fc region. In another embodiment, the anti-HER2 antibody or antibody fragment further includes six or more mutations in the Fc region. In another embodiment, the anti-HER2 antibody or antibody fragment further includes six mutations in the Fc region.

[0063] In another embodiment, one or more connectors are pyrolytic or non-pyrolytic connectors. In other embodiments, one or more connectors are bifunctional or multifunctional connectors.

[0064] In another embodiment, the TLR agonist comprises according to Figure 1 The structure of structure 2. In another embodiment, the TLR agonist comprises a structure according to structure 2 selected from the group consisting of compounds AXC-745, AXC-746, and AXC-753. In another embodiment, the TLR agonist comprises a structure according to any one of the following: compounds AXC-745, AXC-746, and AXC-753, said 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 according to Figure 1 The structure of structure 3. 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 comprising the structure according to AXC-837 or AXC-847 compound, said compound further comprising a linker. In another embodiment, the TLR agonist comprising the structure according to structure 3, said compound further comprising a linker. In another embodiment, the TLR agonist comprising the structure according to structure 3 further comprising a linker.

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

[0067] In another embodiment, the TLR agonist comprises according to Figure 1The structure of structure 5. In 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-8 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 the structure according to structure 5 is selected from the group consisting of compounds 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. In another embodiment, the TLR agonist comprises a structure according to any of the following: 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 Compounds AXC-888, AXC-889, AXC-890, AXC-891, AXC-892, AXC-893, AXC-895, AXC-896, AXC-897, AXC-898, AXC-901, AXC-903, AXC-904, AXC-905, AXC-906, AXC-907, AXC-908, AXC-909, AXC-911, AXC-912, AXC-913, AXC-914, AXC-915, or AXC-916. In another embodiment, the TLR agonist comprises a structure according to structure 5 that further includes 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 NO:1-13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises the amino acid sequence of at least two of SEQ ID NO:1-13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises a) SEQ ID NO:1 or 2; and b) any of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises the heavy chain of a) SEQ ID NO:1 or 2; and b) any of SEQ ID NO: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 of SEQ ID NO: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 NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO:2 and SEQ ID NO:3. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO:2 and SEQ ID NO:4. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO:2 and SEQ ID NO:5. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO:2 and SEQ ID NO:6. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO:2 and SEQ ID NO:7. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO:2 and SEQ ID NO:8. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO:2 and SEQ ID NO:9. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO:2 and SEQ ID NO:10. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO:2 and SEQ ID NO:11. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO:2 and SEQ ID NO:12. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO:2 and SEQ ID NO:13.In another embodiment, the present invention provides an anti-HER2 antibody or antibody fragment wherein a non-naturally encoded amino acid is incorporated in a site-specific manner at position 114 according to the Kabat number.

[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, said TLR agonist comprising, according to Figure 1 The TLR agonist conjugate (TC) comprises an antiHER2 antibody or antibody fragment conjugated to a TLR agonist selected from the compounds listed in Tables 3-7, 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, and the TC further comprises a chemotherapy agent or immunotherapy agent. In another embodiment, the present invention provides a TLR agonist conjugate (TC) comprising an antiHER2 antibody or antibody fragment conjugated to a TLR agonist selected from the compounds listed in Tables 3-7, 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, and the TC further comprises a chemotherapy agent or immunotherapy 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, said TLR agonist comprising, according to Figure 1The TLR agonist conjugate (TC) comprises an antiHER2 antibody or antibody fragment conjugated to a TLR agonist selected from the compounds listed in Tables 3-7, 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, and the TC further comprises a pharmaceutical conjugate. In other embodiments, the pharmaceutical conjugate is an antibody-pharmaceutical conjugate. In another embodiment, the present invention provides a TLR agonist conjugate (TC) comprising an antiHER2 antibody or antibody fragment conjugated to a TLR agonist selected from the compounds listed in Tables 3-7, 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, and the TC further comprises a pharmaceutical conjugate. In other embodiments, the pharmaceutical conjugate is an antibody-pharmaceutical conjugate. In other embodiments, TC further comprises cytokines or cytotoxins.

[0071] In another embodiment, the present invention provides a method of treating a subject or patient suffering from cancer, disease, condition, indication, or ailment, the method comprising administering to the subject or patient a therapeutically effective amount of the composition of the present invention or TC. In some embodiments, the tumor or cancer is a HER2-positive tumor or cancer. In some embodiments, the tumor, cancer, indication, disease, ailment, or ailment is a HER2-positive tumor, cancer, indication, disease, ailment, or ailment. In some 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 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 of the present invention or TC further comprising a chemotherapeutic agent or immunotherapeutic agent. In some embodiments, the TC is administered co-administered with at least one chemotherapeutic agent. Chemotherapy agents may be selected from the group consisting of: temozolomide, gemcitabine, doxorubicin, cyclophosphamide, paclitaxel, cisplatin, fluoropyrimidine, taxanes, anthracyclines, lapatinib, capecitabine, letrozole, pertuzumab, docetaxel, IFN-α. In another embodiment of the present invention, the TC is administered co-administered with at least one chemotherapeutic agent.

[0073] In another embodiment, the present invention provides a method for 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 a composition of the present invention or TC 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 the TC of the present invention. In other embodiments, the cells are tumor cells or cancer cells. In some 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 some embodiments, the tumor or cancer is a HER2-positive tumor or cancer. In some embodiments, the tumor, cancer, indication, disease, ailment, or condition to be treated is a HER2-positive tumor, cancer, indication, disease, ailment, or condition.

[0075] This invention provides a method for inhibiting or reducing tumor or cancer growth, comprising contacting the tumor with an effective amount of the TC of the present invention to stimulate the immune system near the tumor in a patient. This invention provides a method for inhibiting or reducing tumor or cancer growth, comprising contacting the tumor with an effective amount of the polyethylene glycol-modified TC of the present invention or a stable dimer or polymer of TC. In one embodiment, the TC is unpolyglycolated or monopolyglycolated. In one embodiment, the TC is dipolyglycolated. In one embodiment, the TC has more than one and / or different TLR agonist molecules attached thereto. Another embodiment of the invention provides a method for modulating the immune response against tumor cells using the TC of the present invention.

[0076] In some embodiments, the present invention provides a method of treating cancer using TC. In some embodiments, the TC of the present invention can be used to treat or prevent cancer-related diseases, conditions, and ailments, including conditions directly or indirectly related to cancer, such as angiogenesis and precancerous conditions, such as developmental abnormalities. In some embodiments, the tumor is a liquid tumor or a solid tumor. In some embodiments, the condition to be treated is cancer. 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 any cancer or disease or ailment related to these cancers. Cancer is a cancer that originates in epithelial cells, which are cells that cover the body surface, produce hormones, and make up glands. As non-limiting examples, 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, melanoma of the skin or eye, anal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethral cancer, renal pelvis cancer, ureteral cancer, endometrial cancer, cervical cancer, pituitary cancer, central nervous system (CNS) vegetations, primary CNS lymphoma, brainstem glioma, and spinal axis tumor. In some cases, cancer is skin cancer, such as basal cell carcinoma, squamous cell carcinoma, melanoma, non-melanoma, or actinic keratosis. In some embodiments, the invention also relates to a method of treating acute leukemia in mammals, comprising administering a therapeutically effective amount of the present invention's TC to said mammal. The invention also provides a method for inhibiting the proliferation of acute leukemia blast cells, comprising administering a therapeutically effective dose of the present invention's TC to a mammal suffering from acute leukemia.

[0077] In another embodiment, the TC disclosed herein can be used to modulate an immune response. Modulation of the immune response may include stimulating, activating, enhancing, strengthening, or upregulating the immune response. Modulation of the immune response may also include suppressing, inhibiting, blocking, reducing, or downregulating the 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 the use of the compositions of the present invention in the manufacture of pharmaceuticals.

[0080] In another embodiment, the invention provides a method comprising, according to Figure 1Immunostimulatory antibody conjugates (ISACs) comprising any of the TLR agonists of any structure. In another embodiment, the present invention provides immunostimulatory antibody conjugates (ISACs) comprising any 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 compounds 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 invention provides having according to Figure 1 Salts of any compound with the structure of TC. In another embodiment, the invention provides salts of any compound in Tables 3, 4, 5, 6, and 7. In another embodiment, the invention provides pharmaceutical compositions of compositions, compounds, and TC disclosed according to the invention, or salts thereof. In other embodiments, the pharmaceutical composition or salt further comprises a pharmaceutically acceptable excipient. Attached Figure Description

[0082] Figure 1 The general structure of the TLR agonist applicable to the invention is described.

[0083] Figure 2 The structures of various TC conjugates were described.

[0084] Figure 3 The structures of other TC conjugates were described.

[0085] Figure 4 The bioactivity of selected TC conjugates in cell proliferation assays was described.

[0086] Figure 5A and 5B The TLR7 activity of various TLR7 agonists was described.

[0087] Figure 6 The TLR7 activity of various TLR7 agonists attached to the linker was described.

[0088] Figure 7 The TLR7 activity of additional TLR7 agonists and TLR7 agonists attached to the linker was described.

[0089] Figure 8 The TLR7 activity of additional TLR7 agonists and TLR7 agonists attached to the linker was described.

[0090] Figure 9 The TLR7 activity of different TLR7 agonists attached to linkers (drug linkers or DL) was described compared to that of non-natural amino acid pAF (DL-pAF).

[0091] Figures 10A-10C An HPLC chromatogram of an unconjugated antiHER2 antibody with a non-natural amino acid at amino acid position HA114 was depicted. Figure 10A ), and at amino acid position HA114 with the TLR agonist AXC-875 ( Figure 10B ) and AXC-880 ( Figure 10C HPLC chromatogram of conjugated antiHER2 antibody.

[0092] Figure 11A-11C Comparison of tumor cell lines with high expression of SKOV3 HER2 ( Figure 11A Tumor cell lines with intermediate / low expression of JIMT-1HER2 ( Figure 11B ); and tumor cell lines with low HER2 expression (A431) Figure 11C Tumor-dependent ISAC activity of various payload linkers conjugated with anti-HER2 antibodies.

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

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

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

[0096] Figure 15A and 15BComparison of tumor cell lines with high SKBR3 HER2 expression ( Figure 15A ) and the HCC1806 tumor cell line with very low HER2 expression ( Figure 15B The tumor-dependent ISAC activity of three (3) payload linkers conjugated with anti-HER2 antibody was evaluated, with HER2-AXC-879 showing the best ISAC activity. Detailed Implementation

[0097] This document discloses therapeutic target (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 this invention may comprise a TLR agonist linked to a non-natural amino acid in the targeting moiety. Methods for preparing such TCs comprising non-natural amino acids incorporated into the targeting moiety peptide are also included.

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

[0099] A further or alternative embodiment is a method for detecting the presence of a polypeptide in a patient, the method comprising administering a polypeptide containing at least one heterocyclic non-natural amino acid, and, relative to a homologous naturally occurring amino acid polypeptide, the resulting heterocyclic non-natural amino acid polypeptide modulates the immunogenicity of the 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 therefore, they can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the methods and compositions described herein, which will be limited only by the appended claims.

[0101] As used herein and in the appended claims, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural references.

[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 this invention pertains. While any methods, apparatus, and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention described herein, preferred methods, apparatus, 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 invention described herein. Publications discussed herein are provided solely for their publication prior to the filing date of this application. Nothing herein should be construed as an admission that the inventors described herein have no right to claim prior disclosure by virtue of a 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 generate a β-hydroxy carbonyl compound—aldol.

[0105] As used herein, the term "affinity marker" refers to a marker that can reversibly or irreversibly bind to another molecule, modifying, disrupting, or forming a compound with it. For example, affinity markers include enzymes and their substrates, or antibodies and their antigens.

[0106] The terms “alkoxy,” “alkamino,” and “alkthio” (or thioalkoxy) are used in their conventional sense and refer to alkyl groups that are attached to a molecule by an oxygen, amino, or sulfur atom, respectively.

[0107] Unless otherwise stated, the term "alkyl" itself, or as part of another molecule, means a straight-chain or branched or cyclic hydrocarbon group or a combination thereof, which may be fully saturated, monounsaturated or polyunsaturated, and may include divalent and polyvalent groups (i.e., C1-C1) having a specified number of carbon atoms. 10 (Indicates one to ten carbons). Examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers of n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl is an alkyl group having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotonyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and more advanced homologs and isomers. Unless otherwise stated, the term "alkyl" also means those alkyl derivatives, including those defined in more detail herein, such as "heteroalkyl," "haloalkyl," and "homoalkyl."

[0108] The term "alkylene" itself, or as part of another molecule, refers to a divalent group derived from an alkane, such as (-CH2-). nWhere 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" refers to a shorter-chain alkyl or alkylene group, typically having 8 or fewer carbon atoms. Unless otherwise stated, 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 mimics 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 pyrrolidone and selenocysteine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, for example, an α-carbon bound to a hydrogen, carboxyl, amino, and R group. Such analogs may have modified R groups (e.g., oroleucine) or modified peptide backbones while retaining the same basic chemical structure as naturally occurring amino acids. Non-limiting examples of amino acid analogs include homoserine, oroleucine, methionine sulfoxide, and methionine methylsulfonium.

[0110] Amino acids may be referred to in this article by their names, their commonly known three-letter symbols, or the single-letter symbols recommended by the IUPAC-IUB Biochemistry Nomenclature Committee. Nucleotides, on the other hand, may be referred to by their generally accepted single-letter codes.

[0111] "Amino-terminal modifying groups" refer to any molecule that can be attached to a terminal amino group. For example, such terminal amino groups can be located at the ends of polymeric molecules, including but not limited to peptides, polynucleotides, and polysaccharides. Terminal modifying groups include, but are not limited to, various water-soluble polymers, peptides, or proteins. For example only, terminal modifying groups include polyethylene glycol or serum albumin. Terminal modifying 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] As used herein, “antibody” means a protein composed of one or more polypeptides, which are substantially encoded by all or part of an antibody gene. Immunoglobulin genes include, but are not limited to, genes for the constant regions of κ, λ, α, γ (IgG1, IgG2, IgG3, and IgG4), δ, ε, and μ, as well as numerous genes for the variable regions of immunoglobulins. Antibodies as used herein are intended to include both full-length antibodies and antibody fragments, and include antibodies naturally occurring in any organism or engineered antibodies (e.g., variants).

[0113] The term "antibody fragment" refers to any antibody form other than the full-length form. Antibody fragments in this article include antibodies as smaller components present in full-length antibodies, as well as engineered antibodies. Antibody fragments include, but are not limited to, Fv, Fc, Fab and (Fab')2, single-chain Fv (scFv), biantibodies, triantibodies, tetraantibodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, and variable regions, as well as alternative scaffold non-antibody molecules, bispecific antibodies, etc. (Maynard and Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9:395-402). Another functional substructure is the single-chain Fv (scFv), which consists of variable regions of the immunoglobulin heavy and light chains covalently linked by peptide linkers (S-zHu et al., 1996, Cancer Research, 56, 3055-3061). These small (Mr 25,000) proteins typically retain specificity and affinity for antigens in a single polypeptide and can provide suitable building blocks for larger antigen-specific molecules. Unless otherwise specified, statements and claims using the terms “antibody” or “multiple antibodies” specifically include “antibody fragments” and “multiple 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 bioactive molecules. Bioactive molecules can be conjugated to antibodies via linkers, polymers, or other covalent bonds.

[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 comprising 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 sharing adjacent carbon atom pairs) groups. Aromatic groups may be unsubstituted or substituted. Non-limiting examples of "aromatic" or "aryl" groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, anthraceneyl, and phenanthracenyl. Substituents in each of the above 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, arylthoxy, arylalkyl), it includes aryl rings and heteroaryl rings as defined above. Thus, the term "arylalkyl" or "alkylaryl" 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 (oxygen atom, for example only). Examples of such aryl groups include, but are not limited to, phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthoxy)propyl, etc.

[0117] As used herein, the term "arylene" refers to a divalent aryl group. Non-limiting examples of "arylene" include phenylene, pyridylene, pyrimidinylene, and thiopheneylene. Substituents of arylene groups are selected from the group of acceptable substituents described herein.

[0118] A "bifunctional polymer," also known as a "bifunctional connector," is a polymer containing two functional groups that can specifically react with other parts to form covalent or non-covalent bonds. These parts may include, but are not limited to, natural or non-natural amino acids or side groups on peptides containing said natural or non-natural amino acids. Other parts that may be attached to the bifunctional connector or bifunctional polymer may be the same or different parts. For example, a bifunctional connector may have a functional group reactive with a group on a first peptide and another functional group reactive with a group on a second peptide, thereby forming a conjugate comprising the first peptide, the bifunctional connector, and the second peptide. Many procedures and connector molecules for linking various compounds to peptides are known. See, for example, European Patent Application No. 188,256; U.S. Patents 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 entirety. A “multifunctional polymer,” also known as a “multifunctional connector,” is a polymer containing 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 bonds. Bifunctional or multifunctional polymers may have any desired length or molecular weight and may be selected to provide a specific desired spacing or conformation between one or more molecules linked to and bound to the compound or the compound itself.

[0119] As used herein, the term "bioavailability" refers to the rate and extent to which a substance or its active portion becomes available from a pharmaceutical dosage form and at the site of action or in systemic circulation. An increase in bioavailability refers to an increase in the rate and extent to which a substance or its active portion becomes available from a pharmaceutical dosage form and at the site of action or in systemic circulation. For example, an increase in bioavailability may be indicated by an increase in the concentration of a substance or its active portion in the blood compared to other substances or active portions. Methods for evaluating an increase in bioavailability are known in the art and can be used to evaluate the bioavailability of any peptide.

[0120] When used herein, the terms “biologically active molecule,” “biologically active portion,” or “biologically active agent” mean any substance that can affect any physical or biochemical property of a biological system, pathway, molecule, or interaction relating to a living organism, including but not limited to viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans. In particular, as used herein, a biologically active molecule includes, but is not limited to, any substance intended for the diagnosis, cure, relief, treatment, or prevention of disease in humans or other animals, or for the additional enhancement of physical or mental health in 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. The 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, fungicides, antivirals, anti-inflammatory agents, antitumor agents, cardiovascular agents, anxiolytics, hormones, growth factors, steroids, microbial toxins, etc.

[0121] "Modulating bioactivity" refers to increasing or decreasing the reactivity of a peptide, altering its selectivity, or enhancing or decreasing its substrate selectivity. By comparing the bioactivity of non-natural peptides with that of natural peptides, the bioactivity of modified peptides can be analyzed.

[0122] As used herein, the term “biomaterial” refers to biologically derived materials, including but not limited to materials obtained from bioreactors and / or recombinant methods and technologies.

[0123] As used herein, the term "biophysical probe" refers to a probe capable of detecting or monitoring structural changes in a molecule. Such molecules include, but are not limited to, proteins, and "biophysical probes" can be used to detect or monitor interactions between proteins and other macromolecules. Examples of biophysical probes include, but are not limited to, spin-labeled molecules, fluorophores, and photoactivated groups.

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

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

[0126] As used herein, the term "carbonyl" refers to a group containing a portion 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. Furthermore, such groups can be part of linear, branched, or cyclic molecules.

[0127] The term "carboxyl-terminal modifying 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 polymer molecule, wherein such polymer molecules include, but are not limited to, peptides, polynucleotides, and polysaccharides. Terminal modifying groups include, but are not limited to, various water-soluble polymers, peptides, or proteins. For example only, terminal modifying groups include polyethylene glycol or serum albumin. Terminal modifying groups can be used to modify the therapeutic properties of polymer molecules, including but not limited to extending the serum half-life of peptides.

[0128] As used herein, the term "chemically cleavable group," also known as "chemically unstable group," refers to a group that breaks or cleaves upon exposure to acids, bases, oxidants, reducing agents, chemical initiators, or free radical initiators.

[0129] As used herein, “cofolding” refers to a refolding process, reaction, or method involving at least two molecules, whereby the two molecules interact with each other, resulting in the transformation of an unfolded or misfolded molecule into a correctly folded molecule. By way of example only, “cofolding” involves at least two polypeptides, whereby the two polypeptides interact with each other, resulting in the transformation of an unfolded or misfolded polypeptide into a naturally occurring correctly folded polypeptide. Such polypeptides may contain a natural amino acid and / or at least one non-natural amino acid.

[0130] As used herein, “conjugate” refers to a polypeptide directly or via a linker to (e.g., covalently linked) a compound or compound-linker described herein, such as according to Figure 1Compounds or salts of any structure or any structure in 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 the target molecule. The targeting moiety may include, for example, antibodies, peptides, ligands, receptors, or their binding portions. The target biomolecule may be a biological receptor or other cellular structure, such as a tumor antigen. As used herein, the terms “conjugate of the present invention,” “targeting moiety conjugate,” “targeting conjugate,” “targeting moiety-active molecule conjugate,” or “TC” refer to a targeting polypeptide or a portion thereof, analogue, or derivative thereof that binds to a target present on a cell or its subunit, conjugated to a bioactive molecule, a portion thereof, or its analogue (including, but not limited to, TLR7 and / or TLR8 agonists). As used herein, the terms “tumor-targeting moiety conjugate,” “tumor-targeting moiety-bioactive molecule conjugate,” or “BTC” refer to a tumor-targeting polypeptide or a portion thereof, analogue, or derivative thereof that binds to a target present on a tumor cell or its subunit, conjugated to a bioactive molecule, a portion thereof, or its analogue (including, but not limited to, TLR7 and / or TLR8 agonists). Unless otherwise stated, the terms "compounds of the present invention" and "compositions of the present invention" are used as alternatives to the term "conjugates of the present invention".

[0131] The term "conservatively modified variant" applies to natural and non-natural amino acids, as well as natural and non-natural nucleic acid sequences and combinations thereof. For a specific nucleic acid sequence, a "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 sequence, or, when the natural and non-natural nucleic acids do not encode natural and non-natural amino acid sequences, substantially the same sequence. 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 without changing the encoded polypeptide. Such nucleic acid variations are "silent variants," a type of conserved modification variant. Therefore, for example, each natural or non-natural nucleic acid sequence encoding a natural or non-natural polypeptide described herein also describes every possible silent variant of the natural or non-natural nucleic acid. Those skilled in the art will recognize that every codon in a natural or non-natural nucleic acid (except for AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule. Therefore, each silent variant of a natural or non-natural nucleic acid encoding a natural or non-natural polypeptide is implicit in each described sequence.

[0132] Regarding amino acid sequences, alterations, additions, or deletions of a single natural or non-natural amino acid, or a small percentage of natural or non-natural amino acids, in the coding sequence of a nucleic acid, peptide, polypeptide, or protein are considered "variants of conserved modifications," where said alterations result in the deletion of an amino acid, the addition of an amino acid, or the substitution of a natural or non-natural amino acid by a chemically similar amino acid. Conservative representations of functionally similar natural amino acids are well known in the art. Such variants of conserved modifications are attached to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the methods and compositions described herein.

[0133] Conservative substitutions of functionally similar amino acids are known to those skilled in the art. The following eight groups each contain amino acids that are conservedly substituted for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). (See, for example, Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993)).

[0134] Unless otherwise stated, the terms "cycloalkyl" and "heterocycloalkyl" on their own or in combination with other terms refer to the cyclic form of "alkyl" and "heteroalkyl," respectively. Thus, cycloalkyl or heterocycloalkyl includes saturated, partially unsaturated, and fully unsaturated ring linkages. Additionally, for heterocycloalkyl, the heteroatom may occupy the position where the heterocycle is attached to the rest of the molecule. The heteroatom may include, but is not limited to, oxygen, nitrogen, or sulfur. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, etc. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, etc. Additionally, the term includes polycyclic structures, including but not limited to bicyclic and tricyclic cyclic structures. Similarly, the term "heterocyclic alkylene" itself or as part of another molecule indicates a divalent group derived from a heterocyclic alkyl group, and the term "cycloalkylene" itself or as part of another molecule indicates a divalent group 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 arranged in a ring. The outer ring contains water-soluble groups; at the center of the ring is a relatively nonpolar cavity capable of accommodating small molecules.

[0136] As used in this article, the term "cytotoxic" refers to compounds that damage cells.

[0137] As used herein, a denaturing agent (or denaturant) is any compound or substance that causes reversible unfolding of a polymer. For example, a denaturant can cause reversible unfolding of a protein. The strength of a denaturant will be determined by the nature and concentration of the specific denaturant. Examples of denaturants include, but are not limited to, liquid-dissolving agents, detergents, organic solvents, water-miscible solvents, phospholipids, or combinations thereof. Non-limiting examples of liquid-dissolving agents include, but are not limited to, urea, guanidine, and sodium thiocyanate. Non-limiting examples of detergents may include, but are not limited to, strong detergents such as sodium dodecyl sulfate, or polyoxyethylene ethers (e.g., Tween or Triton detergents), sodium dodecyl sarcosinate (Sarkosyl); mild nonionic detergents (e.g., digitalis saponins); mild cationic detergents such as N->2,3--(dioleoyloxy)-propyl-N,N,N-trimethylammonium; mild ionic detergents (e.g., sodium cholate or sodium deoxycholate); or zwitterionic detergents, including but not limited to 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 (especially C2-C4 alkanols such as ethanol or isopropanol), or lower alkanediols (C2-C4 alkanediols such as ethylene glycol), which can be used as denaturing agents. 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. Furthermore, such groups can be part of linear, branched, or cyclic molecules.

[0139] As used herein, the term "detectable marker" means a marker 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, ketaldehydes, ketonic acids, ketone esters, and ketone thioesters. Additionally, 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 may include substituents that will (by example only) produce an ester, ketone, aldehyde, thioester, or amide at either of the two moieties.

[0141] As used herein, the term "medicine" means any substance used to prevent, diagnose, alleviate, treat, or cure a disease or ailment.

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

[0143] The term "enhancement / enhancing" refers to increasing or prolonging the desired effect in terms of efficacy or duration. For example, "enhancing" the effect of a therapeutic agent means increasing or prolonging its effect in treating a disease, condition, or ailment in terms of efficacy or duration. As used herein, "enhancing effective amount" refers to an amount sufficient to enhance the effect of a therapeutic agent in treating a disease, condition, or ailment. When used in a patient, the effective amount for this purpose will depend on the severity and course of the disease, condition, or ailment, prior treatments, the patient's health status and response to the drug, and the judgment of the treating physician.

[0144] As used herein, the term “eukaryote” refers to organisms belonging to the 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 protozoa.

[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 in this article, the term "fluorophore" refers to a molecule that emits photons upon excitation and thus 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 molecular part or unit at which a chemical reaction occurs. These terms are synonymous to some extent in the field of chemistry and are used herein to indicate a molecular part 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, etc.

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

[0151] As used herein, the term "heteroalkyl" refers to a straight-chain, branched, or cyclic hydrocarbon group or combination thereof consisting of an alkyl group and at least one heteroatom selected from the group consisting of O, N, Si, and S, wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N, S, and Si may be located at any internal position of the heteroalkyl group or at a position where the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2,-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. In addition, at most two heteroatoms can be consecutive, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

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

[0153] Similarly, the term "heteroalkylene" refers to a divalent group 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 can occupy any one or both at the chain terminus (including, but not limited to, alkylenedioxy, alkylamino, alkyldiamino, aminooxyalkylene, etc.). Furthermore, 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 "heteroarylene" refers to an aryl group containing at least one heteroatom selected from N, O, and S; wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atom may optionally be quaternized. Heteroaryl groups may be substituted or unsubstituted. Heteroaryl groups can be attached to the remainder of the molecule via heteroatoms. Non-limiting examples of heteroaryl groups include 1-pyrrolithyl, 2-pyrrolithyl, 3-pyrrolithyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isooxazolyl, 4-isooxazolyl, 5-isooxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinel, 2-benzimidazolyl, 5-indolyl, 1-isoquinolinyl, 5-isoquinolinyl, 2-quinoxolinyl, 5-quinoxolinyl, 3-quinolinyl, and 6-quinolinyl.

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

[0156] As used herein, the term "identical" means that two or more sequences or subsequences are identical. Furthermore, the term "substantially identical" as used herein means that, when measured using comparison algorithms or by manual alignment and visual inspection, two or more sequences have a certain percentage of identical contiguous units after comparison through a comparison window or specified region and alignment to achieve maximum correspondence. For example only, two or more sequences may be considered "substantially identical" if the contiguous units are approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical in a specified region. Such percentages describe the "percentage of identity" of two or more sequences. Sequence identity can exist in regions of at least approximately 75-100 contiguous units, in regions of approximately 50 contiguous units, or, when not specified, throughout the entire sequence. This definition also relates to complementary sequences of the test sequences. For example, two or more polypeptide sequences are identical when the amino acid residues are the same across a specified region, and are “substantially identical” if the amino acid residues are approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical. Identity can be found across a region of at least about 75 to about 100 amino acids, a region of about 50 amino acids, or, when not specified, across the entire sequence of the polypeptide. Furthermore, by way of example only, two or more polynucleotide sequences are considered identical when the nucleic acid residues are the same across a specified region, and are “substantially identical” if the nucleic acid residues are approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical. Identity can be present in regions of at least about 75 to about 100 nucleic acid lengths of the polynucleotide sequence, in regions of about 50 nucleic acid lengths, or throughout the entire sequence when not specified.

[0157] For sequence comparison, a reference sequence is typically used as a comparison with a test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, with subsequence coordinates specified if necessary, and the sequence algorithm program parameters 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 percentage of sequence identity between the test sequence and the reference sequence.

[0158] As used herein, the term "immunogenicity" refers to an antibody response to the administration of a therapeutic drug. Immunogenicity of therapeutic non-natural amino acid peptides can be obtained using quantitative and qualitative assays for detecting antibodies against non-natural amino acid peptides in biological fluids. These assays include, but are not limited to, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), luminescent immunoassay (LIA), and fluorescence immunoassay (FIA). Analyzing the immunogenicity of therapeutic non-natural amino acid peptides involves comparing the antibody response following administration of the therapeutic non-natural amino acid peptide with the antibody response following administration of the therapeutic natural amino acid peptide.

[0159] As used herein, the term "isolation" means the separation of a target component from a non-target component and the removal of the target component from the non-target component. The isolated substance may be in a dry or semi-dry state, or in solution form, including but not limited to aqueous solutions. The isolated component may be in a homogeneous state, or the isolated component may be part of a pharmaceutical composition containing additional pharmaceutically acceptable carriers and / or excipients. Purity and homogeneity may be determined using analytical chemistry techniques, including but not limited to polyacrylamide gel electrophoresis or high-performance liquid chromatography. Additionally, when the component of interest is isolated and is the predominant species present in the formulation, the component is described herein as substantially purified. As used herein, the term "purified" may mean that the target component is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% pure, or a greater percentage pure. By way of example only, a nucleic acid or protein is "isolated" when it does not contain at least some of the cellular components it is associated with in its native state, or when the nucleic acid or protein has been concentrated to a level greater than its in vivo or in vitro production concentration. Moreover, for example, a gene is considered isolated when it is separated from an open reading frame flanking the gene and encoding a protein other than the gene of interest.

[0160] As used herein, the term "marker" refers to a substance incorporated into a compound that is readily detectable, thereby enabling the detection and / or monitoring of its physical distribution.

[0161] As used herein, the term "link" or "connector" refers to a bond or chemical moiety formed by a chemical reaction between the functional group of a connector and another molecule. Such bonds can include, but are not limited to, covalent and non-covalent bonds, and such chemical moiety can include, but is not limited to, esters, carbonates, imine phosphates, hydrazones, acetals, orthoesters, peptide links, and oligonucleotide links. Hydrolytically stable links mean that the link is substantially stable in water and does not react with water at a useful pH, including but not limited to prolonged duration under physiological conditions, perhaps even indefinitely. Hydrolytically unstable or degradable links mean that the link is degradable in water or in aqueous solutions, including, for example, blood. Enzymatically unstable or degradable links mean that the link can be degraded by one or more enzymes. By way of example only, PEG and related polymers can include degradable links in the polymer backbone or in linking groups between the polymer backbone and one or more terminal functional groups of the polymer molecule. Such degradable links include, but are not limited to, ester links formed by the reaction of PEG carboxylic acid or activated PEG carboxylic acid with an alcohol group on a bioactive agent, wherein such ester groups are typically hydrolyzed 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 amines and aldehydes; phosphate linkages formed by reacting alcohols with phosphate groups; hydrazone linkages as products of the reaction of hydrazides and aldehydes; acetal linkages as products of the reaction of aldehydes and alcohols; orthoester linkages as products of the reaction of formates and alcohols; peptide linkages formed by, but not limited to, amine groups at the ends of polymers such as PEGs and carboxyl groups of peptides; and oligonucleotide linkages formed by, but not limited to, phosphoramidite groups at the ends of polymers and 5' hydroxyl groups of oligonucleotides. Linkers include, but are not limited to, short linear, branched, multi-armed, or dendritic polymer molecules, such as polymers. In some embodiments of the 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 agents from these linking groups include, for example, irradiation of photoinstantaneous bonds and acid-catalyzed hydrolysis. For example, U.S. Patent No. 4,671,958 includes a description of an immunoconjugate containing a linker, which is cleaved at a target site in vivo by 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 peptide and the molecule to which it is linked. Given the numerous methods reported for attaching various radiodiagnostic compounds, radiotherapy compounds, drugs, toxins, and other agents to antibodies, those skilled in the art will be able to determine suitable methods for attaching a given agent or molecule to a peptide.

[0162] As used herein, the term "modification" refers to the presence of a change in a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. Such a change or modification can be obtained by post-synthetic modification of a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide; or by co-translation or post-translational modification of a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. The form "modified or unmodified" means that the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide under discussion is optionally modified; that is, the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide under discussion can be modified or unmodified.

[0163] As used herein, the term "regulated serum half-life" refers to a positive or negative change in the cyclic half-life of a modified bioactive molecule relative to its unmodified form. For example, modified bioactive molecules include, but are not limited to, natural amino acids, non-natural amino acids, natural amino acid peptides, or non-natural amino acid peptides. 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 between serum concentration and time allows for the calculation of serum half-life. For example, regulated serum half-life can be a prolongation of serum half-life, enabling improvements in dosing regimens or avoidance of toxic effects. Such a prolongation 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 prolongation of the serum half-life of any peptide are well known to those skilled in the art.

[0164] As used herein, the term "regulated therapeutic half-life" refers to a positive or negative change in the half-life of a therapeutically effective amount of a modified bioactive molecule relative to its unmodified form. For example, modified bioactive molecules include, but are not limited to, natural amino acids, non-natural amino acids, natural amino acid peptides, or non-natural amino acid peptides. For example, therapeutic half-life is measured by measuring the pharmacokinetic and / or pharmacodynamic properties of the molecule at various time points after administration. An increase in therapeutic half-life can enable the achievement of a specific beneficial dosing regimen, a specific beneficial total dose, or the avoidance of undesirable effects. For example, a prolonged therapeutic half-life can be caused by increased potency, increased or decreased binding of the modified molecule to its target, increased or decreased other parameters or mechanisms of action of the unmodified molecule, or increased or decreased degradation of the molecule by an enzyme (e.g., protease, by way of example only). Methods for evaluating the prolongation of the therapeutic half-life of any peptide are well known to those skilled in the art.

[0165] "Non-natural amino acids" refer to amino acids that are not one of the 20 common amino acids, pyrolysine, or selenocysteine. Other terms that may be used synonymously with "non-natural amino acids" include "non-naturally encoded amino acids," "non-natural amino acids," "non-naturally occurring amino acids," and their various forms connected by hyphens and without hyphens. The term "non-natural amino acids" includes, but is not limited to, amino acids that are naturally occurring through modification of naturally encoded amino acids (including, but not limited to, the 20 common amino acids, pyrolysine, and selenocysteine), but which themselves are not incorporated into the growing polypeptide chain through 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. Additionally, the term "non-natural amino acids" includes, but is not limited to, amino acids that are not naturally occurring and can be synthesized or obtained by modifying non-natural amino acids. In some embodiments, the non-natural amino acid includes lysine analogues, such as N6-azidoethoxy-L-lysine (AzK), N6-propyneethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetraazine lysine, or allyloxycarbonyl lysine. In some embodiments, the non-natural amino acid comprises a sugar moiety. 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 said amino acids also include those in which the naturally occurring N- or O-bond between the amino acid and the sugar is replaced by a covalent bond not typically found in nature—including, but not limited to, alkenes, oximes, thioethers, amides, etc. Examples of such amino acids also include sugars that are not typically found in naturally occurring proteins, such as 2-deoxy-glucose and 2-deoxygalactose. Specific examples of non-natural amino acids include, but are not limited to, p-acetyl-L-phenylalanine, p-propynyloxy-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, phosphonylserine, phosphonyltyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propynyloxy-L-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine, and p-azidomethyl-phenylalanine.In some implementations, the non-natural 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 in single-stranded or double-stranded form, and polymers thereof. 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 a reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides; (ii) oligonucleotide analogs, including but not limited to PNA (peptide nucleic acid), DNA analogs (phosphate thioesters, aminophosphate esters, etc.) used in antisense technologies; and (iii) conserved variants of these (including but not limited to degenerate codon substitutions) and complementary sequences, as well as explicitly indicated sequences. For example, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

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

[0168] As used herein, the term “pharmaceutically acceptable” means, but is not limited to, a salt, carrier, or diluent, a substance that does not eliminate the biological activity or properties of a compound and is relatively non-toxic, i.e., the substance can be administered to an individual without causing undesirable biological effects or interacting in a harmful manner with any component of a composition containing it.

[0169] As used herein, the terms "polyalkylene glycol" or "poly(olefin glycol)" refer to linear or branched polymeric polyether polyols. Such polyalkylene glycols include, but are not limited to, polyethylene glycol, polypropylene glycol, polybutane glycol, and their derivatives. Other exemplary embodiments are listed, for example, in commercial supplier catalogues, such as Shearwater Corporation's catalogue "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001). By way of example only, such polymeric polyether polyols have an average molecular weight between about 0.1 kDa and about 100 kDa. For example, such polymeric polyether polyols include, but are not limited to, those with a molecular weight between about 100 Da and about 100,000 Da. The molecular weight of the polymer can 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, and about 30,000 Da. a. Approximately 25,000 Da, approximately 20,000 Da, approximately 15,000 Da, approximately 10,000 Da, approximately 9,000 Da, approximately 8,000 Da, approximately 7,000 Da, approximately 6,000 Da, approximately 5,000 Da, approximately 4,000 Da, approximately 3,000 Da, approximately 2,000 Da, approximately 1,000 Da, approximately 900 Da, approximately 800 Da, approximately 700 Da, approximately 600 Da, approximately 500 Da, 400 Da, approximately 300 Da, approximately 200 Da, and approximately 100 Da. In some embodiments, the molecular weight of the polymer is between approximately 100 Da and approximately 50,000 Da. In some embodiments, the molecular weight of the polymer is between approximately 100 Da and approximately 40,000 Da. In some embodiments, the molecular weight of the polymer is between approximately 1,000 Da and approximately 40,000 Da. 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 branched PEG can be between approximately 1,000 Da and approximately 100,000 Da, including but not limited to approximately 100,000 Da, approximately 95,000 Da, approximately 90,000 Da, approximately 85,000 Da, approximately 80,000 Da, approximately 75,000 Da, approximately 70,000 Da, approximately 65,000 Da, approximately 60,000 Da, approximately 55,000 Da, approximately 50,000 Da, and approximately 45,000 Da. The molecular weights of branched PEG are approximately 1,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecular weight of branched PEG is between 1,000 Da and 50,000 Da. In some embodiments, the molecular weight of branched PEG is between 1,000 Da and 40,000 Da. In some embodiments, the molecular weight of branched PEG is between 5,000 Da and 40,000 Da. In some embodiments, the molecular weight of the branched PEG is between about 5,000 Da and about 20,000 Da. In other embodiments, the molecular weight of the branched 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 polymers of amino acid residues. That is, the description of a polypeptide is equally applicable to the description of a peptide and the description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-natural amino acids. Furthermore, the terms “polypeptide,” “peptide,” and “protein” include amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0172] The term "post-translational modification" refers to any modification of a natural or non-natural amino acid that occurs after it has been incorporated into the polypeptide chain via translation. Such modifications include, but are not limited to, co-translational in vivo modifications, co-translational in vitro modifications (e.g., in cell-free translation systems), 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, i.e., 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. Prodrugs are typically drug precursors that are converted into the active or more active form after administration to a subject and subsequent absorption through a process such as conversion via a metabolic pathway. Some prodrugs have chemical groups present on the prodrug that result in less activity and / or impart solubility or another property to the drug. Once the chemical groups have been cleaved and / or modified from the prodrug, the active drug is produced. Prodrugs are converted into the active drug in the body through enzymatic or non-enzymatic reactions. Prodrugs can provide improved physiological and chemical properties, such as better solubility; enhanced delivery characteristics, such as specific targeting of 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) bioavailability via oral administration, unlike the parent drug; and (iii) improved solubility in pharmaceutical compositions compared to the parent drug. Prodrugs include pharmacologically inactive or less active derivatives of active pharmaceutical ingredients. Prodrugs can be designed to modulate the amount of a drug or bioactive molecule reaching a desired site of action by manipulating properties such as physiochemical, biopharmaceutical, or pharmacokinetic properties. In an unrestricted manner, an example of a prodrug is a non-natural amino acid polypeptide administered in ester (“prodrug”) form to facilitate transmembrane transport (where water solubility is detrimental to mobility), but which is then metabolized and hydrolyzed into a carboxylic acid, the active entity, once inside the cell where water solubility is favorable. Prodrugs can be designed as reversible drug derivatives to act as regulators enhancing drug transport to site-specific tissues.

[0174] As used herein, the term "preventative effective dose" refers to the amount of a composition containing at least one non-natural amino acid peptide or at least one modified non-natural amino acid peptide that is applied prophylactically to a patient, which will alleviate to some extent one or more symptoms of the disease, condition, or ailment being treated. In such prophylactic application, this dose may depend on the patient's health status, weight, etc. Such a preventative effective dose is considered to be within the scope of the art through routine experiments, 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 portion that prevents the chemically reactive functional group from reacting under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group being protected. For example only, (i) if the chemically reactive group is an amine or hydrazide, the protecting group may be selected from tert-butoxycarbonyl (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 benzyl or alkyl, such as methyl, ethyl, or tert-butyl.

[0176] For example, the blocking / protecting groups can be selected from:

[0177]

[0178] In addition, protecting groups include, but are not limited to, photoinstantaneous 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 are incorporated herein by reference in their entirety.

[0179] The term "recombinant host cell," also known as "host cell," refers to a cell containing exogenous polynucleotides, wherein the methods for inserting the exogenous polynucleotides into the cell include, but are not limited to, direct uptake, transduction, f-crossing, or other methods known in the art for generating recombinant host cells. By way of example only, such exogenous polynucleotides may be non-integrating vectors, including but not limited to plasmids, or may be integrated into the host genome.

[0180] As used herein, the term "redox activator" refers to a molecule that oxidizes or reduces another molecule, thereby reducing or oxidizing the redox activator itself. Examples of redox activators include, but are not limited to, ferrocene, quinones, and Ru. 2+ / 3+ Complexes, Co 2+ / 3+ Complexes and Os 2+ / 3+ Complex.

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

[0182] As used herein, “refolding” describes any process, reaction, or method that transforms an incorrectly folded or unfolded state into a native or correctly folded conformation. By way of example only, refolding transforms a disulfide-bonded polypeptide from an incorrectly folded or unfolded state into a native or correctly folded conformation relative to the disulfide bond. Such a disulfide-bonded polypeptide can be a native amino acid polypeptide or a non-native amino acid polypeptide.

[0183] As used herein, the term "safety" or "safety profile" refers to the number of possible drug-related side effects relative to the number of times the drug has been administered. For example, a drug that has been administered multiple times and has produced only mild or no side effects is considered to have excellent safety. Methods for evaluating the safety profile of any peptide are known in the art.

[0184] As used herein, the phrase “selective hybridization” or “specific hybridization” refers to a molecule binding to, double-stranding with, or hybridizing with a specific nucleotide sequence in a complex mixture (including, but not limited to, total cellular or library DNA or RNA) under strict hybridization conditions.

[0185] The phrase "strict hybridization conditions" refers to the hybridization of sequences of DNA, RNA, PNA, or other nucleic acid mimics, or combinations thereof, under conditions of low ionic strength and high temperature. For example, under strict conditions, a probe will hybridize with its target sequence in a complex mixture of nucleic acids (including, but not limited to, total cellular or library DNA or RNA), but not with other sequences in the complex mixture. Strict conditions are sequence-dependent and will vary under different conditions. For example, longer sequences hybridize specifically at higher temperatures. Strict hybridization conditions include, but are not limited to: (i) a thermal melting point (Tm) about 5-10 °C lower than the specific sequence at defined ionic strengths and pH; (ii) a salt concentration of about 0.01 M to about 1.0 M at a range of 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 a temperature of at least about 60 °C for long probes (including but not limited to more than 50 nucleotides); (iii) the addition of a destabilizing agent, including but not limited to formamide; (iv) incubation at 42 °C with 50% formamide, 5X SSC and 1% SDS, or incubation at 65 °C with 5X SSC and about 1% SDS, followed by washing in 0.2X SSC and incubation at 65 °C with about 0.1% SDS for about 5 minutes to about 120 minutes. For example only, the detection of selective or specific hybridization includes, but is not limited to, a positive signal at least twice the background. A broad guide to nucleic acid hybridization can be found in Tijssen’s 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 used as a subject of treatment, observation, or experimentation. For example only, a subject may be, but is not limited to, mammals, including but not limited to humans.

[0187] As used herein, the term "substantially purified" means that the target component is substantially or substantially free of other components that typically accompany or interact with the target component prior to purification. For example only, a target component may be "substantially purified" when a formulation of the target component 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% (on a dry weight basis) of contaminating components. Therefore, a "substantially purified" target component may 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 higher. For example only, natural or non-natural amino acid peptides may be purified from native cells, or, in the case of recombinant natural or non-natural amino acid peptides, from host cells. For example, when the formulation 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 substances, the formulation of the natural or non-natural amino acid polypeptide may be "substantially purified". For example, when the natural or non-natural amino acid polypeptide is recombinantly generated from host cells, the natural or non-natural amino acid polypeptide may be present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the cell dry weight. For example, when a natural or non-natural amino acid polypeptide is recombinantly produced from a host cell, the natural or non-natural amino acid polypeptide may be present in the culture medium at cell dry weights of 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 lower. For example, “substantially purified” natural or non-natural amino acid peptides may 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 higher, as determined by appropriate methods, including but not limited to SDS / PAGE analysis, RP-HPLC, SEC and capillary electrophoresis.

[0188] The term "substituent," also known as "non-interfering substituent," refers to a group that can replace another group on a molecule. Such groups include, but are not limited to, halogenated, C1-C... 10 Alkyl, C2-C 10 alkenyl, C2-C10 alkynyl group, C1-C 10 Alkoxy, C5-C 12 Aryl alkyl, 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 Alkyl sulfonyl group, -(CH2) m -O-(C1-C 10 Alkyl), where 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 Alkyl thioalkyl, -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 groups), wherein each m is 1 to 8, -C(O)NR2, -C(S)NR2, -SO2NR2, -NRC(O)NR2, -NRC(S)NR2, their salts, etc. Each R group in the foregoing list includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl or alkylaryl. Where substituents are described in detail by their conventional chemical formulas, they are written from left to right, and they also cover chemically identical substituents resulting from writing the structure from right to left; for example, -CH2O- is equivalent to -OCH2-.

[0189] For example only, alkyl and heteroalkyl substituents (including those groups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, ynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) include, but are not limited to: -OR, =O, =NR, =N-OR, -NR2, -SR, -halogen, -SiR3, -OC(O)R, -C(O)R, -CO2R, -CONR2, -OC(O)NR2, -NRC(O)R, -NRC(O)NR2, -NR(O)2R, -NR-C(NR2)=NR, -S(O)R, -S(O)2R, -S(O)2NR2, -NRSO2R, -CN, and -NO2. Each R group in the 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 combine with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR2 is expected to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl.

[0190] For example, the substituents of 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, -CO2R, -CONR2, -OC(O)NR2, -NRC(O)R, -NRC(O)NR2, -NR(O)2R, -NR-C(NR2)=NR, -S(O)R, -S(O)2R, -S(O)2NR2, -NRSO2R, -CN, -NO2, -R, -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy and fluoro(C1-C4)alkyl, ranging from zero to the total number of open valences on the aromatic ring system; and each R group in the aforementioned list includes, but is not limited to, hydrogen, alkyl, heteroalkyl, aryl and heteroaryl.

[0191] As used herein, the term "therapeutic effective amount" refers to the 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 with a disease, condition, or ailment, sufficient to cure or at least partially prevent or alleviate one or more symptoms of the treated disease, condition, or ailment. The effectiveness of the composition depends on various factors, including but not limited to the severity and course of the disease, condition, or ailment; prior therapy; the patient's health status and response to the medication; and the judgment of the treating physician. By way of example only, a therapeutic effective amount can be determined through routine experiments, including but not limited to dose-escalation clinical trials.

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

[0193] As used herein, the terms “toxic moiety” or “toxic group” refer to compounds that can cause injury, impairment, or death. Toxic components include, but are not limited to, auristatin, DNA minor groove binding agents, DNA minor groove alkylating agents, enediyne, lexitropsin, pyroxine, taxane, puromycin, TLR agonists, maytansinoids, vinca alkaloids, AFP, MMAF, MMAE, AEB, AEB, AEB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizomycin, cyanomorpholino-doxorubicin, TLR agonist-10, echinomycin, comprbetatstatin, chalicheamicin, maytansin, DM-1, fusiformin, podophyllotoxins (e.g., etoposide, teniposide), baccatin and its derivatives, and anti-tubulin agents. Agent), cryptophysin, comprbetastatin, auristatin E, vincristine, vinblastine, vinorelbine, VP-16, camptothecin, epothilone A, epothilone B, nocodazole, colchicine, colchicine amine, estradiol, cimadolic acid, discormolide, maytansine, eleutherobin Mechlorethamine, cyclophosphamide, melphalan, carmustine, lomustine, semustine, streptozocin, chloruracil, uracil mustard, nitrogen mustard, ifosfamide, chlorbutazone mustard, piperobromophenyl, triethylene melamine, triethylene thiophosphate, busulfan, dacarbazine and temozolomide, ytarabine, cytosine arabinoside, fluorouracil, fluorouracil, 6-thioguanine, 6-mercaptopurine, pentostatin, 5-fluorouracil, methotrexate, 10-propargyl-5,8-dideazafolate, 5,8-dideazatetrahydrofolic acidFolic acid, leucovorin, fludarabine phosphate, pentostatin, gemcitabine, Ara-C, paclitaxel, docetaxel, deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine, buquina, antibiotics (e.g., anthracycline), gentamicin, cefotaxime, 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, sildenafil, delavirdine, didanosine, efavirenz, entecavir, fosfonet, ganciclovir, ibatabin, isoprotocin, idoxuridine, inosine, lopinavir, metisavirin, methisazone, nesavirin, etc.), Nexavir, nevirapine, oseltamivir, penciclovir, stavudine, trifluridine, truvada, valacyclovir, zanamivir, etc., daunorubicin hydrochloride, doxorubicin, rubidomycin, cerubidine, idarubicin, doxorubicin, epirubicin and morpholino derivatives, phenoxazinone bicyclic peptides (e.g., bleomycin), basic glycopeptides (e.g., bleomycin), anthraquinone glycosides (e.g., purcamycin, mithramycin) Anthraquinones (e.g., mitoxantrone), aziridine pyrroloindole-diones (e.g., mitomycin), macrocyclic immunosuppressants (e.g., cyclosporine, FK-506, tacrolimus, prograf, rapamycin, etc.), navelbene, CPT-11, anastrozole, letrazole, capecitabine, raloxifene, cyclophosphamide, ifosfamide, droloxafine, allocolchicine, halichondrin B, colchicine, colchicine derivatives, maytansine, rhizoxin, paclitaxel, paclitaxel derivatives, docetaxel, thiocolchicine, triphenylmethylcysteine.The list includes cysterin, vincristine sulfate, cisplatin, carboplatin, hydroxyurea, N-methylhydrazine, epidophyllotoxin, procarbazine, mitoxantrone, folate, and tegafur. "Taxane" includes paclitaxel and any active taxane derivatives or prodrugs.

[0194] As used herein, the term "treatment" includes reducing, alleviating, or improving symptoms of a disease or condition; preventing additional symptoms; improving or preventing the underlying metabolic cause of symptoms; inhibiting a disease or condition, such as preventing its progression; reducing a disease or condition; causing a disease or condition to subside; alleviating the condition caused by a disease or condition; or terminating the symptoms of a disease or condition. The term "treatment" includes, but is not limited to, preventative and / or therapeutic treatment.

[0195] As used herein, the term "water-soluble polymer" refers to any polymer that is soluble in aqueous solvents. Such water-soluble polymers include, but are not limited to, polyethylene glycol, polyethylene glycol propionaldehyde, and their mono-C1-C2 polymers. 10Alkoxy or aryloxy derivatives (described in U.S. Patent No. 5,252,714, which is incorporated herein by reference), monomethoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran derivatives (including dextran sulfate), polypropylene glycol, polypropylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols, heparin, heparin fragments, polysaccharides, oligosaccharides, polysaccharides, cellulose and cellulose derivatives (including, but not limited to, methylcellulose and carboxymethylcellulose), serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycols and their derivatives, copolymers of polyalkylene glycols and their derivatives, polyvinyl ethyl ethers and α-β-poly[(2-hydroxyethyl)-DL-asparagine, etc., or mixtures thereof. For example, conjugation of such water-soluble polymers with natural or non-natural amino acid peptides can lead to changes including, but not limited to, the following: increased water solubility; prolonged or modulated serum half-life; prolonged or modulated therapeutic half-life relative to the unmodified form; increased bioavailability; modulated biological activity; prolonged circulation time; modulated immunogenicity; and modulated physical association characteristics, including but not limited to aggregation and multimerization, altered receptor binding, binding to activity modulators or other targeted peptides, altered binding to one or more binding partners, and altered dimerization or multimerization of the targeted peptide receptor. Furthermore, such water-soluble polymers may or may not possess their own biological activity and can be used as connectors for attaching targeted peptides to other substances, including but not limited to one or more targeted peptides or one or more bioactive molecules.

[0196] Unless otherwise stated, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA technology and pharmacology within the art should be employed.

[0197] The compounds described herein (including, but not limited to, non-natural amino acids, non-natural amino acid peptides, modified non-natural amino acid peptides, and reagents used to generate the aforementioned compounds) include isotopically labeled compounds that are identical to those listed in the different chemical formulas and structures described herein, but in fact, one or more atoms are replaced by atoms having atomic masses or mass numbers different from those commonly found in nature. Examples of isotopes that can be incorporated into the compounds described herein include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, such as... 2 H, 3 H, 13 C 14 C 15 N、 18 O、 17 O、 35 S, 18 F,36 Cl. Certain isotope-labeled compounds described herein (e.g., those doped with radioactive isotopes, such as...) 3 H and 14 Compounds of C are useful in drug and / or substrate tissue distribution assays. Furthermore, compounds such as deuterium (i.e.,...) 2 Isotope substitution of H can provide certain therapeutic advantages due to greater metabolic stability, such as prolonged in vivo half-life or reduced dose requirements.

[0198] Some of the compounds described herein (including, but not limited to, non-natural amino acids, non-natural amino acid peptides, and modified non-natural amino acid peptides, as well as reagents used to generate the aforementioned compounds) have asymmetric carbon atoms and can therefore exist as enantiomers or diastereomers. Based on their physicochemical differences, diastereomer mixtures can be separated into their individual diastereomers by known methods, such as chromatography and / or stepwise crystallization. Enantiomers can be separated by reacting an enantiomer mixture with a suitable optically active compound (e.g., an alcohol) to convert it into a diastereomer mixture, thus separating the diastereomers, and by converting (e.g., hydrolyzing) the individual diastereomers into their respective pure enantiomers. All such isomers (including diastereomers, enantiomers, and mixtures thereof) are considered part of the compositions described herein.

[0199] In other or further embodiments, the compounds described herein (including, but not limited to, non-natural amino acids, non-natural amino acid peptides, and modified non-natural amino acid peptides, and reagents for generating the aforementioned compounds) are used in the form of a prodrug. In other or further embodiments, the compounds described herein (including, but not limited to, non-natural amino acids, non-natural amino acid peptides, and modified non-natural amino acid peptides, and reagents for generating the aforementioned compounds) are metabolized upon administration to a desired organism to produce metabolites, which are then used to produce the desired effect, including the desired therapeutic effect. In further or additional embodiments, these are active metabolites of non-natural amino acids and "modified or unmodified" non-natural amino acid peptides.

[0200] The methods and formulations described herein include the use of N oxides, crystalline forms (also known as polymorphs), or pharmaceutically acceptable salts of non-natural amino acids, non-natural amino acid peptides, and modified non-natural amino acid peptides. In some embodiments, the non-natural amino acids, non-natural amino acid peptides, and modified non-natural amino acid peptides may be present as tautomers. All tautomers are included within the scope of the non-natural amino acids, non-natural amino acid peptides, and modified non-natural amino acid peptides provided herein. Furthermore, the non-natural amino acids, non-natural amino acid peptides, and modified non-natural amino acid peptides described herein may exist in both non-solventized 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 peptides, and modified non-natural amino acid peptides provided herein are also considered to be disclosed herein.

[0201] Some compounds described herein (including, but not limited to, non-natural amino acids, non-natural amino acid peptides, and modified non-natural amino acid peptides, as well as reagents used to generate the aforementioned compounds) may exist in several tautomeric forms. All such tautomeric forms are considered part of the compositions described herein. Furthermore, for example, all enol-ketone forms of any compound described herein (including, but not limited to, non-natural amino acids, non-natural amino acid peptides, and modified non-natural amino acid peptides, as well as reagents used to generate the aforementioned compounds) are considered part of the compositions described herein.

[0202] Some of the compounds described herein (including, but not limited to, non-natural amino acids, non-natural amino acid peptides, and modified non-natural amino acid peptides, as well as reagents used to generate any of the aforementioned compounds) are acidic and can form salts with pharmaceutically acceptable cations. Some of the compounds described herein (including, but not limited to, non-natural amino acids, non-natural amino acid peptides, and modified non-natural amino acid peptides, as well as reagents used to generate the aforementioned 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 they 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. Salts are recovered by using at least one of the following techniques: filtration, precipitation with a non-solvent followed by filtration, solvent evaporation, or, in the case of aqueous solutions, lyophilization.

[0203] 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 an organic base, pharmaceutically acceptable salts of the non-natural amino acid polypeptides disclosed herein can be formed. Alternatively, salts of starting materials or intermediates can be used to prepare the salt form of the disclosed non-natural amino acid polypeptides. The non-natural amino acid polypeptides described herein can be prepared into pharmaceutically acceptable acid addition salts (a type of pharmaceutically acceptable salt) by reacting the free base form of the non-natural amino acid polypeptides described herein with a pharmaceutically acceptable inorganic or organic acid. Alternatively, the non-natural amino acid polypeptides described herein can be prepared into pharmaceutically acceptable base addition salts (a type of pharmaceutically acceptable salt) by reacting the free acid form of the non-natural amino acid polypeptides described herein with a pharmaceutically acceptable inorganic or organic base.

[0204] Pharmaceutically acceptable types of salts include, but are not limited to: (1) acid addition salts, which form 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- Naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-en-1-carboxylic acid, glucoheponic acid, 4,4'-methylenebis-(3-hydroxy-2-en-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, dodecyl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, mucoconic acid, etc. are formed; (2) 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, the salts are formed. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucosamine, etc. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, etc.

[0205] Various methods, including but not limited to ion exchange chromatography, ion chromatography, capillary electrophoresis, inductively coupled plasma atomic absorption spectrometry, atomic absorption spectrometry, mass spectrometry, or any combination thereof, can be used to analyze and identify the corresponding counterions of pharmaceutically acceptable salts of non-natural amino acid peptides. Furthermore, the therapeutic activity of such pharmaceutically acceptable salts of non-natural amino acid peptides can be tested using the techniques and methods described in the examples.

[0206] It should be understood that a reference to a salt includes its solvation form or its crystalline form, particularly a solvate or polymorph. Solvates contain both stoichiometric and non-stoichiometric solvents and are often formed during crystallization with pharmaceutically acceptable solvents such as water, ethanol, etc. When the solvent is water, a hydrate is formed, or when the solvent is an alcohol, an alcoholysis is formed. Polymorphs comprise different crystalline arrangements of the same elemental composition of a compound. Polymorphs typically exhibit different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal forms, optical and electrical properties, stability, and solubility. Various factors, such as recrystallization solvents, crystallization rates, and storage temperatures, can cause the single-crystal form to predominate.

[0207] Using a variety of techniques, including but not limited to thermal analysis, X-ray diffraction, spectroscopy, vapor adsorption, and microscopy, screening and characterization of drug-acceptable salt polymorphs and / or solvates of non-natural amino acid peptides can be performed. 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, locate 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, UVIS, and NMR (liquid and solid). Various microscopy techniques include, but are not limited to, polarized light microscopy, scanning electron microscopy (SEM) with energy-dispersive X-ray analysis (EDX), environmental scanning electron microscopy with EDX (in gaseous or water vapor atmospheres), IR microscopy, and Raman microscopy.

[0208] While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous changes, variations, and substitutions will now be apparent to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice the invention. It is contemplated that the following claims define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

[0209] TLR agonist junction derivatives

[0210] At one level, this document describes tools (methods, compositions, techniques) for generating and using targeted peptides comprising at least one non-natural amino acid or a modified non-natural amino acid having a carbonyl, dicarbonyl, oxime, or hydroxylamine group, or similar. Such targeted peptides comprising non-natural amino acids may contain other functionalities, including but not limited to polymers; water-soluble polymers; polyethylene glycol derivatives; second proteins or peptides or peptide analogs; antibodies or antibody fragments; and any combination thereof. It should be noted that the various functionalities mentioned above are not intended to imply that a member of one functionality cannot be classified as a member of another. In practice, overlap will exist depending on the specific circumstances. For example, water-soluble polymers overlap with polyethylene glycol derivatives in scope; however, the overlap is not complete, and therefore both functionalities are referred to above.

[0211] On the one hand, this document describes methods for selecting and designing TLR agonist linker derivatives and targeting peptides, and the modification of said targeting peptides using the methods, compositions, and techniques described herein. New TLR agonist linker derivatives and targeting peptides can be designed de novo, for example only, including being designed as part of a high-throughput screening process (in which case multiple peptides can be designed, synthesized, characterized, and / or tested), or designed based on investigator interests. New TLR linker derivatives and targeting peptides can also be designed based on the structures of known or partially characterized peptides. For example only, TLR agonists are a subject of extensive scientific research; new compounds can be designed based on the structures of TLR agonists. The principles for selecting which amino acids to substitute and / or modify are described separately. The document also describes the selection of which modifications to employ and how they can be used to meet the needs of experimenters or end users. Such needs may include, but are not limited to, manipulating the therapeutic efficacy of the peptide, improving the safety profile of the peptide, adjusting the pharmacokinetics, pharmacology, and / or pharmacodynamics of the peptide, such as, for example only, increasing water solubility, bioavailability, prolonging serum half-life, prolonging therapeutic half-life, modulating immunogenicity, modulating biological activity, or prolonging circulation time. In addition, by way of example only, such modifications include providing additional functionality to the peptide, incorporating antibodies, and any combination of the aforementioned modifications.

[0212] This article also describes TLR agonist linker derivatives and targeting peptides that have or can be modified to contain oxime, carbonyl, dicarbonyl, or hydroxylamine groups. Methods for the production, purification, characterization, and use of such TLR agonist linker derivatives and targeting peptides are also described.

[0213] TLR agonist linker derivatives or targeting peptides may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more carbonyl or dicarbonyl, oxime, hydroxylamine, or their protected forms. TLR agonist linker derivatives or targeting peptides may be the same or different; for example, in derivatives containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different reactive groups, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different sites may be present.

[0214] As described herein, this disclosure provides a targeting polypeptide coupled to another molecule having the formula "targeting polypeptide-LM", where L is a linking group or chemical bond, and M is any other molecule, including but not limited to another targeting polypeptide. In some embodiments, L is stable in vivo. In some embodiments, L is hydrolyzable in vivo. In some embodiments, L is metastable in vivo.

[0215] Using standard linkers and procedures known to those skilled in the art, a targeting peptide and M can be linked together via L. In some embodiments, the targeting peptide and M are directly fused, and L is the bond. In other embodiments, the targeting peptide and M are fused via the linker group L. For example, in some embodiments, the targeting peptide and M are linked together by peptide bonds, optionally via peptide or amino acid spacers. In some embodiments, the targeting peptide and M are linked together by chemical conjugation, optionally via the linker group (L). In some embodiments, L is directly conjugated to each of the targeting peptide 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 target polypeptide is conjugated to M by reacting a nucleophilic reactive moiety on the target polypeptide with an electrophilic reactive moiety on Y, or by reacting an electrophilic reactive moiety on the target polypeptide with a nucleophilic reactive moiety on M. In embodiments, when L is a group linking the target polypeptide and M together, the target polypeptide and / or M can be conjugated to L by reacting a nucleophilic reactive moiety on the target polypeptide and / or M with an electrophilic reactive moiety on L, or by reacting an electrophilic reactive moiety on the target 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, acid anhydride, ester, succinimide ester, alkyl halide, sulfonate, maleimide, haloacetyl, and isocyanate groups. In embodiments where the targeted peptide and M are conjugated together via a reaction of a carboxylic acid with an amine, an activator can be used to form an activated ester of the carboxylic acid.

[0217] The activated ester of the carboxylic acid can be, for example, N-hydroxysuccinimide (NHS), toluenesulfonate (Tos), methanesulfonate, trifluoromethanesulfonate, 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-oxy-tripyrrolidinephosphonium hexafluorophosphate (PyBOP), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethylurea cationic hexafluorophosphate (HATU) and o-benzotriazol-N,N,N',N'-tetramethylurea cationic hexafluorophosphate (HBTU).

[0218] In some embodiments, the targeting peptide comprises a nucleophilic reactive group (e.g., an amino, thiol, or hydroxyl group of a lysine, cysteine, or serine side chain) capable of conjugating to an electrophilic reactive group on M or L. In some embodiments, the targeting peptide comprises an electrophilic reactive group (e.g., a carboxylic acid ester group of an Asp or Glu side chain) capable of conjugating to a nucleophilic reactive group on M or L. In some embodiments, the targeting peptide is chemically modified to comprise a reactive group capable of conjugating directly to M or L. In some embodiments, the targeting peptide is modified at the N-terminus or C-terminus to comprise a natural or non-natural amino acid having a nucleophilic side chain. In exemplary embodiments, the N-terminal or C-terminal amino acid of the targeting peptide 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 peptide may be modified to comprise a lysine residue. In some embodiments, the N-terminal or C-terminal amino acid of the targeting peptide is modified to comprise a natural or non-natural amino acid having an electrophilic side chain, such as Asp and Glu. In some embodiments, the internal amino acids of the targeting peptide are replaced by natural or non-natural amino acids having nucleophilic side chains as described above. In exemplary embodiments, the substituted internal amino acids of the targeting peptide are selected from the group consisting of lysine, ornithine, serine, cysteine, and homocysteine. For example, the internal amino acids of the targeting peptide may be replaced by lysine residues. In some embodiments, the internal amino acids of the targeting peptide are replaced by 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 the target peptide or to L. In some embodiments, M comprises a nucleophilic reactive group (e.g., amine, thiol, hydroxyl) capable of conjugating to an electrophilic reactive group on the target peptide or L. In some embodiments, M comprises an electrophilic reactive group (e.g., carboxyl group, activated form of carboxyl group, compound having a leaving group) capable of conjugating to a nucleophilic reactive group on the target peptide or L. In some embodiments, M is chemically modified to comprise a nucleophilic reactive group capable of conjugating to an electrophilic reactive group on the target peptide or L. In some embodiments, M is chemically modified to comprise an electrophilic reactive group capable of conjugating to a nucleophilic reactive group on the target peptide or L.

[0220] In some embodiments, conjugation can be achieved using organosilanes, such as aminosilanes treated with glutaraldehyde; silanol-based carbonyl diimidazole (CDI) activation; or dendritic polymers. Various dendritic polymers are known in the art, including poly(amidoamine) (PAMAM) dendritic polymers synthesized via a divergent method starting from an ammonia or ethylenediamine initiator core; a subclass of PAMAM dendritic polymers based on a triaminoethyleneimine core; radially layered poly(amidoamine-silicone) dendritic polymers (PAMAMOS), which are reverse monomolecular micelles composed of a hydrophilic, nucleophilic polyamidoamine (PAMAM) interior and a hydrophobic organosilicon (OS) exterior; poly(propyleneimine) (PPI) dendritic polymers, which are typically polyalkylamines with primary amine end groups, while the dendritic polymer interior is composed of many tertiary tripropyleneamines; polyacrylamine (POPAM) dendritic polymers; diaminobutane (DAB) dendritic polymers; amphiphilic dendritic polymers; micellar dendritic polymers, which are water-soluble hyperbranched polyphenylene monomolecular micelles; polylysine dendritic polymers; and dendritic polymers based on a polybenzyl ether hyperbranched backbone.

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

[0222] In some embodiments, click chemistry can be used for conjugation. Click reactions are broad in scope and easy to perform, using only readily available reagents and are insensitive to oxygen and water. In some embodiments, the click reaction is a cycloaddition reaction that forms a triazole group between an alkynyl group and an azide group. In some embodiments, the click reaction uses a copper or ruthenium catalyst. Suitable methods for carrying out 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); Tornee et al., J. Org. Chem. 67:3057 (2002); Manetsch et al., J. Am. Chem. Soc. 126:12809 (2004); Lewis 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 to conjugates such as streptavidin / biotin or avidin / biotin or lectin / carbohydrate was also considered.

[0224] In some embodiments, the targeting peptide and / or M is functionalized with an organic derivatizing agent to include nucleophilic or electrophilic reactive groups. This derivatizing agent is capable of reacting with selected side chains or N-terminal or C-terminal residues of the targeting amino acid on the targeting peptide and with functional groups on M. Reactive groups on the targeting peptide and / or M include, for example, aldehydes, amino groups, esters, thiols, α-haloacetyl groups, maleimide groups, or hydrazide groups. Derivatizing agents include, for example, maleimide benzoyl sulfosuccinimide ester (conjugated via cysteine ​​residues), N-hydroxysuccinimide (conjugated via lysine residues), glutaraldehyde, succinic anhydride, or other agents known in the art. Alternatively, the targeting peptide and / or M can be indirectly interconnected via an intermediate carrier such as a polysaccharide or peptide carrier. Examples of polysaccharide carriers include glycosaminoglycans. Suitable examples of peptide carriers include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and mixed polymers of these amino acids and other amino acids such as serine, thereby imparting the desired solubility to the resulting loaded carrier.

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

[0226] Histidine residues are derivatized by reaction with diethyl pyrocarbonate at pH 5.5–7.0, as this agent is relatively specific to histidine side chains. Bromobenzoyl bromide is also useful; the reaction is preferably carried out in 0.1 M sodium dimethylarsinate at pH 6.0.

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

[0228] Arginine residues are modified by reaction with one or more conventional reagents, such as phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires the reaction to be carried out under basic conditions because the guanidine functional group has a high pKa. Furthermore, these reagents can react with lysine groups as well as the ε-amino group of arginine.

[0229] Tyrosine residues can be specifically modified, with particular interest in introducing spectral markers into tyrosine residues through reactions with aromatic diazo compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyrosine species and 3-nitro derivatives, respectively.

[0230] The carboxyl side group (aspartic or glutamic) is selectively modified by reacting with a carbodiimide (RN=C=N-R'), where R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholino-4-ethyl)carbodiimide or 1-ethyl-3-(4-azacation-4,4-dimethylpentyl)carbodiimide. Furthermore, the aspartic and glutamic residues are converted to asparagine acyl and glutamine acyl residues by reacting with ammonium ions.

[0231] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of serine or threonyl residues, methylation of the α-amino groups of the 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 chemically or enzymatically coupling glycosides to peptides. Sugars can be attached to (a) arginine and histidine, (b) a free carboxyl group, (c) a free thiol group, such as the thiol group of cysteine, (d) a free hydroxyl group, such as the hydroxyl group of serine, threonine, or hydroxyproline, (e) an aromatic residue, such as a residue of tyrosine or tryptophan, or (f) an amide group of glutamine. These methods are described in WO1987 / 05330 and can be found 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 peptide and M are conjugated together by reacting a nucleophilic reactive portion on the targeting peptide with an electrophilic reactive portion on M. In alternative embodiments, the targeting peptide and M are conjugated together by reacting an electrophilic reactive portion on the targeting peptide with a nucleophilic portion on M. In an exemplary embodiment, L is an amide bond formed when an amine (e.g., an ε-amine of a lysine residue) on the targeting peptide reacts with a carboxyl group on M. In alternative embodiments, the targeting peptide and / or M are derivatized with a derivatizing agent prior to conjugation.

[0234] In some embodiments, L is a linker group. In some embodiments, L is a bifunctional linker and contains only two reactive groups before conjugation with the target peptide and M. In embodiments where both the target peptide and M have electrophilic reactive groups, L contains two identical or two different nucleophilic groups (e.g., amine, hydroxyl, thiol) before conjugation with the target peptide and M. In embodiments where both the target peptide and M have nucleophilic reactive groups, L contains two identical or two different electrophilic groups (e.g., carboxyl, activated form of carboxyl, compound with leaving group) before conjugation with the target peptide and M. In embodiments where one of the target peptides or M has a nucleophilic reactive group and the other of the target peptide or M has an electrophilic reactive group, L contains one nucleophilic reactive group and one electrophilic group before conjugation with the target peptide and M.

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

[0236]

[0237] In this embodiment, 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-restricted combinations of nucleophilic and electrophilic groups

[0239]

[0240]

[0241]

[0242]

[0243] In some embodiments, A and B may include olefinic 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 pyridyl dithiols, aryl azides, diaziridine, carbodiimides, and acyl 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 peptide conjugation with the composition, the hydrophobic linking group comprises at least two reactive groups (A and B), as described herein and as follows:

[0245]

[0246] In some embodiments, the hydrophobic linker comprises a maleimide or iodoacetyl group 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 maleimide or iodoacetyl group may be coupled to the thiol moiety on the target peptide or M, and the carboxylic acid or activated carboxylic acid may be coupled to the amine on the target peptide or M. Any coupling agent known to those skilled in the art, such as 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 linker comprises an aliphatic chain having 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 linker group is hydrophilic, such as polyalkylene glycol. Prior to peptide conjugation with the composition, the hydrophilic linker group comprises at least two reactive groups (A and B), as described herein and as follows:

[0249]

[0250] In some embodiments, the connector is polyethylene glycol (PEG). In some embodiments, the PEG has a molecular weight of about 100 Daltons to about 10,000 Daltons, for example, about 500 Daltons to about 5,000 Daltons. In some embodiments, the PEG has a molecular weight of about 10,000 Daltons to about 40,000 Daltons.

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

[0252] In some embodiments, the linker group is composed of an amino acid, dipeptide, tripeptide, or polypeptide, wherein the amino acid, dipeptide, tripeptide, or polypeptide contains at least two activating groups as described herein. In some embodiments, the linker group (L) comprises a portion selected from the group consisting of: amino, ether, thioether, maleimide, 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 length. 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. The chain atoms and linker can be selected according to their expected solubility (hydrophilicity) to provide a more soluble conjugate. In some embodiments, L provides a functional group that can be cleaved by enzymes or other catalysts or by hydrolytic conditions found in target tissues, organs, or cells. In some embodiments, the length of L is long enough to reduce the possibility of steric hindrance.

[0254] In some embodiments, L is stable in biological fluids such as blood or blood fractions. In some embodiments, L remains 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 remains 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 contain functional groups capable of undergoing in vivo hydrolysis. In some exemplary embodiments, L remains stable in serum for at least about 72 hours. Non-limiting examples of functional groups that do 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 biohydrolyzable. In these embodiments, L contains a functional group capable of undergoing biohydrolysis. Non-limiting examples of functional groups capable of undergoing biohydrolysis include esters, acid anhydrides, and thioesters. For example, the following compound is capable of undergoing biohydrolysis because it contains an ester group:

[0257]

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

[0259] In some embodiments, L is metastable in vivo. In these embodiments, L optionally contains functional groups that can be chemically or enzymatically cleaved in vivo over a period of time (e.g., functional groups that are acid-labile, reductively unstable, or enzymatically unstable). In these embodiments, L may contain, for example, an hydrazone moiety, a disulfide moiety, or a cathepsin-cleavable moiety. When L is metastable, and without being bound by any particular theory, the targeted peptide-LM conjugate is stable in the extracellular environment, for example, stable in serum during the aforementioned time period, but unstable in the intracellular environment or under conditions simulating the intracellular environment, thus cleaving upon entry into the cell. In some implementations, when L is metastable, L remains 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, for example, 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 following structure:

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

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

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

[0265] The azide-containing polymer derivatives of the present invention can be prepared by various methods known in the art and / or disclosed herein. In one method described 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 pair with any of a number of suitable counterions, including sodium, potassium, tert-butylammonium, etc.), the polymer backbone having a first end bonded to a first functional group and a second end bonded to a suitable leaving group. The leaving group undergoes nucleophilic substitution and is partially replaced by an azide, thereby providing the desired azide-containing polymer.

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

[0267] As shown, a suitable polymer backbone for use in this invention has the formula X-polymer-LY, wherein the polymer is poly(ethylene glycol), X is a functional group that does not react with an azide group, and Y is a suitable leaving group. Examples of suitable functional groups include, but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl, amine, aminooxy, protected amine, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine and vinylpyridine, and ketones. Examples of suitable leaving groups include, but are not limited to, chloride, bromide, iodide, methanesulfonate, tresylate and toluenesulfonate.

[0268] In another method for preparing the azide-containing polymer derivative of the present invention, an azide-functionalized linker 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 is chemically functionalized and will selectively react with chemical functionalities on the polymer to form an azide-containing polymer derivative product, wherein the azide is separated from the polymer backbone by a linking group.

[0269] An exemplary reaction scheme is shown below:

[0270] X-polymer-Y+N-connector-N=N=N→PG-X-polymer-connector-N=N=N where:

[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 azide functionality but will react effectively and selectively with N functional groups.

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

[0273] In the case of polymeric diamines, more specific examples are shown below, wherein one of the amines is protected by a protecting group moiety such as tert-butyl-Boc, and the resulting monoprotected polymeric diamine reacts with a linker moiety having an azide functional group: BocHN-polymer--NH2+HO2C-(CH2)3-N=N=N.

[0274] In this context, various activators, 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-containing linker. After successful amide bond formation, the resulting N-tert-butyl-Boc protected azide-containing derivative can be directly used to modify bioactive molecules or further processed to mount 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 mount other useful functionalities, such as maleimide groups, activated disulfides, activated esters, etc., to produce valuable heterobifunctional reagents.

[0275] Heterofunctional derivatives are particularly useful when it is desirable to attach different molecules to each end of a polymer. For example, ω-N-amino-N-azido polymers would allow molecules with activated electrophilic groups such as aldehydes, ketones, activated esters, and activated carbonates to attach to one end of the polymer, and molecules with acetylene groups to attach to the other end of the polymer.

[0276] In another embodiment of the invention, A is an aliphatic linker having 1-10 carbon atoms or a substituted aryl ring having 6-14 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 peptides can be linked by linker peptides, wherein the linker peptides are 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 peptide molecules to be linked together. Multiple targeting peptides and modified targeting peptides can be interconnected in a head-to-head configuration via their N-termini by using such linkers or by direct chemical bonding between the respective N-termini of each peptide. For example, two targeting peptides can be linked to form a dimer by chemical bonding between their N-terminal amino groups or modified N-terminal amino groups. Moreover, linker molecules designed to contain multiple chemical functional groups for bonding to the N-terminus of each targeting peptide can be used to link multiple targeting peptides at their respective N-termini. Alternatively, multiple targeting peptides can be linked by bonding between amino acids other than N-terminal or C-terminal amino acids. Examples of covalent bonds that can be used to form dimers and polymers of the targeting peptides described herein include, but are not limited to, disulfide bonds, thiol bonds, or mercaptan bonds. Alternatively, certain enzymes (such as sorting enzymes) can be used to form covalent bonds between the target peptide and the linker (including at the N-terminus of the target peptide).

[0278] Linkers can have a wide range of molecular weights or molecular lengths. Linkers with larger or smaller molecular weights can be used to provide the desired spatial relationship or conformation between the targeting peptide and the linker entity, or between the linker entity and its binding partner (if any). Linkers with longer or shorter molecular lengths can also be used to provide the desired spatial or flexibility between the targeting peptide and the linker entity, or between the linker 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, acylhydrazine, hydroxylamine, or carbonyl-containing moiety at at least a first end of the polymer backbone; and b) at least a second functional group at a second end of the polymer backbone. The second functional group may be the same as or different from the first functional group. In some embodiments, the second functional group does not react with the first functional group. In some embodiments, the present 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 exemplary embodiments, the polymer is linked to a targeting peptide or a modified targeting peptide via a connector. For example, the connector may contain one or two amino acids that bind to a polymer, such as an albumin-binding portion, at one end and to any available site on the peptide backbone at the other end. Other exemplary connectors include hydrophilic connectors, such as chemical moieties containing at least five non-hydrogen atoms, of which 30%–50% are N or O. Further exemplary connectors that can link polymers to targeting peptides or modified targeting peptides are disclosed in US2012 / 0295847 and WO / 2012 / 168430, each of which is incorporated herein by reference in its entirety.

[0281] Optionally, multiple targeting peptides or modified targeting peptide molecules can be linked by a linker peptide, wherein the linker peptide 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 long, or longer, wherein optionally the N-terminus of one targeting peptide is fused to the C-terminus of the linker peptide, and the N-terminus of the linker peptide is fused to the N-terminus of another targeting peptide. Further exemplary linker peptides 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" and "electrophilic reagent" refer to atoms or groups of atoms capable of accepting electron pairs to form covalent bonds. The term "electrophilic group" as used herein includes, but is not limited to, compounds containing halides, carbonyl groups, and epoxides. Common electrophilic reagents can be halides, such as phosgene, 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 "nucleophile" and "nucleophile reagent" refer to atoms or groups of atoms that have electron pairs capable of forming covalent bonds. This type of group can be an ionizable group that reacts as an anionic group. The term "nucleophile" as used herein includes, but is not limited to, hydroxyl groups, primary amines, secondary amines, tertiary amines, and thiols.

[0284] Table 2 provides a variety of starting electrophiles and nucleophiles that can be combined to generate desired functional groups. The information provided is intended to be illustrative and is not limited to the synthetic techniques described herein.

[0285] Table 2: Examples of covalent bonding and its precursors

[0286]

[0287]

[0288] Generally speaking, carbon electrophiles are susceptible to attack by complementary nucleophiles, including carbon nucleophiles. The aggressive nucleophile brings 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, and aryl Grignard reagents, organolithium, organozinc, alkyl-, alkenyl, aryl-, and alkynyl-tin reagents (organostananes), and 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 phosphononium salts, enols, and enolides; these carbon nucleophiles have the advantage of being relatively readily generated 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 formed between the two reagents.

[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, and aminoureas. These non-carbon nucleophiles, when used in combination with carbon electrophiles, typically form heteroatom bonds (CXC), where X is a heteroatom, including but not limited to oxygen, sulfur, or nitrogen.

[0291] In some cases, the polymers used in this invention are terminated at one end with a hydroxyl or methoxy group, i.e., X is hydrogen or CH3 (“methoxyPEG”). Alternatively, the polymers may be terminated with reactive groups, thereby forming bifunctional polymers. Typical reactive groups may include those commonly used to react with functional groups present in 20 common amino acids (including, but not limited to, maleimide groups, activated carbonates (including, but not limited to, p-nitrophenyl esters), activated esters (including, but not limited to, N-hydroxysuccinimide, p-nitrophenyl esters), and aldehydes), as well as functional groups that are inert to the 20 common amino acids but specifically react with complementary functional groups (including, but not limited to, azide groups, alkynyl groups). Note that the other end of the polymer represented by Y in the above formula will be attached directly or indirectly to the target peptide via a naturally occurring or non-naturally encoded amino acid. For example, Y may be an amide, carbamate, or urea linked to an amino group of the peptide (including, but not limited to, the ε-amine or N-terminus of lysine). Alternatively, Y may be a maleimide linked to a thiol group (including, but not limited to, the thiol group of cysteine). Alternatively, Y can be a linkage to residues that are not commonly found among the 20 common amino acids. For example, an azide group on the polymer can react with an alkyne group on the targeting peptide to form a Huisgen[3+2] cycloaddition product. Alternatively, an alkyne group on the polymer can react with an azide group present in the targeting peptide to form a similar product. In some embodiments, where applicable, a strong nucleophile (including, but not limited to, hydrazides, acylhydrazides, hydroxylamines, and aminoureas) can react with an aldehyde or ketone group present in the targeting peptide to form a hydrazone, oxime, or aminourea (which in some cases can be further reduced by treatment with a suitable reducing agent). Alternatively, the strong nucleophile can be incorporated into the targeting peptide via a non-naturally encoded amino acid and used to preferentially react with a ketone or aldehyde group present in the water-soluble polymer.

[0292] Any molecular weight of polymer can be used as needed, including but not limited to approximately 100 Daltons (Da) to 100,000 Da or greater (including but not limited to sometimes 0.1-50 kDa or 10-40 kDa). The molecular weight of the polymer can have a wide range, including but not limited to between approximately 100 Da and approximately 100,000 Da or greater. Polymers can be between approximately 100 Da and approximately 100,000 Da, including but not limited to 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, etc. 0 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the polymer is between about 100 Da and about 50,000 Da. Branched polymers may also be used, including but not limited to polymer molecules with a molecular weight in the range of 1-100 kDa (including but not limited to 1-50 kDa or 5-20 kDa) per chain. The molecular weight of each chain of the branched polymer may be, including but not limited to, between about 1,000 Da and about 100,000 Da or greater. The molecular weight of each chain of the branched polymer can 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, and 50,000 Da. The molecular weights are 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecular weight of each chain of the branched polymer is 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, but are not limited to, the Shearwater Polymers directory and the Nektar Therapeutics directory, which are incorporated herein by reference.

[0293] In some embodiments, the present invention provides polymer derivatives containing azide and acetylene, comprising a water-soluble polymer backbone having an average molecular weight of about 800 Da to about 100,000 Da. The polymer backbone of the water-soluble polymer may 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 the practice of the present invention, and the use of the terms PEG or poly(ethylene glycol) is intended to cover and include all such molecules. The term PEG includes, but is not limited to, any form of polyethylene glycol, including bifunctional PEG, multi-arm PEG, derivatized PEG, branched PEG, branched PEG, pendant PEG (i.e., PEG or related polymers having one or more functional groups pendant over the polymer backbone), or PEG having degradable bonds therein.

[0294] Besides these forms of polymers, polymers can also be prepared using weak or degradable bonds in the main chain. For example, polymers can be prepared by subjecting ester bonds in the polymer main chain to hydrolysis. As shown below, this hydrolysis causes the polymer to break down into lower molecular weight fragments: -polymer-CO2-polymer-+H2O→polymer-CO2H+HO-polymer-

[0295] Many polymers are also suitable for this invention. In some embodiments, a water-soluble polymer backbone having 2 to about 300 ends is particularly useful in this 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 in the range of about 800 Da to about 100,000 Da, and typically about 6,000 Da to about 80,000 Da. The molecular weight of each chain in the polymer backbone can be between approximately 100 Da and approximately 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, The molecular weights are 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 100 Da and about 50,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 100 Da and about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 1,000 Da and about 40,000 Da. 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 one embodiment of the invention, the intact polymer-conjugate prior to hydrolysis exhibits minimal degradation after application, such that the hydrolysis of cleavable bonds effectively controls the slow rate of release of the active target peptide into the bloodstream, in contrast to its enzymatic degradation prior to release into systemic circulation.

[0297] Suitable physiologically cleavable bonds include, but are not limited to, esters, carbonates, carbamates, sulfates, phosphates, acyloxyalkyl ethers, acetals, and ketals. Such conjugates should have physiologically cleavable bonds that are stable after storage and administration. For example, the polymer-linked or modified targeting peptides should maintain their integrity after the manufacture of the final pharmaceutical composition, after dissolution in a suitable delivery medium (if used), and regardless of the route of administration.

[0298] This invention also includes a phosphate-based linker with tunable stability for intracellular delivery of drug conjugates disclosed in US2017 / 0182181, which is incorporated herein by reference. The phosphate-based linker comprises a monophosphate, diphosphate, triphosphate, or tetraphosphate group (phosphate group) covalently linked to the distal end of a linker arm, said linker arm including a tuning element, optional spacer element, and reactive functional group in the direction from distal to proximal. The phosphate group of the phosphate-based linker is capable of conjugating to a payload, and the reactive functional group is capable of conjugating to a cell-specific targeting ligand such as an antibody. The general structure of the phosphate-based linker is: phosphate group - tuning element - optional spacer element - reactive functional group. Phosphate-based linkers conjugated with a payload generally have the following structure: payload - phosphate group - tuning element - optional spacer element - functional reactive group, and when conjugated with a target ligand, they generally have the following structure: payload - phosphate group - tuning element - optional spacer element - target ligand. These phosphate-based linkers exhibit differentiated and tunable stability in the blood relative to the intracellular environment (e.g., lysosomal compartment). The rate at which the phosphate group cleaves in the intracellular environment to release the payload in its native or active form can be influenced by the tuning element structure and further action is mediated by the substitution of the phosphate group and whether the phosphate group is a monophosphate, diphosphate, triphosphate, or tetraphosphate. Furthermore, these phosphate-based linkers provide the ability to construct conjugates such as antibody-drug conjugates, in which the conjugates have a reduced tendency to form aggregates compared to conjugates with the same payload conjugated to antibodies or target ligands using phosphate-based linkers not disclosed herein.

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

[0300] TLR agonist derivatives with a linker containing a hydroxylamine (also known as an aminooxy) group allow for the reaction with a variety of electrophilic groups to form conjugates (including, but not limited to, with PEG or other water-soluble polymers). Like hydrazides, acylhydrazides, and aminoureas, the enhanced nucleophilicity of the aminooxy group enables it to react efficiently and selectively with a wide range 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 reaction with a hydrazine group results in a corresponding hydrazone, the reaction of an aminooxy group with a carbonyl or dicarbonyl group typically produces an oxime, such as, for example, a ketone, aldehyde, or other functional group with similar chemical reactivity. In some embodiments, TLR agonist derivatives having a linker comprising an azide, alkyne, or cycloalkyne allow molecules to be linked via cycloaddition reactions (e.g., 1,3-dipolar cycloaddition, azide-alkyne Huisgen cycloaddition, etc.). (Described in U.S. Patent No. 7,807,619, which is incorporated herein by reference in respect of the extent of the reaction).

[0301] Therefore, in some embodiments, this document describes TLR agonist derivatives having a linker comprising hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, ketone-amine, ketone-yne, and ene-diketone hydroxylamine groups, hydroxylamine-like groups (having similar reactivity to and structurally similar to hydroxylamine groups), masked hydroxylamine groups (which can be readily converted to hydroxylamine groups), or protected hydroxylamine groups (having similar reactivity to hydroxylamine groups after deprotection). In some embodiments, the TLR agonist derivatives having a linker comprise azides, alkynes, or cycloalkynes.

[0302] Such TLR agonist linker derivatives or targeting peptides may be in the form of salts or may be incorporated into non-natural amino acid peptides, polymers, polysaccharides or polynucleotides, and optionally undergo post-translational modifications.

[0303] In some embodiments, the compounds of formulas (I)-(VII) are stable in aqueous solution under weakly acidic conditions for at least one month. In some embodiments, the compounds of formulas (I)-(VII) are stable under weakly acidic conditions for at least two weeks. In some embodiments, the compounds of formulas (I)-(VII) are stable under weakly acidic conditions for at least five days. In some embodiments, such acidic conditions are pH 2 to 8.

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

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

[0306] In some embodiments, the TLR agonist linker derivatives, targeting peptides, TCs, linkers, and reagents described herein, including compounds of formulas (I)-(VII), are stable in aqueous solutions under weakly acidic conditions (including, but not limited to, pH 2-8). In other embodiments, such compounds remain stable under weakly acidic conditions for at least one month. In other embodiments, such compounds remain stable under weakly acidic conditions for at least two weeks. In other embodiments, such compounds remain stable under weakly acidic conditions for at least five days.

[0307] Another aspect of the compositions, methods, techniques, and strategies described herein is a method for studying or using any of the aforementioned “modified or unmodified” non-natural amino acid-targeting peptides. This aspect, by way of example only, includes therapeutic, diagnostic, assay-based, industrial, cosmetic, plant biology, environmental, energy production, consumer, and / or military applications that would benefit from targeting peptides comprising “modified or unmodified” non-natural amino acid peptides or proteins.

[0308] This invention provides TC molecules comprising at least one non-natural amino acid. In some embodiments of the invention, the TC having at least one non-natural amino acid comprises at least one post-translational modification. In one embodiment, the at least one post-translational modification comprises attachment of a molecule comprising a second reactive group to at least one non-natural amino acid comprising a first reactive group using chemical methods known to those skilled in the art suitable for a particular reactive group. The molecule includes, but is not limited to, markers, dyes, linkers, another TC polypeptide, polymers, water-soluble polymers, polyethylene glycol derivatives, photocrosslinking agents, radionuclides, cytotoxic compounds, drugs, affinity markers, photoaffinity markers, reactive compounds, resins, a second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelating agents, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, antisense polynucleotides, sugars, cyclodextrins, repressive ribonucleic acid, biomaterials, nanoparticles, spin. The following are considered compounds or substances: a label, a fluorophore, a metallic moiety, a radioactive moiety, a novel functional group, a group covalently or non-covalently interacting with other molecules, a photocaged moiety, a photochemically excitable moiety, a photoisomerizing moiety, biotin, a biotin derivative, a biotin analog, a moiety doped with heavy atoms, a chemically cleavable group, a photocleavable group, an extended side chain, a carbon-linked sugar, a redox activator, an aminothioic acid, a toxic moiety, an isotope-labeled moiety, a biophysical probe, a phosphorescent group, a chemiluminescent group, an electron-dense group, a magnetic group, an intercalation group, a chromophore, an energy transfer agent, a bioactive agent, a detectable label, a small molecule, a quantum dot, a nano-emitter, a radionuclide, a radioactive emitter, a neutron trapper, or any combination thereof or any other desired compound or substance. For example, the first reactive group is an alkynyl moiety (including, but not limited to, the non-natural amino acid p-propynyloxyphenylalanine, where propynyl is sometimes also referred to as the acetylene moiety), the second reactive group is an azide moiety, and a [3+2] cycloaddition chemistry method is utilized. In another example, the first reactive group is an azide moiety (including, but not limited to, p-azido-L-phenylalanine or pAZ as sometimes referred to in this specification, among non-natural amino acids), and the second reactive group is an alkynyl moiety. In some 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) comprising at least one post-translational modification is used, wherein said at least one post-translational modification comprises a sugar moiety. In some embodiments, the post-translational modification is performed in vivo in eukaryotic cells or in non-eukaryotic cells. A linker, polymer, water-soluble polymer, or other molecule can attach the molecule to the peptide. 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 peptide.

[0309] In some embodiments, the TC protein includes at least one post-translational modification performed in vivo by a host cell, wherein the post-translational modification is generally not performed by another host cell type. In some embodiments, the protein includes at least one post-translational modification performed in vivo by eukaryotic cells, wherein the post-translational modification is generally not performed by non-eukaryotic cells. Examples of post-translational modifications include, but are not limited to, glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitate addition, phosphorylation, glycolipid bonding modification, etc.

[0310] In some embodiments, TC comprises one or more non-naturally encoded amino acids for glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitate addition, phosphorylation, or glycolipid-linked modification of the peptide. In some embodiments, TC comprises one or more non-naturally encoded amino acids for glycosylation of the peptide. In some embodiments, TC comprises one or more naturally encoded amino acids for glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitate addition, phosphorylation, or glycolipid-linked modification of the peptide. In some embodiments, TC comprises one or more naturally encoded amino acids for glycosylation of the peptide.

[0311] In some embodiments, TC comprises the addition and / or substitution of one or more glycosylated non-naturally encoded amino acids of the enhancing peptide. In some embodiments, TC comprises the deletion of one or more glycosylated amino acids of the enhancing peptide. In some embodiments, TC comprises the addition and / or substitution of one or more glycosylated non-naturally encoded amino acids of different amino acids in the enhancing peptide. In some embodiments, TC comprises the deletion of one or more glycosylated amino acids of different amino acids in the enhancing peptide. In some embodiments, TC comprises the addition and / or substitution of one or more glycosylated non-naturally encoded amino acids of different amino acids in the enhancing peptide. In some embodiments, TC comprises the addition and / or substitution of one or more glycosylated non-naturally encoded amino acids of different amino acids in the enhancing peptide. In some embodiments, TC comprises the addition and / or substitution of one or more glycosylated non-naturally encoded amino acids of different amino acids in the enhancing peptide. In some embodiments, TC comprises the addition and / or substitution of one or more glycosylated non-naturally encoded amino acids of different amino acids in the enhancing peptide. In some embodiments, TC comprises the addition and / or substitution of one or more glycosylated non-naturally encoded amino acids of different amino acids in the enhancing peptide.

[0312] In one embodiment, post-translational modification includes attaching an oligosaccharide to asparagine via a GlcNAc-asparagine linker (including, but not limited to, wherein the oligosaccharide comprises (GlcNAc-Man)2-Man-GlcNAc-GlcNAc, etc.). In another embodiment, post-translational modification includes attaching an oligosaccharide (including, but not limited to, Gal-GalNAc, Gal-GlcNAc, etc.) to serine or threonine via a GalNAc-serine, GalNAc-threonine, GlcNAc-serine, or GlcNAc-threonine linker. In some embodiments, the protein or peptide of the present invention may comprise a secretion or localization sequence, an epitope tag, a FLAG tag, a multihistidine 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, pectic acid 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 (bacteriophage), Bgl2 (yeast), and transposon-derived signal sequences bla. Any such sequence can be modified to provide the desired results regarding the peptide, 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 may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more non-natural amino acids. The non-natural amino acids may be the same or different; for example, the protein may have one, two, three, four, five, six, seven, eight, nine, ten or more different sites containing one, two, three, four, five, six, seven, eight, nine, ten or more different non-natural amino acids. In some embodiments, at least one, but less than all, of a specific amino acid present in the native form of the protein is replaced by a non-natural amino acid.

[0314] This invention provides methods and compositions based on TCs containing at least one non-naturally encoded amino acid. Introducing at least one non-naturally encoded amino acid into the TC allows for the application of conjugation chemistry 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 present amino acids. In some embodiments, the TC containing the 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. This invention provides an efficient method for selectively modifying proteins with PEG derivatives or TLR-linker derivatives, the method involving the selective incorporation of non-genetically encoded amino acids into proteins in response to a codon, the non-genetically encoded amino acids including but not limited to those containing functional groups or substituents not found in the 20 naturally incorporated amino acids, the functional groups or substituents including but not limited to ketones, azides, or acetylene moieties; these amino acids are then modified with a suitable reactive PEG derivative. Once incorporated, the amino acid side chains can be modified using chemical methods known to those skilled in the art to suit the specific functional groups or substituents present in the non-naturally encoded amino acid. A variety of known chemical methods are applicable to this invention for incorporating water-soluble polymers into proteins. Such methods include, but are not limited to, Huisgen [3+2] cycloaddition reactions (see, for example, Padwa, A. Comprehensive Organic Synthesis, Vol. 4, (1991) ed., Trost, BM, Pergamon, Oxford, pp. 1069-1109; and Huisgen, R. 1,3-Dipolar Cycloaddition Chemistry, (1984) ed., Padwa, A., Wiley, New York, pp. 1-176), which each have, but are not limited to, acetylene or azide derivatives.

[0315] Because the Huisgen [3+2] cycloaddition method involves cycloaddition rather than nucleophilic substitution, it allows for the modification of proteins with extremely high selectivity. This reaction can be carried out at room temperature in aqueous conditions with excellent regioselectivity (1,4>1,5) by adding a catalytic amount of Cu(I) salt to the reaction mixture. See, for example, Tornee 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 via [3+2] cycloaddition include virtually any molecule having a suitable functional group or substituent, including but not limited to azido or acetylene derivatives. These molecules can be added, respectively, to non-natural amino acids having an acetylene group, including but not limited to p-propynyloxyphenylalanine; or to non-natural amino acids having an azido group, including but not limited to p-azido-phenylalanine.

[0316] The five-membered ring formed by the Huisgen[3+2] cycloaddition is generally irreversible in reducing environments and stable against hydrolysis for extended periods in aqueous environments. Therefore, the active PEG derivatives or TLR-connector derivatives of this 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] This invention also provides water-soluble and hydrolytically stable derivatives of PEG derivatives or TLR linker derivatives, and related hydrophilic polymers having one or more acetylene or azide moieties. PEG polymer derivatives containing an acetylene moiety are highly selective for coupling with an azide moiety in response to the selective introduction of a select codon into the protein. Similarly, PEG polymer derivatives containing an azide moiety are highly selective for coupling with an acetylene moiety in response to the selective introduction of a select codon into the protein. More specifically, the azide moiety includes, but is not limited to, alkyl azides, aryl azides, and derivatives of these azides. Derivatives of alkyl and aryl azides may include other substituents, provided that the acetylene-specific reactivity is maintained. The acetylene moiety includes alkyl and aryl acetylenes and their respective derivatives. Derivatives of alkyl and aryl acetylenes may include other substituents, provided that the azide-specific reactivity is maintained.

[0318] This invention provides conjugates of substances having multiple functional groups, substituents, or portions with other substances, including but not limited to: markers; dyes; polymers; water-soluble polymers; polyethylene glycol derivatives; photocrosslinking agents; radionuclides; cytotoxic compounds; drugs; affinity markers; photoaffinity markers; reactive compounds; resins; a second protein or polypeptide or polypeptide analog; antibodies or antibody fragments; metal chelators; cofactors; fatty acids; carbohydrates; polynucleotides; DNA; RNA; antisense polynucleotides; sugars; water-soluble dendritic polymers; cyclodextrins; inhibitory ribonucleic acid; biomaterials; nanoparticles; spin markers; fluorophores; metal-containing portions; radioactive portions; and new functional groups. Groups; groups that interact covalently or non-covalently with other molecules; light-shielding moieties; photochemically excitable moieties; photoisomerizing moieties; biotin; biotin derivatives; biotin analogs; moieties incorporating heavy atoms; chemically cleavable groups; photocleavable groups; extended side chains; carbon-linked sugars; redox activators; aminothioic acids; toxic moieties; isotope-labeled moieties; biophysical probes; phosphorescent groups; chemiluminescent groups; electron-dense groups; magnetic groups; intercalation groups; chromophores; energy transfer agents; bioactive agents; detectable markers; small molecules; quantum dots; nanoemitters; radionuclides; radioactive emitters; neutron trappers; or any combination of the above or any other desired compounds or substances. The invention also includes conjugates of substances having an azide or acetylene moiety with PEG polymer derivatives having the corresponding acetylene or azide moiety. For example, PEG polymers containing an azide moiety can be coupled to bioactive molecules in proteins at positions containing non-genetically encoded amino acids with acetylene functional groups. The coupling of PEG and bioactive molecules includes, but is not limited to, Huisgen[3+2] cycloaddition products.

[0319] It is well established in the art that PEG can be used to modify the surfaces of biomaterials (see, for example, U.S. Patent 6,610,281; Mehvar, R., J. Pharm Pharm Sci., 3(1):125-136 (2000), which is incorporated herein by reference). The invention also includes biomaterials comprising a surface having one or more reactive azide or acetylene sites and one or more of the azide- or acetylene-containing polymers of the invention, said polymers being coupled to said surface via Huisgen[3+2] cycloaddition bonds. Biomaterials and other substances can also be coupled to azide- or acetylene-activated polymer derivatives via bonds other than azide or acetylene bonds, such as by bonds comprising carboxylic acid, amine, alcohol, or thiol moieties, such that the azide or acetylene moieties are available for subsequent reactions.

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

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

[0322] More specifically, in the case of acetylene-containing PEG derivatives, water-soluble polymers having at least one active hydroxyl moiety undergo a reaction that replaces a halogen or other activated leaving group from the acetylene-containing precursor. Alternatively, water-soluble polymers having at least one active nucleophilic or electrophilic moiety undergo a reaction with a linker having acetylene at one end, such that a covalent bond is formed between the PEG polymer and the linker, and the acetylene moiety is positioned at the end of the polymer. The use of halogen moieties, activated leaving groups, nucleophilic and electrophilic moieties, and the preparation and use of PEG derivatives or TLR-linker derivatives in the context of organic synthesis are well established by those skilled in the art.

[0323] This 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-connector derivatives, connectors, or another TC polypeptide. Azide- and acetylene-containing PEG derivatives or TLR-connector derivatives can be used to modify surface and molecular properties, where biocompatibility, stability, solubility, and non-immunogenicity are important, while providing a more selective means of attaching PEG derivatives or TLR-connector derivatives to proteins than previously known in the art.

[0324] General recombinant nucleic acid methods used in conjunction with the present invention

[0325] In many embodiments of the invention, recombinant methods are used to isolate, clone, and frequently modify the nucleic acid encoding the target polypeptide of interest in TC. Such embodiments are used, including but not limited to, in the production of protein expression or in the generation of variants, derivatives, expression cassettes, or other sequences of the target polypeptide derived from TC. In some embodiments, the sequence encoding the polypeptide of the invention is operatively linked to a heterologous promoter.

[0326] Nucleotide sequences encoding target peptides containing non-naturally encoded amino acids (TCs) can be synthesized based on the amino acid sequence of the parent peptide, and then said nucleotide sequence can be modified to achieve the introduction (i.e., incorporation or substitution) or removal (i.e., deletion or substitution) of relevant amino acid residues. The nucleotide sequence can be suitably modified by site-directed mutagenesis according to conventional methods. Alternatively, the nucleotide sequence can be prepared by chemical synthesis (including, but not limited to, using an oligonucleotide synthesizer), wherein the oligonucleotide is designed based on the amino acid sequence of the desired peptide, and preferably those codons favorable in the host cell in which the recombinant peptide will be generated are selected. For example, several small oligonucleotides encoding multiple parts of the desired peptide can be synthesized and assembled by PCR, ligation, or chain reaction. See, for example, Barany et al., Proc. Natl. Acad. Sci. 88:189-193 (1991); U.S. Patent 6,521,427, which is incorporated herein by reference.

[0327] This invention utilizes conventional techniques in the field of recombinant genetics. Basic texts disclosing the general methods used in this 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 (edited by Ausubel et al., 1994).

[0328] This invention also relates to eukaryotic host cells, non-eukaryotic host cells, and organisms for in vivo incorporation of non-natural amino acids via orthogonal tRNA / RS pairs. Genetic engineering (including, but not limited to, transformation, transduction, or transfection) of host cells using the polynucleotides or constructs of this invention, said constructs comprising the polynucleotides of this invention, including but not limited to the vectors of this invention, which may 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 this invention. These methods include: fusion of recipient cells with DNA-containing bacterial protoplasts, electroporation, particle bombardment, and infection with viral vectors (discussed further below), etc. Bacterial cells can be used to amplify the quantity of plasmids containing the DNA constructs of this invention. Bacteria are grown to the logarithmic phase, and plasmids within the bacteria can be isolated using a variety of methods known in the art (see, for example, Sambrook). Additionally, kits for purifying plasmids from bacteria are commercially available (see, for example, EasyPrep). TM FlexiPrep TM Both are from Pharmacia Biotech; StrataClean is from Stratagene. TM ; and QIAprep from Qiagen TMThe isolated and purified plasmids are then further manipulated to produce other plasmids for transfecting cells or incorporating into relevant 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. Vectors optionally contain a universal expression cassette containing at least one independent terminator sequence, sequences (including but not limited to shuttle vectors) that allow the cassette to replicate in eukaryotes or prokaryotes or both, and selection markers for prokaryotic and eukaryotic systems. Vectors are adapted 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.). For example, the ATCC provides a catalogue of bacteria and phages that can be used for cloning, such as the ATCC Catalogue of Bacteria and Phages (edited by Gherna et al., 1992), published by the ATCC. Additional fundamental procedures and basic theoretical considerations for sequencing, cloning, and other aspects of molecular biology are also found in Recombinant DNA Second Edition, Scientific American Books (NY), by Watson et al. (1992). Furthermore, virtually any nucleic acid (and almost any labeled nucleic acid, whether standard or non-standard) is available by custom or standard order from a variety of commercial sources, such as Midland Certified Reagent Company (Midland, TX, available at mcrc.com), The Great American Gene Company (Ramona, CA, available at genco.com), ExpressGen Inc. (Chicago, IL, available at expressgen.com), Operan Technologies Inc. (Alameda, CA), and many others.

[0330] Choose a codon

[0331] The selected codons of this invention extend the genetic codon framework of the protein biosynthetic machinery. For example, selected codons include, but are not limited to, unique three-base codons; nonsense codons, such as stop codons, including but not limited to the amber codon (UAG), ochre codon, or opal codon (UGA); non-natural codons; codons of four or more bases; rare codons, etc. It will be apparent to those skilled in the art that a wide range of selected 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 TC.

[0332] In one embodiment, the method involves the use of a select codon, which is a stop codon, for in vivo incorporation of one or more non-natural amino acids. For example, an O-tRNA that recognizes a stop codon (including, but not limited to, UAG) is generated and aminoacylated by an O-RS containing the desired non-natural amino acid. This O-tRNA is not recognized by naturally occurring host aminoacyl-tRNA synthetases. Stop codons, including, but not limited to, TAG, can be introduced at sites of interest in the polypeptide of interest using conventional site-directed mutagenesis. See, for example, Sayers, JR et al. (1988), 5'-3' Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res, 16:791-802. When the O-RS, O-tRNA, and nucleic acid encoding the polypeptide of interest are combined in vivo, a non-natural amino acid is incorporated in response to the UAG codon, resulting in a polypeptide containing the non-natural amino acid at the designated position.

[0333] Non-natural amino acids can be incorporated into the body without significantly disrupting eukaryotic host cells. For example, since the repression efficiency of the UAG codon depends on the competition between O-tRNA (including but not limited to amber repressor tRNA) and eukaryotic release factors (including but not limited to eRF) (which bind to the stop codon and initiate the release of growth peptides from the ribosome), the repression efficiency can be modulated by (including but not limited to) increasing the expression levels of O-tRNA and / or repressor tRNA.

[0334] Rare codons can also encode non-natural amino acids. For example, the rare arginine codon AGG has been shown to efficiently insert Ala into synthetic tRNA with alanine acylation when the concentration of arginine is reduced in in vitro protein synthesis reactions. See, for example, Ma et al., Biochemistry, 32:7939 (1993). In this case, the synthetic tRNA competes with naturally occurring tRNAArg, which is a minor species in E. coli. Some organisms do not use all triplet codons. The unspecified codon AGA in Micrococcus luteus has been used to insert amino acids into in vitro transcribed / translated extracts. See, for example, Kowal and Oliver, Nucl. Acid. Res., 25:4685 (1997). Components of the present invention can be generated to utilize these rare codons in vivo.

[0335] Codon selection also includes extended codons, including but not limited to codons with four or more bases, such as four, five, six or more bases. Examples of tetrabase codons include, but are not limited to, AGGA, CUAG, UAGA, CCCU, etc. Examples of pentabase codons include, but are not limited to, AGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC, etc. Features of the invention include the use of extended codons based on frameshift suppression. Four or more base codons can insert, but are not limited to, one or more non-natural amino acids into the same protein. For example, in the presence of mutated O-tRNA (including, but not limited to, special frameshift-suppressed tRNAs with an anticodon loop, such as an anticodon loop of at least 8-10 nt), four or more base codons are read as a single amino acid. In other embodiments, the anticodon loop can be decoded, including but not limited to, codons with at least four bases, at least five bases, or at least six bases or more. Since there are 256 possible tetrabase codons, codons with 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, tetrabasic codons have been used to incorporate non-natural amino acids into proteins using in vitro biosynthetic methods. See, for example, Ma et al., (1993) Biochemistry, 32:7939; and Hohsaka et al., (1999) J. Am. Chem. Soc., 121:34. Using CGGG and AGGU, NBD derivatives of 2-naphthylalanine and lysine were simultaneously incorporated into streptavidin in vitro via two chemically acylated frameshift repressor tRNAs. See, for example, Hohsaka et al., (1999) J. Am. Chem. Soc., 121:12194. In in vivo studies, Moore et al. examined the ability of tRNALeu derivatives with the NCUA anticodon to repress the UAGN codon (N can be U, A, G, or C) and found that the tetrad UAGA could be decoded by tRNALeu with the UCUA anticodon with efficiencies ranging from 13% to 26%, with almost no decoding in the 0 or -1 boxes. 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 this invention to reduce missense reads and frameshift suppression at other unwanted sites.

[0337] For a given system, the codon selection may also include one of the natural three-base codons, where the endogenous system does not use (or rarely uses) the natural three-base codon. For example, this includes systems lacking tRNAs that recognize the natural three-base codon, and / or systems where the three-base codon is a rare codon.

[0338] The codons may optionally include unnatural base pairs. 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 properties of the third base pair include: stable and selective base pairing, efficient enzymatic incorporation into DNA with high fidelity via polymerase, and efficient continuation primer extension after the synthesis of the newly formed 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 phosphorylated to form the corresponding triphosphates. Furthermore, the added genetic information is stable and not destroyed by cellular enzymes. Previous efforts by Benner and others have utilized hydrogen bonding patterns different from those in canonical Watson-Crick pairs, the most notable example being the iso-C:iso-G pair. See, for example, Switcher 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 to some extent with the natural bases and cannot be enzymatically replicated. Kool and colleagues demonstrated that hydrophobic stacking interactions between bases can drive base pair formation instead of hydrogen bonds. See Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and Guckian and Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. To develop non-natural base pairs that meet all the above requirements, Schultz, Romesberg, and colleagues systematically synthesized and investigated a series of non-natural hydrophobic base pairs. They found that the PICS:PICS self-pair is more stable than the natural base pairs and can be efficiently incorporated into DNA via the Klenow fragment (KF) of E. 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 are used as chain terminators for further replication. Recently, mutant DNA polymerases have been evolved that can be used to replicate the PICS self-pair. Additionally, the 7AI self-pair can be replicated. See, for example, Tae et al., (2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py, has also been developed, which forms a stable pair upon binding to Cu(II). See, Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Because extended codons and non-natural codons are inherently orthogonal to natural codons, the method of this invention can utilize this property to generate orthogonal tRNAs targeting them.

[0340] Translation bypass systems can also be used to incorporate non-natural amino acids into desired polypeptides. In a translation bypass system, a large sequence is incorporated into a gene but not translated into a protein. This sequence contains structures that act as cues to induce the ribosome to skip the sequence and resume translation downstream of the insertion.

[0341] Nucleic acid molecules encoding targeting peptides of interest, such as TC, can be readily mutated to introduce cysteine ​​residues at any desired location on the peptide. Cysteine ​​residues are widely used to introduce active molecules, water-soluble polymers, proteins, or a variety of other molecules onto proteins of interest. Suitable methods for incorporating cysteine ​​residues into desired locations on peptides are known to those skilled in the art, such as the methods described in U.S. Patent No. 6,608,183 (which is incorporated herein by reference) and standard mutagenesis techniques.

[0342] III. Non-naturally encoded amino acids

[0343] Many non-naturally encoded amino acids are suitable for use in this invention. Any number of non-naturally encoded amino acids can be introduced into the TC. Generally, the introduced non-naturally encoded amino acids are substantially chemically inert 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, ketone, aldehyde, and aminooxy groups) that react efficiently and selectively with functional groups not found in the 20 common amino acids to form stable conjugates. For example, a targeted polypeptide containing a non-naturally encoded amino acid with an azide functional group can react with polymers (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 α-amino acids is shown below (Formula I):

[0345] I

[0346]

[0347] Non-naturally encoded amino acids are generally any structure having the chemical formulas listed above and are applicable to this invention, wherein the R group is any substituent other than those used in the twenty natural amino acids. Because the non-naturally encoded amino acids of this invention generally differ from natural amino acids only in their side-chain structure, they form amide bonds with other amino acids (including, but not limited to, naturally or non-naturally encoded ones) in the same manner as they form 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-, azide-, hydroxyl-, hydrazine, cyano-, halo-, acylhydrazine, alkenyl, alkynyl, ether, thiol, seleno-, sulfonyl-, borate, borate, boronic acid, phosphate, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino, etc., or any combination thereof. Other non-natural amino acids of interest applicable to this invention include, but are not limited to, amino acids containing photoactivated crosslinking agents, spin-labeled amino acids, fluorescent amino acids, metal-bound amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that interact covalently or non-covalently with other molecules, photo-masked amino acids and / or photoisomerized amino acids, amino acids containing biotin or biotin analogs, glycosylated amino acids such as sugar-substituted serine, other carbohydrate-modified amino acids, ketone-containing amino acids, amino acids containing polyethylene glycol or polyether, 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 with more than about 5 or more than about 10 carbon atoms), carbon-linked sugar-containing amino acids, redox-active amino acids, amino acids containing aminothioic acids, and amino acids containing one or more toxic moieties.

[0348] Exemplary non-naturally encoded amino acids applicable to this invention and usable in reactions with water-soluble polymers include, but are not limited to, those having reactive groups such as carbonyl, aminooxy, hydrazine, acylhydrazine, aminourea, azide, and alkyne. In some embodiments, the non-naturally encoded amino acid comprises a sugar moiety. 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 those in which the naturally occurring N- or O-bond between the amino acid and the sugar is replaced by a covalent bond (including, but not limited to, alkenes, oximes, thioethers, amides, etc.) not typically found in nature. Examples of such amino acids also include sugars not typically found in naturally occurring proteins, such as 2-deoxy-glucose, 2-deoxy-galactose, etc.

[0349] Many of the non-naturally encoded amino acids described 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 may optionally be synthesized as described herein or using standard methods known to those skilled in the art. For organic synthesis techniques, see, for example, Fessendon and Fessendon, Organic Chemistry (1982, 2nd edition, Willard Grant Press, Boston Mass.); March, Advanced Organic Chemistry (3rd edition, 1985, Wiley and Sons, New York); and Carey and Sundberg, 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, the non-natural amino acids suitable for use in this invention optionally also comprise modified main-chain structures, including but not limited to those shown in Formulas II and III:

[0350]

[0351] Wherein Z typically includes OH, NH2, SH, NH-R′, or SR′; X and Y may be the same or different, typically including 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 non-natural amino acids having Formula I, along with hydrogen. For example, the non-natural amino acids of the present invention optionally contain substitutions in the amino or carboxyl groups as shown in Formulas II and III. Non-natural amino acids of this type include, but are not limited to, α-hydroxy acids, α-thio acids, α-aminothiocarbamates, including but not limited to, side chains or non-natural side chains corresponding to the twenty common natural amino acids. 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, GABA, etc. Other structural substitutes include cyclic amino acids, such as proline analogs and 3-, 4-, 6-, 7-, 8-, and 9-membered cyclic proline analogs, β- and γ-amino acids, such as substituted β-alanine and γ-aminobutyric acid.

[0352] Many non-natural amino acids are based on natural amino acids, such as tyrosine, glutamine, and phenylalanine, and are suitable for use in this invention. Tyrosine analogues 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 groups (including but not limited to acetyl groups), benzoyl groups, amino groups, hydrazine, hydroxyl groups, thiol groups, carboxyl groups, isopropyl groups, methyl groups, and C6-C groups. 20Straight-chain or branched hydrocarbons, saturated or unsaturated hydrocarbons, O-methyl, polyether, nitro, alkynyl, etc. Additionally, multi-substituted aromatic rings are also considered. Glutamine analogs applicable to 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 applicable to the present invention include, but are not limited to, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine, wherein the substituents include (but are not limited to) hydroxyl, methoxy, methyl, allyl, aldehyde, azide, iodo, bromo, ketone (including but not limited to acetyl), benzoyl, alkynyl, etc. Specific examples of non-natural amino acids applicable to this 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-propynyloxy-phenylalanine, etc. Examples of structures of various non-natural amino acids applicable to the present invention are provided, for example, in WO 2002 / 085923 entitled “In vivo incorporation of unnatural amino acids”. For further methionine analogues, see also Kiick et al., (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudingerligation, PNAS 99:19-24, which is incorporated herein by reference. International application PCT / US06 / 47822 entitled “Compositions Containing, Methods Involving, and Uses of Non-natural Amino Acids and Polypeptides” describes the reductive alkylation and reductive amination of aromatic amine moieties (including, but not limited to, p-amino-phenylalanine), which is incorporated herein by reference.

[0353] In another embodiment of the invention, a TC polypeptide having one or more non-naturally encoded amino acids is covalently modified. Selective chemical reactions orthogonal to different functionalities of biological systems are considered important tools in chemical biology. As relatively new to synthetic chemistry libraries, these bioorthogonal reactions inspire new strategies for compound library synthesis, protein engineering, functional proteomics, and cellular surface chemical remodeling. Azides have played a prominent role as unique chemical handles for bioconjugation. Staudinger linkages have been used with phosphine to tag azidos in glycoconjugates introduced into cells via metabolism. Staudinger linkages can be performed in living animals without physiological harm; however, Staudinger reactions are not without their risks. Required phosphines are susceptible to aerobic oxidation, and optimizing them to improve water solubility and increase reaction rates has proven to be a comprehensive challenge.

[0354] An alternative mode of bioorthogonal reactivity for azido groups is the [3+2] cycloaddition reaction with alkynes described by Huisgen. In its classical form, this reaction has limited applicability in biological systems due to the high temperature (or pressure) requirement for a reasonable reaction rate. Sharpless and colleagues overcame this obstacle by developing a copper(I) catalytic form known as “click chemistry,” which proceeds readily at physiological temperatures and in functionally rich biological environments. This discovery enables 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 excluding applications where cells must remain viable. Catalyst-free Huisgen cycloadditions of alkynes activated by electron-withdrawing substituents have been reported to occur at ambient temperatures. However, these compounds undergo Michael reactions with biological nucleophiles.

[0355] In one embodiment, a composition is provided comprising a targeting peptide of TC containing a non-natural amino acid (such as p-(propynyloxy)-phenylalanine). Various compositions containing p-(propynyloxy)-phenylalanine are also provided, and these include, but are not limited to, proteins and / or cells. On one hand, the composition containing the non-natural amino acid p-(propynyloxy)-phenylalanine further comprises orthogonal tRNA. The non-natural amino acid may be bonded to the orthogonal tRNA (including, but not limited to, covalent bonding), 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 of the orthogonal tRNA, etc.

[0356] The chemical moieties that can be incorporated into proteins via non-natural amino acids offer a variety of advantages and manipulability for proteins. For example, the unique reactivity of ketone functional groups allows for selective in vivo and in vitro modification of proteins using any of a number of hydrazine- or hydroxylamine-containing reagents. Heavy-atom non-natural amino acids, for instance, can be used for phase determination of X-ray structural data. The site-specific introduction of heavy atoms using non-natural amino acids also provides selectivity and flexibility in selecting the location of heavy atoms. 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 temporal control of the time to provide photoreactive groups through arbitrary excitation. In one instance, (including, but not limited to) using nuclear magnetic resonance and vibrational spectroscopy, the methyl group of a non-natural amino acid can be replaced with an isotopically labeled group (including, but not limited to, methyl) as a probe for local structure and kinetics. For example, alkynyl or azide functional groups allow molecules to selectively modify proteins via [3+2] cycloaddition reactions.

[0357] The non-natural amino acid incorporated into the amino terminus of the polypeptide can consist of an R group and a second reactive group, wherein the R group is any substituent other than those used in the twenty natural amino acids, and the second reactive group is different from the NH2 group typically found in α-amino acids. A similar non-natural amino acid can be incorporated into the C-terminus, wherein the second reactive group is different from the COOH group typically found in α-amino acids.

[0358] The non-natural amino acids of this invention can be selected or designed to provide additional features not available in the twenty natural amino acids. For example, non-natural amino acids may optionally be designed or selected to, for instance, modify the biological properties of the protein in which they are incorporated. For example, by including non-natural amino acids in a protein, the following properties may optionally be modified: toxicity, biodistribution, solubility, stability (e.g., resistance to heat, hydrolysis, oxidation, 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.

[0359] In some embodiments, the present invention provides TC linked to a water-soluble polymer, 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 Publications 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 entirety. Non-naturally encoded amino acids are also described in U.S. Patents 7,083,970 and 7,045,337, which are incorporated herein by reference in their entirety.

[0360] Some embodiments of the present invention utilize TC polypeptides substituted with acetylphenylalanine amino acids at one or more positions. The synthesis of 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 amino acids can be prepared similarly by those skilled in the art. Furthermore, non-limiting exemplary synthesis of non-natural amino acids included herein is proposed in U.S. Patent No. 7,083,970, which is incorporated herein by reference in its entirety.

[0361] Amino acids with electrophilic reactive groups allow molecules to be linked via a variety of reactions, including nucleophilic addition. Such electrophilic reactive groups include carbonyl groups (including ketones and dicarbonyls), carbonyl-like groups (which have similar reactivity to carbonyl groups (including ketones and dicarbonyls) and are structurally similar to carbonyl groups), masked carbonyl groups (which can be readily converted to carbonyl groups (including ketones and dicarbonyls)), or protected carbonyl groups (which, upon deprotection, have similar reactivity to carbonyl groups (including ketones and dicarbonyls)). Such amino acids include those with the structure of formula (IV):

[0362]

[0363] in:

[0364] A is optional, and when present, is a lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenyl, substituted lower alkenyl, alynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkylene arylene, substituted alkylene arylene, arylalkylene or substituted arylalkylene;

[0365] B is optional and, when present, is a connector selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenyl, substituted lower alkenyl, 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')-, wherein each R' is independently H, an alkyl group or a substituted alkyl group;

[0366] J is

[0367] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0368] Each R” is independently hydrogen, alkyl, substituted alkyl, or protecting group, or when there is more than one R” group, the two R” may optionally form a heterocyclic alkyl group;

[0369] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0370] R2 is optional, and when present, it is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0371] R3 and R4 are each independently hydrogen, halogen, lower alkyl or substituted lower alkyl, or R3 and R4 or the two R3 groups optionally form cycloalkyl or heterocycloalkyl.

[0372] Alternatively, the -ABJR groups together form a bicyclic or tricyclic cycloalkyl or heterocyclic alkyl group comprising at least one carbonyl group (including dicarbonyl), a protected carbonyl group (including protected dicarbonyl), or a masked carbonyl group (including masked dicarbonyl).

[0373] Alternatively, the -JR groups together form a monocyclic or bicyclic cycloalkyl or heterocyclic alkyl group comprising at least one carbonyl group (including dicarbonyl), a protected carbonyl group (including protected dicarbonyl), or a masked carbonyl group (including masked dicarbonyl).

[0374] The conditions are that B exists when A is phenylene and each R3 is H; and B is not -NHC(O)(CH2CH2)- when A is -(CH2)4- and each R3 is H; and R is not methyl when A and B are absent and each R3 is H.

[0375] Additionally, structures with formula (V) are included:

[0376]

[0377] in:

[0378] A is optional, and when present, is a lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenyl, substituted lower alkenyl, alynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkylene arylene, substituted alkylene arylene, arylalkylene or substituted arylalkylene;

[0379] B is optional and, when present, is a connector selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenyl, substituted lower alkenyl, 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')-, wherein each R' is independently H, an alkyl group or a substituted alkyl group;

[0380] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0381] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0382] R2 is optional, and when present, it is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0383] The conditions are that B exists when A is phenylene; and B is not -NHC(O)(CH2CH2)- when A is -(CH2)4-; and R is not methyl when neither A nor B exists.

[0384] In addition, amino acids having the structure of formula (VI) are included:

[0385]

[0386] in:

[0387] B is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenyl, substituted lower alkenyl, 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')-, wherein each R' is independently H, an alkyl group or a substituted alkyl group;

[0388] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0389] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0390] R2 is optional, and when present, it is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0391] Each R a Independently selected from the group consisting of: H, halogens, alkyl groups, substituted alkyl groups, -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, an alkyl group, or a substituted alkyl group.

[0392] In addition, it includes the following amino acids:

[0393]

[0394] Such compounds are optionally amino-protected, carboxyl-protected, or salts thereof. Additionally, any of the following non-natural amino acids may be incorporated into non-natural amino acid polypeptides.

[0395] In addition, the following amino acids having the structure of formula (VII) are included:

[0396]

[0397] in

[0398] B is optional and, when present, is a connector selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenyl, substituted lower alkenyl, 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')-, wherein each R' is independently H, an alkyl group or a substituted alkyl group;

[0399] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0400] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0401] R2 is optional, and when present, it is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0402] Each R a Independently selected from the group consisting of: H, halogens, alkyl groups, substituted alkyl groups, -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;

[0403] The condition is that when A is -(CH2)4-, B is not -NHC(O)(CH2CH2)-.

[0404] In addition, it includes the following amino acids:

[0405]

[0406] Such compounds are optionally amino-protected, optionally carboxyl-protected, optionally both amino-protected and carboxyl-protected, or salts thereof. Additionally, these non-natural amino acids and any of the following non-natural amino acids may be incorporated into non-natural amino acid polypeptides.

[0407] In addition, the following amino acids having the structure of formula (VIII) are included:

[0408]

[0409] Wherein A is optional, and when present, is a lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenyl, substituted lower alkenyl, alynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkylene aryl, substituted alkylene aryl, arylalkylene or substituted arylalkylene;

[0410] B is optional and, when present, is a connector selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenyl, substituted lower alkenyl, 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')-, wherein each R' is independently H, an alkyl group or a substituted alkyl group;

[0411] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0412] R2 is optional and, when present, is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0413] In addition, the following amino acids having the structure of formula (IX) are included:

[0414]

[0415] B is optional and, when present, is a connector selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenyl, substituted lower alkenyl, 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')-, wherein each R' is independently H, an alkyl group or a substituted alkyl group;

[0416] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0417] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0418] R2 is optional, and when present, it is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0419] Each R a Independently selected from the group consisting of: H, halogens, alkyl groups, substituted alkyl groups, -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, an alkyl group, or a substituted alkyl group.

[0420] In addition, it includes the following amino acids:

[0421] Such compounds are optionally amino-protected, optionally carboxyl-protected, optionally both amino-protected and carboxyl-protected, or salts thereof. Additionally, these non-natural amino acids and any of the following non-natural amino acids may be incorporated into non-natural amino acid polypeptides.

[0422] In addition, the following amino acids having the structure of formula (X) are included:

[0423]

[0424] Wherein B is optional, and when present, is a connector selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenyl, substituted lower alkenyl, 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')-, wherein each R' is independently H, an alkyl group or a substituted alkyl group;

[0425] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0426] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0427] R2 is optional, and when present, it is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0428] Each R a Independently selected from the group consisting of: H, halogens, alkyl groups, substituted alkyl groups, -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.

[0429] In addition, it includes the following amino acids:

[0430]

[0431] Such compounds are optionally amino-protected, optionally carboxyl-protected, optionally both amino-protected and carboxyl-protected, or salts thereof. Additionally, these non-natural amino acids and any of the following non-natural amino acids may be incorporated into non-natural amino acid polypeptides.

[0432] In addition to monocarbonyl structures, the non-natural amino acids described herein may include groups such as dicarbonyl, dicarbonyl-like groups, masked dicarbonyl, and protected dicarbonyl.

[0433] For example, amino acids having the structure of formula (XI) include the following:

[0434]

[0435] Wherein A is optional, and when present, is a lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenyl, substituted lower alkenyl, alynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkylene aryl, substituted alkylene aryl, arylalkylene or substituted arylalkylene;

[0436] B is optional and, when present, is a connector selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenyl, substituted lower alkenyl, 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')-, wherein each R' is independently H, an alkyl group or a substituted alkyl group;

[0437] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0438] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0439] R2 is optional and, when present, is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0440] In addition, the following amino acids having the structure of formula (XII) are included:

[0441]

[0442] B is optional and, when present, is a connector selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenyl, substituted lower alkenyl, 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')-, wherein each R' is independently H, an alkyl group or a substituted alkyl group;

[0443] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0444] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0445] R2 is optional, and when present, it is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0446] Each R a Independently selected from the group consisting of: H, halogens, alkyl groups, substituted alkyl groups, -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, an alkyl group, or a substituted alkyl group.

[0447] In addition, it includes the following amino acids:

[0448] Such compounds are optionally amino-protected, optionally carboxyl-protected, optionally both amino-protected and carboxyl-protected, or salts thereof. Additionally, these non-natural amino acids and any of the following non-natural amino acids may be incorporated into non-natural amino acid polypeptides.

[0449] In addition, the following amino acids having the structure of formula (XIII) are included:

[0450]

[0451] Wherein B is optional, and when present, is a connector selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenyl, substituted lower alkenyl, 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')-, wherein each R' is independently H, an alkyl group or a substituted alkyl group;

[0452] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0453] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0454] R2 is optional, and when present, it is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0455] Each R aIndependently selected from the group consisting of: H, halogens, alkyl groups, substituted alkyl groups, -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.

[0456] In addition, it includes the following amino acids:

[0457] Such compounds are optionally amino-protected, optionally carboxyl-protected, optionally both amino-protected and carboxyl-protected, or salts thereof. Additionally, these non-natural amino acids and any of the following non-natural amino acids may be incorporated into non-natural amino acid polypeptides.

[0458] In addition, the following amino acids having the structure of formula (XIV) are included:

[0459]

[0460] in:

[0461] A is optional, and when present, is a lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenyl, substituted lower alkenyl, alynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkylene arylene, substituted alkylene arylene, arylalkylene or substituted arylalkylene;

[0462] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0463] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0464] R2 is optional, and when present, it is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0465] 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.

[0466] In addition, the following amino acids having the structure (XIV-A) are included:

[0467]

[0468] in:

[0469] A is optional, and when present, is a lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenyl, substituted lower alkenyl, alynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkylene arylene, substituted alkylene arylene, arylalkylene or substituted arylalkylene;

[0470] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0471] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0472] R2 is optional, and when present, it is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0473] L is an alkylene, a substituted alkylene, N(R') (alkylene) or N(R') (substituted alkylene), where R' is H, an alkyl, a substituted alkyl, a cycloalkyl or a substituted cycloalkyl.

[0474] In addition, the following amino acids having the structure (XIV-B) are included:

[0475]

[0476] in:

[0477] A is optional, and when present, is a lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenyl, substituted lower alkenyl, alynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkylene arylene, substituted alkylene arylene, arylalkylene or substituted arylalkylene;

[0478] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0479] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0480] R2 is optional, and when present, it is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0481] L is an alkylene, a substituted alkylene, N(R') (alkylene) or N(R') (substituted alkylene), where R' is H, an alkyl, a substituted alkyl, a cycloalkyl or a substituted cycloalkyl.

[0482] In addition, the following amino acids having the structure of formula (XV) are included:

[0483]

[0484] in:

[0485] A is optional, and when present, is a lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenyl, substituted lower alkenyl, alynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkylene arylene, substituted alkylene arylene, arylalkylene or substituted arylalkylene;

[0486] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0487] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0488] R2 is optional, and when present, it is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0489] 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 Independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 and R 9 They can be combined to form =O or cycloalkyl, or any adjacent R 8 The groups can form cycloalkyl groups together.

[0490] In addition, the following amino acids having the structure (XV-A) are included:

[0491]

[0492] in:

[0493] A is optional, and when present, is a lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenyl, substituted lower alkenyl, alynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkylene arylene, substituted alkylene arylene, arylalkylene or substituted arylalkylene;

[0494] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0495] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0496] R2 is optional, and when present, it is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0497] 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 Independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 and R 9 They can be combined to form =O or cycloalkyl, or any adjacent R 8 The groups can form cycloalkyl groups together.

[0498] In addition, the following amino acids having the structure (XV-B) are included:

[0499]

[0500] in:

[0501] A is optional, and when present, is a lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenyl, substituted lower alkenyl, alynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkylene arylene, substituted alkylene arylene, arylalkylene or substituted arylalkylene;

[0502] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0503] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0504] R2 is optional, and when present, it is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0505] 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 Independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 and R 9 They can be combined to form =O or cycloalkyl, or any adjacent R 8 The groups can form cycloalkyl groups together.

[0506] In addition, the following amino acids having the structure of formula (XVI) are included:

[0507]

[0508] in:

[0509] A is optional, and when present, is a lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenyl, substituted lower alkenyl, alynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkylene arylene, substituted alkylene arylene, arylalkylene or substituted arylalkylene;

[0510] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0511] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0512] R2 is optional, and when present, it is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0513] 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.

[0514] In addition, the following amino acids having the structure (XVI-A) are included:

[0515]

[0516] in:

[0517] A is optional, and when present, is a lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenyl, substituted lower alkenyl, alynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkylene arylene, substituted alkylene arylene, arylalkylene or substituted arylalkylene;

[0518] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0519] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0520] R2 is optional, and when present, it is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0521] L is an alkylene, a substituted alkylene, N(R') (alkylene) or N(R') (substituted alkylene), where R' is H, an alkyl, a substituted alkyl, a cycloalkyl or a substituted cycloalkyl.

[0522] In addition, the following amino acids having the structure (XVI-B) are included:

[0523]

[0524] in:

[0525] A is optional, and when present, is a lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenyl, substituted lower alkenyl, alynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkylene arylene, substituted alkylene arylene, arylalkylene or substituted arylalkylene;

[0526] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0527] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0528] R2 is optional, and when present, it is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0529] L is an alkylene, a substituted alkylene, N(R') (alkylene) or N(R') (substituted alkylene), where R' is H, an alkyl, a substituted alkyl, a cycloalkyl or a substituted cycloalkyl.

[0530] In addition, amino acids having the structure of formula (XVII) are included:

[0531]

[0532] in:

[0533] A is optional, and when present, is a lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenyl, substituted lower alkenyl, alynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkylene arylene, substituted alkylene arylene, arylalkylene or substituted arylalkylene;

[0534] m is -C(R3)-,

[0535] Wherein (a) indicates bonding with group A, and (b) indicates bonding with the corresponding carbonyl group, and 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 cycloalkyl or heterocycloalkyl.

[0536] R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0537] T3 is a bond, C(R)(R), O or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

[0538] R1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and

[0539] R2 is optional and, when present, is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

[0540] In addition, amino acids ...

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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