Compositions containing, methods and uses of antibody-TLR agonist conjugates

Abstract

Abstract Disclosed herein are Trastuzunab-linked TLR-agonist derivative analogs that include at least one non-natural amino acid, and methods for making such non-natural amino acids and polypeptides. The Trastuzunab-linked TLR-agonist derivative analogs can include a wide range of possible functionalities, but typically have at least one oxime, carbonyl, dicarbonyl, and / or hydroxylamine group. Also disclosed herein are non-natural amino acid Trastuzumab-linked TLR-agonist derivative analogs that are further modified post-translationally, methods for effecting such modifications, and methods for purifying such Trastuzumab-linked TLR-agonist derivative analogs. Typically, the modified Trastuzunab- linked TLR-agonist derivative analogs include at least one oxime, carbonyl, dicarbonyl, and / or hydroxylamine group. Further disclosed are methods for using such non-natural amino acid Trastuzumab-linked TLR-agonist derivative analogs and modified non-natural amino acid Trastuzumab-linked TLR-agonist derivative analogs, including therapeutic, diagnostic, and other biotechnology use. Abstract 20 26 20 50 50 29 J un 2 02 6 A b s t r a c t 2 0 2 6 2 0 5 0 5 0 2 9 J u n 2 0 2 6

Description

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional application of Australian Patent Application No. 2024205610, which is a divisional application of Australian Patent Application No. 2020223031, which is an Australian national phase application derived from International Patent Application No. PCT / US2020 / 018015, filed 12 February 2020, which 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 its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on February 7, 2020 is named AMBX_0230_PCT_SL.txt and is 30,527 bytes in size. FIELD OF THE INVENTION

[0003] The present invention disclosure relates to TLR-agonists compounds and TLR-agonist conjugates (TCs) and uses thereof. The invention further pertains to pharmaceutical compositions containing (TCs) as a therapeutic or prophylactic. BACKGROUND OF THE INVENTION

[0004] Targeting molecules or polypeptides such as antibodies and fragments thereof, and TLR agonists compounds can be conjugated together using non-naturally encoded amino acids by site-specific conjugation to produce novel TLR-agonist Conjugates (TC). The novel TCs can be constructed in such a way that during systemic treatment, the circulating TC can target the TLR agonist to the tumor site and stimulate beneficial immune responses locally, thereby minimizing systemic cytokine release syndrome. SUMMARY OF THE INVENTION 2026205050   29 Jun 2026

[0005] The invention relates to targeting polypeptides with one or more non-naturally encoded amino acids conjugated to agonist compounds of TLRs including but not limited to TLR7 and / or TLR8. Such conjugates are referred to herein as TLR-agonist Conjugates (TCs). TCs of the present invention include targeting biological molecules or polypeptides and TLR agonists compounds conjugated together using non-naturally encoded amino acids by sitespecific conjugation to produce novel Biological TLR-agonist Conjugates (BTCs). The targeting biological molecules or polypeptides can be a tumor targeting biological biological molecules or polypeptides.

[0006] The invention, in additional embodiments, further relates to TCs further conjugated to a water soluble polymer that forms stable dimers or multimers.

[0007] The present invention provides methods of inhibiting or reducing growth of a tumor or cancer comprising contacting the tumor with an effective amount of TC of the invention to stimulate the immune system of the patient in proximity to the tumor. The present invention provides methods of inhibiting or reducing growth of a tumor or cancer comprising contacting the tumor with an effective amount of a PEGylated TC, or stable dimer or multimer of the TC of the invention. In one embodiment, the TC is non-pegylated or monopegylated. In one embodiment, the TC is dipegylated. In one embodiment, the TC has more than one and / or different TLR agonist molecules attached to it. In one embodiment, the TC has more than one and / or same TLR agonist molecules attached to it. Another embodiment of the present invention provides methods of using TCs of the present invention to modulate the immune response to tumor cells. In certain embodiments, the TC is co-administered with at least one chemotherapeutic agent and / or at least one immunotherapeutic agent. The chemotherapeutic agent can be selected from the group consisting of temozolomide, gemictabine, doxorubicin, cyclophosphamide, paclitaxel, cisplatin, fluoropyrimidine, taxane, anthracycline, lapatinib, capecitabine, letrozole, pertuzumab, docetaxel, IFN-a. In another embodiment of the present invention, TC is coadministered with at least one chemotherapeutic agent and / or at least one immunotherapeutic agent.

[0008] In some embodiments, the TC comprises a targeting polypeptide including but not limited to an antigen-binding polypeptides (ABP) comprising one or more non-naturally encoded amino acids. In some embodiments, the ABP comprises a complete antibody heavy chain. In some embodiments, the ABP comprises a complete antibody light chain. In some embodiments, the ABP comprises a variable region of an antibody light chain. In some embodiments, the ABP comprises a variable region of an antibody heavy chain. In some embodiments, the ABP 3 2026205050   29 Jun 2026 comprises at least one CDR of an antibody light chain. In some embodiments, the ABP comprises at least one CDR of an antibody heavy chain. In some embodiments, the ABP comprises at least one CDR of a light chain and at least one CDR of a heavy chain. In some embodiments, the ABP comprises a Fab. In some embodiments, the ABP comprises two or more Fabs. In some embodiments, the ABP comprises a (Fab’)2. In some embodiments, the ABP comprises two or more (Fab’)2. In some embodiments, the ABP comprises a scFv. In some embodiments, the ABP comprises two or more scFv. In some embodiments, the ABP comprises a minibody. In some embodiments, the ABP comprises two or more minibodies. In some embodiments, the ABP comprises a diabody. In some embodiments, the ABP comprises two or more diabodies. In some embodiments, the ABP comprises a variable region of a light chain and a variable region of a heavy chain. In some embodiments, the ABP comprises a complete light chain and a complete heavy chain. In some embodiments, the ABP comprises one or more Fc domain or portion thereof. In some embodiments, the ABP comprises a combination of any of the above embodiments. In some embodiments, the ABP comprises a homodimer, heterodimer, homomultimer or heteromultimer of any of the above embodiments. In some embodiments, the ABP comprises a polypeptide that binds to a binding partner wherein the binding partner comprises an antigen, a polypeptide, a nucleic acid molecule, a polymer, or other molecule or substance. In some embodiments, the ABP is associated with a non-antibody scaffold molecule or substance. In some embodiments, the antigen is a tumor antigen.

[0009] Toll- like receptors (TLRs) detect a wide range of conserved pathogen-associated molecular patterns (PAMPs). They play an important role of sensing invading pathogens and subsequent initiation of innate immune responses. There are 10 known members of the TLR family in human, which are type I transmembrane proteins featuring an extracellular leucine-rich domain and a cytoplasmic tail that contains a conserved Toll / interleukin (IL)- l receptor (TIR) domain. Within this family, TLR3, TLR7, TLR8, and TLR9 are located within endosomes. TLR7 and TLR8 can be activated by binding to a specific small molecule ligand (i.e., TLR7 agonist or TLR8 agonist) or its native ligand (i.e., single- stranded RNA, ssRNA). Following binding of an agonist to TLR7 or TLR8, the receptor in its dimerized form is believed to undergo a structural change leading to the subsequent recruitment of adapter proteins at its cytoplasmic domain, including the myeloid differentiation primary response gene 88 (MyD88). Following the initiation of the receptor signalling cascade via the MyD88 pathway, cytoplasmic transcription factors such as interferon regulatory factor 7 (IRF-7) and nuclear factor kappa B (NF-kB) are activated. These transcription factors then translocate to the nucleus and initiate the transcription 4 2026205050   29 Jun 2026 of various genes, e.g., IFN-alpha and other antiviral cytokine genes. TLR7 is predominately expressed on plasmacytoid cells, and on B cells. Altered responsiveness of immune cells might contribute to the reduced innate immune responses in cancer patients. Agonist-induced activation of TLR7 and / or TLR8 conjugated to a targeting moiety such as an antibody or fragment thereof may therefore represent a novel approach for the treatment of cancer. Treatment with TC comprising a TLR7 or TLR8 agonist represents a promising solution to provide greater efficacy with better tolerability. Suitable TLR7 and / or TLR8 agonists for use in the present invention to make TCs are found in the following US Patents, each of which is incorporated by reference herein: U.S. Patent No. 6,825,350; U.S. Patent No. 6,656,389; U.S. Patent No. 6,656,398; U.S. Patent No. 6,683,088; U.S. Patent No. 6,756,382; U.S. Patent No. 6,825,350; U.S. Patent No. 6,667,312; U.S. Patent No. 6,677,347; U.S. Patent No. 7,598,382; U.S. Patent No. 8,673,932.

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

[0011] In some embodiments, one or more engineered bonds are created with one or more non-natural amino acids. The intramolecular bond may be created in many ways, including but not limited to, a reaction between two amino acids in the protein under suitable conditions (one or both amino acids may be a non-natural amino acid); a reaction with two amino acids, each of which may be naturally encoded or non-naturally encoded, with a linker, polymer, or other molecule under suitable conditions, etc.

[0012] In some embodiments, one or more amino acid substitutions in the TC polypeptide may be with one or more naturally occurring or non-naturally occurring amino acids. In some embodiments the amino acid substitutions in the TC may be with naturally occurring or non-naturally occurring amino acids, provided that at least one substitution is with a non-naturally encoded amino acid. In some embodiments, one or more amino acid substitutions in the TC polypeptide may be with one or more naturally occurring amino acids, and additionally at least one substitution is with a non-naturally encoded amino acid. In some embodiments the TC polypeptide may be an antibody or antibody fragment. In some embodiments the TC polypeptide may be a tumor targeting polypeptide. 2026205050   29 Jun 2026

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

[0014] In some embodiments, the non-naturally encoded amino acid comprises a carbonyl group. In some embodiments, the non-naturally encoded amino acid has the structure: (CH2)nR1COR2 R3HN' / ^''''COR4 wherein n is 0-10; R1 is an alkyl, aryl, substituted alkyl, or substituted aryl; R2 is H, an alkyl, aryl, substituted alkyl, and substituted aryl; and R3 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R4 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.

[0015] In some embodiments, the non-naturally encoded amino acid comprises an aminooxy group. In some embodiments, the non-naturally encoded amino acid comprises a hydrazide group. In some embodiments, the non-naturally encoded amino acid comprises a hydrazine group. In some embodiments, the non-naturally encoded amino acid residue comprises a semicarbazide group.

[0016] In some embodiments, the non-naturally encoded amino acid residue comprises an azide group. In some embodiments, the non-naturally encoded amino acid has the structure: (CH2)nR1X(CH2)mN3 R2HN^ ^COR3 wherein n is 0-10; R1 is an alkyl, aryl, substituted alkyl, substituted aryl or not present; X is O, N, S or not present; m is 0-10; R2 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.

[0017] In some embodiments, the non-naturally encoded amino acid comprises an alkyne group. In some embodiments, the non-naturally encoded amino acid has the structure: (CH2)nR1X(CH2)mCCH r2hn^^xcor3 2026205050   29 Jun 2026 wherein n is 0-10; R1 is an alkyl, aryl, substituted alkyl, or substituted aryl; X is O, N, S or not present; m is 0-10, R2 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.

[0018] In some embodiments, the polypeptide is a TC that comprises 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 modification, linker, polymer, or biologically active molecule.

[0019] The present invention also provides isolated nucleic acids comprising a polynucleotide that encode the targeting polypeptides of TC and the present invention provides isolated nucleic acids comprising a polynucleotide that hybridizes under stringent conditions to the polynucleotides. The present invention also provides isolated nucleic acids comprising a polynucleotide that encode the targeting polypeptides wherein the polynucleotide comprises at least one selector codon. It is readily apparent to those of ordinary skill in the art that a number of different polynucleotides can encode any polypeptide of the present invention.

[0020] In some embodiments, the selector codon is selected from the group consisting of an amber codon, ochre codon, opal codon, a unique codon, a rare codon, a five-base codon, and a four-base codon.

[0021] The present invention also provides methods of making a TC polypeptide linked to a water soluble polymer or linked to one or more TC polypeptides to form a homodimer or homomultimer. In some embodiments, the method comprises contacting an isolated TC polypeptide comprising a non-naturally encoded amino acid with a water soluble polymer or a linker comprising a moiety that reacts with the non-naturally encoded amino acid. In some embodiments, the non-naturally encoded amino acid incorporated into the TC polypeptide is reactive toward a water soluble polymer or a linker that is otherwise unreactive toward any of the 20 common amino acids. In some embodiments, the non-naturally encoded amino acid incorporated into the TC polypeptide is reactive toward a linker, polymer, or biologically active molecule that is otherwise unreactive toward any of the 20 common amino acids.

[0022] In some embodiments, the TC polypeptide linked to the water soluble polymer or a linker is made by reacting a TC polypeptide comprising a carbonyl-containing amino acid with a poly(ethylene glycol) molecule or a linker comprising an aminooxy, hydrazine, hydrazide or semicarbazide group. In some embodiments, the aminooxy, hydrazine, hydrazide or semicarbazide group is linked to the poly(ethylene glycol) molecule or a linker through an amide 7 2026205050   29 Jun 2026 linkage. In some embodiments, the aminooxy, hydrazine, hydrazide or semicarbazide group is linked to the poly(ethylene glycol) molecule or a linker through a carbamate linkage.

[0023] In some embodiments, the TC polypeptide linked to the water soluble polymer is made by reacting a poly(ethylene glycol) molecule or a linker comprising a carbonyl group with a polypeptide comprising a non-naturally encoded amino acid that comprises an aminooxy, hydrazine, hydrazide or semicarbazide group.

[0024] In some embodiments, the TC polypeptide linked to the water soluble polymer or a linker is made by reacting a TC comprising an alkyne-containing amino acid with a poly(ethylene glycol) molecule comprising an azide moiety. In some embodiments, the azide or alkyne group is linked to the poly(ethylene glycol) molecule or a linker through an amide linkage.

[0025] In some embodiments, the TC polypeptide linked to the water soluble polymer or a linker is made by reacting an TC polypeptide comprising an azide-containing amino acid with a poly(ethylene glycol) molecule comprising an alkyne moiety. In some embodiments, the azide or alkyne group is linked to the poly(ethylene glycol) molecule or a linker through an amide linkage.

[0026] In some embodiments, the poly(ethylene glycol) molecule or a linker has a molecular weight of between about 0.1 kDa and about 100 kDa. In some embodiments, the poly(ethylene glycol) molecule or a linker has a molecular weight of between 0.1 kDa and 50 kDa. In some embodiments, the poly(ethylene glycol) molecule or a linker is a branched polymer or linker. In some embodiments, each branch of the poly(ethylene glycol) branched polymer or linker has a molecular weight of between 1 kDa and 100 kDa, or between 1 kDa and 50 kDa.

[0027] In some embodiments, the water soluble polymer linked to the TC polypeptide comprises a polyalkylene glycol moiety. In some embodiments, the non-naturally encoded amino acid residue incorporated into the TC comprises a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine, a semicarbazide group, an azide group, or an alkyne group. In some embodiments, the non-naturally encoded amino acid residue incorporated into the TC polypeptide comprises a carbonyl moiety and the water soluble polymer comprises an aminooxy, hydrazide, hydrazine, or semicarbazide moiety. In some embodiments, the non-naturally encoded amino acid residue incorporated into the TC polypeptide comprises an alkyne moiety and the water soluble polymer comprises an azide moiety. In some embodiments, the non-naturally encoded amino acid residue incorporated into the TC polypeptide comprises an azide moiety and the water soluble polymer comprises an alkyne moiety. The present invention also 8 2026205050   29 Jun 2026 provides compositions comprising a TC polypeptide comprising a non-naturally encoded amino acid and a pharmaceutically acceptable carrier. In some embodiments, the non-naturally encoded amino acid is linked to a water soluble polymer.

[0028] The present invention also provides cells comprising a polynucleotide encoding the targeting polypeptide of the TC comprising a selector codon. In some embodiments, the cells comprise an orthogonal RNA synthetase and / or an orthogonal tRNA for substituting a non-naturally encoded amino acid into the targeting polypeptide of the TC.

[0029] The present invention also provides methods of making the targeting polypeptide of the TC comprising a non-naturally encoded amino acid. In some embodiments, the methods comprise culturing cells comprising a polynucleotide or polynucleotides encoding the targeting polypeptide of the TC, an orthogonal RNA synthetase and / or an orthogonal tRNA under conditions to permit expression of the targeting polypeptide of the TC or variant thereof; and purifying the TC polypeptide from the cells and / or culture medium.

[0030] The present invention also provides methods of increasing therapeutic half-life, serum half-life or circulation time of a TC. The present invention also provides methods of modulating immunogenicity of a TC. In some embodiments, the methods comprise substituting a non-naturally encoded amino acid for any one or more amino acids in naturally occurring targeting polypeptide of the TC and / or linking the targeting polypeptide to a linker, a polymer, a water soluble polymer, or a biologically active molecule.

[0031] The present invention also provides methods of treating a patient in need of such treatment with an effective amount of a TC molecule of the present invention. In some embodiments, the methods comprise administering to the patient a therapeutically-effective amount of a pharmaceutical composition comprising a TC comprising a non-naturally-encoded amino acid and a pharmaceutically acceptable carrier. In some embodiments, the non-naturally encoded amino acid is linked to a water soluble polymer. In some embodiments, the TC is glycosylated. In some embodiments, the TC is not glycosylated.

[0032] The present invention also provides TCs comprising a water soluble polymer or a linker linked by a covalent bond to the TC at a single amino acid. In some embodiments, the water soluble polymer comprises a poly(ethylene glycol) moiety. In some embodiments, the amino acid covalently linked to the water soluble polymer or a linker is a non-naturally encoded amino acid present in the targeting polypeptide of the TC.

[0033] The present invention provides a TC polypeptide comprising at least one linker, polymer, or biologically active molecule, wherein said linker, polymer, or biologically active 9 2026205050   29 Jun 2026 molecule is attached to the polypeptide through a functional group of a non-naturally encoded amino acid ribosomally incorporated into the targeting polypeptide of the TC. In TC conjugates, the PEG or other water soluble polymer, another TC, polypeptide, or biologically active molecule can be conjugated directly to the TC via a linker. In one embodiment the linker is long enough to permit flexibility and allow for dimer formation. In one embodiment the linker is at least 3 amino acids, or 18 atoms, in length so as to permit dimer formation. In some embodiments, the polypeptide is linked to a linker to permit formation of a multimer. In some embodiments, the linker is a bifunctional linker. In some embodiments, the composition and / or TCs of the present invention can comprise multiple linkers. In other embodiments, each linker may include one or more compounds attached. A linker can also comprise alkylene, alkenylene, alkynylene, polyether, polyester, polyamide group(s) and also, polyamino acids, polypeptides, 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, a peptide linker cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease. A cleavable linker may comprise a valine-citrulline linker or a valine-alanine peptide. In some embodiments, the linker can be a dipeptide linker, such as a valine-citrulline or a phenylalanine-lysine linker. A valine-citrulline- or valine-alanine-containing linker can contain a maleimide or succinimide group. A valine-citrulline- or valine-alanine-containing linker can contain a para aminobenzyl alcohol (PABA) group or para-aminobenzyl carbamate (PABC). Other suitable linkers include linkers hydrolyzable at a pH of less than 5.5, such as a hydrazone linker. Additional suitable cleavable linkers include disulfide linkers. In some embodiments, the cleavable linker may include a linker cleaved at the tumor microenvironment such as tumor infiltrating T-cells. In some embodiments, a non-cleavable linker includes, but is not limited to, a maleimidocaproyl linker. The maleimidocaproyl linker can comprise N-maleimidomethylcyclohexane-1-carboxylate, a succinimide group, a pentafluorophenyl group, and / or one or more PEG molecules but is not limited to such. In some embodiments, any one of the compositions, compounds or salts thereof of the present invention, can be linked to a polypeptide by way of a linker. In some embodiments, any one of the compounds or salts thereof disclosed herein, in Tables 3, 4, 5, 6, and 7 can be linked to a polypeptide by way of a linker. In some embodiments, the polypeptide is a targeting polypeptide or biological targeting polypeptide or tumor targeting polypeptide. In some embodiments, the targeting polypeptide is an antibody or antibody fragment. 2026205050   29 Jun 2026

[0034] In some embodiments, the TC polypeptide is monoPEGylated. The present invention also provides a TC comprising a linker, polymer, or biologically active molecule that is attached to one or more non-naturally encoded amino acid wherein said non-naturally encoded amino acid is ribosomally incorporated into the polypeptide at pre-selected sites.

[0035] In some embodiments, the present invention provides a composition comprising one or more targeting polypeptides having one or more non-naturally encoded amino acids incorporated, wherein at least one of the polypeptides is linked to a TLR agonist molecule via a linker covalently bonded to the non-natural amino acid of the polypeptide.

[0036] In another embodiment, the present invention provides a composition wherein the one or more targeting polypeptide is a same or different targeting polypeptide. In another embodiment, the invention provides a composition wherein the one or more targeting polypeptide binds to a cell surface target, or tumor cell target, or cancer cell target. In another embodiment, the one or more targeting polypeptide is a monospecific, bispecific, or multispecific targeting polypeptide.

[0037] In other embodiments, the monospecific, bispecific, or multi-specific targeting polypeptide comprises a drug conjugate or checkpoint inhibitor. Any suitable immune checkpoint inhibitor is contemplated for use with the compositions or TCs of the present invention. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins. In another embodiment, the immune checkpoint inhibitor reduces the interaction between one or more immune checkpoint proteins and their ligands. Inhibitory nucleic acids that decrease the expression and / or activity of immune checkpoint molecules can also be used in the present invention. In some embodiments, the immune checkpoint inhibitor is CTLA4, TIGIT, glucocorticoid-induced TNFR-related protein (GITR), inducible T cell costimulatory (ICOS), CD96, poliovirus receptor-related 2 (PVRL2), PD-1, PD-Ll, PD-L2, LAG-3, B7-H4, killer immunoglobulin receptor (KIR), OX40, OX40-L indoleamine 2,3-dioxygenase 1 (IDO-1), indoleamine 2, 3 -di oxygenase 2 (IDO-2), CEACAM1, CD272, TEVI3, the adenosine A2A receptor, and VISTA protein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA4, PD-1, or PD-Ll .

[0038] In another embodiment, the targeting polypeptide comprises an antibody or antibody fragment. In other embodiments, the targeting polypeptide is an antibody or antibody fragment that binds to an antigen of a cell. In another embodiment the targeting polypeptide is an antibody or antibody fragment that binds to a target selected from the group consisting of HER2, HER3, PD-1, PDL-1, EGFR, TROP2, PSMA, VEGFR, CTLA-4, EpCAM, MUC1, MUC16, c-met, 11 2026205050   29 Jun 2026 GPC3, ENPP3, TIM-1, FOLR1, STEAP1, Mesothelin, 5T4, CEA, CA9, Cadherin 6, ROR1, SLC34A2, SLC39A6, SLC44A4, LY6E, DLL3, ePhA2, GPNMB, SLITRK6, CD3, CD19, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD47, CD52, CD56, CD70, CD96, CD97, CD99, CD117, CD123, CD179, CD223, and CD276. In some embodiments, the targeting polypeptide comprises an antibody or antibody fragment that binds to HER2. In another embodiment, the targeting polypeptide is trastuzumab.

[0039] In another embodiment, the antibody or antibody fragment comprises an IgG, Fab, (Fab’)2, Fv, or single chain Fv (scFv). In some embodiments, the antibody or antibody fragment comprises one or more Fab, (Fab’)2, Fv, or single chain Fv (scFv) mutations. In some embodiments, the antibody or antibody fragment comprises one or more Fc mutations. In other embodiments, the antibody or antibody fragment comprises one to six Fc mutations. In some embodiments, the antibody or antibody fragment comprises two or more Fc mutations. In other embodiments, the antibody or antibody fragment comprises three or more Fc mutations. In some embodiments, the antibody or antibody fragment comprises four or more Fc mutations. In other embodiments, the antibody or antibody fragment comprises five or more Fc mutations. In other embodiments, the antibody or antibody fragment comprises six Fc mutations.

[0040] In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acid incorporated in the heavy chain, light chain, or both the heavy and light chains. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acid incorporated in the heavy chain and light chain. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acid incorporated in the heavy chain, light chain, or both the heavy and light chains and further comprises one or more Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acid incorporated in each of the heavy chain and light chain, the antibody or antibody fragment further comprising one or more Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acid incorporated in the heavy chain, light chain, or both the heavy and light chains and further comprises at least two Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acid incorporated in each of the heavy chain and light chain, the antibody or antibody fragment further comprising at least two Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acid incorporated in the heavy chain, light chain, or both the heavy and light chains and further comprises at least three 12 2026205050   29 Jun 2026 Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acid incorporated in each of the heavy chain and light chain, the antibody or antibody fragment further comprising at least three Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acid incorporated in the heavy chain, 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 acid incorporated in each of the heavy chain and light chain, the antibody or antibody fragment further comprising at least four Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acid incorporated in the heavy chain, 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 acid incorporated in each of the heavy chain and light chain, the antibody or antibody fragment further comprising at least five Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acid incorporated in the heavy chain, light chain, or both the heavy and light chains and further comprises at least six Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acid incorporated in each of the heavy chain and light chain, the antibody or antibody fragment further comprising at least six Fc mutations.

[0041] In another embodiment, the targeting polypeptides comprise one or more non-naturally encoded amino acids selected from the group of para-acetyl phenylalanine, p-nitrophenylalanine, p-sulfotyrosine,   p-carboxyphenylalanine,   o-nitrophenylalanine, m- nitrophenylalanine, p-boronyl phenylalanine, o-boronylphenylalanine, m-boronylphenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-OMe phenylalanine, o-OMe phenylalanine, m-OMe phenylalanine, p-sulfophenylalanine, o-sulfophenylalanine, m-sulfophenylalanine, 5-nitro His, 3-nitro Tyr, 2-nitro Tyr, nitro substituted Leu, nitro substituted His, nitro substituted De, nitro substituted Trp, 2-nitro Trp, 4-nitro Trp, 5-nitro Trp, 6-nitro Trp, 7-nitro Trp, 3-aminotyrosine, 2-aminotyrosine, O-sulfotyrosine, 2-sulfooxyphenylalanine, 3-sulfooxyphenylalanine, o-carboxyphenylalanine, m-carboxyphenylalanine, p-acetyl-L-phenylalanine,  p-propargyl-phenylalanine,  O-methyl-L- tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcP-serine, L-Dopa,  fluorinated phenylalanine,  isopropyl-L- phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-13 2026205050   29 Jun 2026 phosphoserine, phosphonoserine, phosphonotyrosine,     p-iodo-phenylalanine,     p- bromophenylalanine, p-amino-L-phenylalanine, p-propargyloxy-L-phenylalanine, 4-azido-L-phenylalanine, para-azidoethoxy phenylalanine, and para-azidomethyl-phenylalanine. In another embodiment, the non-natural amino acid is selected from a group consisting of para-acetyl-phenylalanine, 4-azido-L-phenylalanine, para-azidoethoxy phenylalanine or para-azidomethyl-phenylalanine. In other embodiments, the non-naturally encoded amino acid is site specifically incorporated into the one or more targeting polypeptide.

[0042] In another embodiment, the TLR agonist is a TLR7 agonist, a TLR8 agonist, or a TLR7 / TLR8 dual agonist. In other embodiments, the TLR agonist is a TLR agonist comprising a molecule structure according to any one of structures 1, 2, 3, 4 or 5 of Figure 1. In another embodiment the TLR agonist is any one of TLR agonists selected from the group of structures according to Tables 3, 4, 5, 6, 7 of the present invention.

[0043] In other embodiments, the targeting polypeptide is conjugated to one or more linker, polymer, or biologically active molecule. In some embodiments, the targeting polypeptide is is directly or indirectly conjugated to one or more linker, polymer, or biologically active molecule. In some embodiments, the one or more linker is a cleavable or non-cleavable linker.

[0044] In some embodiments, the one or more linker is 0.1kDa to 50kDa. In other embodiments, the one or more linker is 0.1kDa to 10kDa. In other embodiments, the one or more linker or polymer is linear, branched, multimeric, or dendrimeric. In another embodiment, the one or more linker or polymer is a bifunctional or multifunctional linker or a bifunctional or multifunctional polymer.

[0045] In other embodiments, the one or more polymer is a water soluble polymer. In other embodiments, the water soluble polymer is polyethylene glycol (PEG). In some embodiments, the PEG has a molecular weight between 0.1kDa and 100kDa. In other embodiments, the PEG has a molecular weight between 0.1kDa and 50kDa. In other embodiments, the PEG has a molecular weight between 0.1kDa and 40kDa. In other embodiments, the PEG has a molecular weight between 0.1kDa and 30kDa. In other embodiments, the PEG has a molecular weight between 0.1kDa and 20kDa. In other embodiments, the PEG has a molecular weight between 0.1kDa and 10kDa. In some embodiments, the poly(ethylene glycol) molecule has a molecular weight of between about 0.1 kDa and about 100 kDa. In some embodiments, the poly(ethylene glycol) molecule has a molecular weight of between 0.1 kDa and 50 kDa. In some embodiments, the poly(ethylene glycol) has a molecular weight of between 1 kDa and 25 kDa, or between 2 and 22 kDa, or between 5 kDa and 20 kDa. For example, the molecular weight of the 14 2026205050   29 Jun 2026 poly(ethylene glycol) polymer may be about 5 kDa, or about 10 kDa, or about 20 kDa, or about 30 kDa. For example, the molecular weight of the poly(ethylene glycol) polymer may be 5 kDa or 10 kDa or 20 kDa, or 30 kDa. In some embodiments the poly(ethylene glycol) molecule is a branched PEG. In some embodiments the poly(ethylene glycol) molecule is a branched 5K PEG. In some embodiments the poly(ethylene glycol) molecule is a branched 10K PEG. In some embodiments the poly(ethylene glycol) molecule is a branched 20K PEG. In some embodiments the poly(ethylene glycol) molecule is a linear PEG. In some embodiments the poly(ethylene glycol) molecule is a linear 5K PEG. In some embodiments the poly(ethylene glycol) molecule is a linear 10K PEG. In some embodiments the poly(ethylene glycol) molecule is a linear 20K PEG. In some embodiments the poly(ethylene glycol) molecule is a linear 30K PEG. In some embodiments, the molecular weight of the poly(ethylene glycol) polymer is an average molecular weight. In certain embodiments, the average molecular weight is the number average molecular weight (Mn). The average molecular weight may be determined or measured using GPC or SEC, SDS / PAGE analysis, RP-HPLC, mass spectrometry, or capillary electrophoresis.

[0046] In another embodiment, at least one linker, polymer, or biologically active molecule is linked to at least one non-naturally encoded amino acids. In some embodiments, the linker is a PEG. In other embodiments, the linker is a PEG with a molecular weight between 0.1kDa and 50 kDa. In other embodiments, the linker is a PEG with a molecular weight between 0.1kDa and 40 kDa. In other embodiments, the linker is a PEG with a molecular weight between 0.1kDa and 30 kDa. In other embodiments, the linker is a PEG with a molecular weight between 0.1kDa and 20 kDa. In other embodiments, the linker is a PEG with a molecular weight between 0.1kDa and 10 kDa. In other embodiments, the linker is a PEG with a molecular weight between 0.1kDa and 5 kDa.

[0047] In another embodiment, the targeting polypeptide comprises one or more amino acid substitution, addition or deletion that increases the stability or solubility of the composition. In another embodiment, the targeting polypeptide comprises one or more amino acid substitution, addition or deletion that enhances / reduces ADCP or ADCC activity. In another embodiment, the targeting polypeptide comprises one or more amino acid substitution, addition or deletion that increases pharmacokinetics of the composition. In other embodiments, the composition comprises one or more amino acid substitution, addition or deletion that increases the expression of the targeting polypeptide in a recombinant host cell or synthesized in vitro.

[0048] In another embodiment, the non-naturally encoded amino acid is reactive toward a linker, polymer, or biologically active molecule that is otherwise unreactive toward any of the 20 15 2026205050   29 Jun 2026 common amino acids in the polypeptide. In another embodiment, the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group. In other embodiments, the non-naturally encoded amino acid comprises a carbonyl group.

[0049] In another embodiment, the targeting polypeptide is linked to a cytotoxic agent or an immunostimulatory agent. In another embodiment, the TC or BTC of the present invention is linked to a cytotoxic agent or an immunostimulatory agent. In another embodiment, the targeting polypeptide comprises a cytotoxic agent or an immunostimulatory agent. In another embodiment, the TC or BTC of the present invention comprises a cytotoxic agent or an immunostimulatory agent.

[0050] In another embodiment, the present invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist comprising a 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 in 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 a structure according to structure 1 of Figure 1. In another embodiment, the TLR agonist comprising a structure according to structure 1 is selected from the group of: AXC-621, AXC-622, AXC-625, AXC-626, AXC-627, AXC-638, AXC-639, AXC-640, AXC-642, AXC-662, AXC-665, AXC-666, AXC-667, AXC-668, AXC-669, AXC-670, AXC-671, AXC-672, AXC-675, AXC-678, AXC-679, AXC-681, AXC-687, AXC-688, AXC-689, AXC-690, AXC-691, AXC-696, AXC-697, AXC-698, AXC-699, AXC-700, AXC-701, AXC-702, AXC-709, AXC-710, AXC-711, AXC-712, AXC-713, AXC-714, AXC-715, AXC-716, AXC-717, AXC-718, AXC-719, AXC-722, AXC-723, AXC-724, AXC-725, AXC-726, AXC-727, AXC-729, AXC-731, AXC-732, AXC-733, AXC-734, AXC-735, AXC-736, AXC-737, AXC-738, AXC-739, AXC-740, AXC-741, AXC-743, AXC-742, AXC-747, AXC-748, AXC-749, AXC-750, AXC-751, AXC-752, AXC-754, AXC-755, AXC-756, AXC-757, AXC-758, AXC-759, AXC-760, AXC-761, AXC-762, AXC-764, AXC-771, AXC-772, AXC-773, AXC-777, AXC-778, AXC-779, AXC-789, AXC-793, AXC-799, AXC-800, AXC-801, AXC-802, AXC-803, AXC-804, AXC-805, AXC-806, AXC-807, AXC-808, AXC-809, AXC-810, AXC-831 and AXC-910 compounds. In another embodiment, present invention provides a the TLR agonist of any one 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, 16 2026205050   29 Jun 2026 AXC-668, AXC-669, AXC-670, AXC-671, AXC-672, AXC-675, AXC-678, AXC-679, AXC-681, AXC-687, AXC-688, AXC-689, AXC-690, AXC-691, AXC-696, AXC-697, AXC-698, AXC-699, AXC-700, AXC-701, AXC-702, AXC-709, AXC-710, AXC-711, AXC-712, AXC-713, AXC-714, AXC-715, AXC-716, AXC-717, AXC-718, AXC-719, AXC-722, AXC-723, AXC-724, AXC-725, AXC-726, AXC-727, AXC-729, AXC-731, AXC-732, AXC-733, AXC-734, AXC-735, AXC-736, AXC-737, AXC-738, AXC-739, AXC-740, AXC-741, AXC-743, AXC-742, AXC-747, AXC-748, AXC-749, AXC-750, AXC-751, AXC-752, AXC-754, AXC-755, AXC-756, AXC-757, AXC-758, AXC-759, AXC-760, AXC-761, AXC-762, AXC-764, AXC-771, AXC-772, AXC-773, AXC-777, AXC-778, AXC-779, AXC-789, AXC-793, AXC-799, AXC-800, AXC-801, AXC-802, AXC-803, AXC-804, AXC-805, AXC-806, AXC-807, AXC-808, AXC-809, AXC-810, AXC-831, or AXC-910 compounds further comprising a linker. In another embodiment, the TLR agonist comprises a structure according to structure 1 further comprising a linker.

[0051] In other embodiments, the TLR agonist comprising a structure according to structure 1 is selected from the group of: AXC-625, AXC-626, AXC-638, AXC-639, AXC-640, AXC-642, AXC-662, AXC-667, AXC-668, AXC-669, AXC-670, AXC-671, AXC-672, AXC-675, AXC-681, AXC-687, AXC-688, AXC-689, AXC-690, AXC-691, AXC-697, AXC-699, AXC-700, AXC-701, AXC-702, AXC-709, AXC-710, AXC-711, AXC-713, AXC-714, AXC-717, AXC-719, AXC-722, AXC-723, AXC-724, AXC-725, AXC-726, AXC-727, AXC-731, AXC-732, AXC-733, AXC-734, AXC-735, AXC-736, AXC-737, AXC-738, AXC-739, AXC-740, AXC-741, AXC-743, AXC-742, AXC-747, AXC-748, AXC-750, AXC-751, AXC-752, AXC-754, AXC-755, AXC-756, AXC-757, AXC-758, AXC-759, AXC-760, AXC-761, AXC-762, AXC-764, AXC-771, AXC-772, AXC-773, AXC-777, AXC-778, AXC-779, AXC-789, AXC-793, AXC-800, AXC-801, AXC-802, AXC-803, AXC-804, AXC-805, AXC-806, AXC-807, AXC-808, AXC-809, AXC-810, AXC-831 and AXC-910 compounds. In other embodiments, the TLR agonist comprising a structure according to structure 1 is selected from the group of: AXC-801, AXC-802, AXC-831 and AXC-910 compounds. In other embodiments, the TLR agonist comprising a structure according to structure 1 selected from the group of: AXC-801, AXC-802, AXC-831 and AXC-910 compounds further comprises a linker.

[0052] In another embodiment, the anti-HER2 antibody or antibody fragment comprises one or more non-naturally encoded amino acid incorporated in the heavy chain, light chain, or both the heavy and light chains. In another embodiment, the one or more non-naturally encoded amino acids is selected from the group of para-acetyl phenylalanine, p-nitrophenylalanine, p17 2026205050   29 Jun 2026 sulfotyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-boronyl phenylalanine, o-boronylphenylalanine, m-boronylphenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-OMe phenylalanine, o-OMe phenylalanine, m-OMe phenylalanine, p-sulfophenylalanine, o-sulfophenylalanine, m-sulfophenylalanine, 5-nitro His, 3-nitro Tyr, 2-nitro Tyr, nitro substituted Leu, nitro substituted His, nitro substituted De, nitro substituted Trp, 2-nitro Trp, 4-nitro Trp, 5-nitro Trp, 6-nitro Trp, 7-nitro Trp, 3-aminotyrosine, 2-aminotyrosine, O-sulfotyrosine, 2-sulfooxyphenylalanine,      3-sulfooxyphenylalanine,      o-carboxyphenylalanine,      m- carboxyphenylalanine, p-acetyl-L-phenylalanine, p-propargyl-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcP-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propargyloxy-L-phenylalanine, 4-azido-L-phenylalanine, para-azidoethoxy phenylalanine, and para-azidomethyl-phenylalanine. In other embodiments, the non-natural amino acid is para-acetyl-phenylalanine, 4-azido-L-phenylalanine, para-azidomethyl-phenylalanine, or para-azidoethoxy phenylalanine.

[0053] In another embodiment, the anti-HER2 antibody or antibody fragment further comprises one or more mutations in the Fc region. In another embodiment, the anti-HER2 antibody or antibody fragment further comprises two or more mutations in the Fc region. In another embodiment, the anti-HER2 antibody or antibody fragment further comprises three or more mutations in the Fc region. In another embodiment, the anti-HER2 antibody or antibody fragment further comprises four or more mutations in the Fc region. In another embodiment, the anti-HER2 antibody or antibody fragment further comprises five or more mutations in the Fc region. In another embodiment, the anti-HER2 antibody or antibody fragment further comprises six or more mutations in the Fc region. In another embodiment, the anti-HER2 antibody or antibody fragment further comprises six mutations in the Fc region.

[0054] In another embodiment, the one or more linker is a cleavable or non-cleavable linker. In other embodiments, the one or more linker is a bifunctional or multifunctional linker.

[0055] In another embodiment, the TLR agonist comprises a structure according to structure 2 of Figure 1. In another embodiment, the TLR agonist comprising a structure according to structure 2 selected from the group of AXC-745, AXC-746, and AXC-753 compounds. In another embodiment, the TLR agonist comprising a structure according to any one of: AXC-745, 18 2026205050   29 Jun 2026 AXC-746, and AXC-753 compounds further comprises a linker. In another embodiment, the TLR agonist comprises a structure according to structure 2 further comprising a linker.

[0056] In another embodiment, the TLR agonist comprises a structure according to structure 3 of Figure 1. In another embodiment, the TLR agonist comprises a structure according to structure 3 is AXC-837 or AXC-847 compound. In another embodiment, the TLR agonist comprises a structure according to AXC-837 or AXC-847 compound further comprises a linker. In another embodiment, the TLR agonist comprises a structure according to AXC-847 compound further comprises a linker. In another embodiment, the TLR agonist comprises a structure according to structure 3 further comprising a linker.

[0057] In another embodiment, the TLR agonist comprises a structure according to structure 4 of Figure 1. In another embodiment, the TLR agonist comprising a structure according to structure 4 is selected from the group of: AXC-844, AXC-842, AXC-843, AXC-845, AXC-846, AXC-836, or AXC-841 compounds. In another embodiment, the TLR agonist comprising a structure according to structure 4 of any one of: AXC-844, AXC-842, AXC-843, AXC-845, AXC-846, AXC-836, or AXC-841 compounds further comprises a linker. In another embodiment, the TLR agonist comprises a structure according to structure 4 further comprising a linker.

[0058] In another embodiment, the TLR agonist comprises a structure according to structure 5 of Figure 1. In another embodiment, the TLR agonist comprising a structure according to structure 5 is selected from the group of: AXC-862, AXC-863, AXC-867, AXC-868, AXC-869, AXC-872, AXC-873, AXC-876, AXC-877, AXC-878, AXC-879, AXC-880, AXC-881, AXC-882, AXC-883, AXC-884, AXC-885, AXC-886, AXC-887, AXC-888, AXC-889, AXC-890, AXC-891, AXC-892, AXC-893, AXC-895, AXC-896, AXC-897, AXC-898, AXC-901, AXC-903, AXC-904, AXC-905, AXC-906, AXC-907, AXC-908, AXC-909, AXC-911, AXC-912, AXC-913, AXC-914, AXC-915, or AXC-916 compounds. In other embodiments, the TLR agonist comprising a structure according to structure 5 is selected from the group of: AXC-862, AXC-863, AXC-867, AXC-868, AXC-869, AXC-873, AXC-876, AXC-879, AXC-880, AXC-882, AXC-889, AXC-893, AXC-896, AXC-897, AXC-901, AXC-907, AXC-909, AXC-913, and AXC-914 compounds. In another embodiment, the TLR agonist comprising a structure according to any one of: AXC-862, AXC-863, AXC-867, AXC-868, AXC-869, AXC-872, AXC-873, AXC-876, AXC-877, AXC-878, AXC-879, AXC-880, AXC-881, AXC-882, AXC-883, AXC-884, AXC-885, AXC-886, AXC-887, AXC-888, AXC-889, AXC-890, AXC-891, AXC-892, AXC-893, AXC-895, AXC-896, AXC-897, AXC-898, AXC-901, AXC-903, AXC-19 2026205050   29 Jun 2026 904, AXC-905, AXC-906, AXC-907, AXC-908, AXC-909, AXC-911, AXC-912, AXC-913, AXC-914, AXC-915, or AXC-916 compounds further comprising a linker. In another embodiment, the TLR agonist comprises a structure according to structure 5 further comprising a linker.

[0059] In another embodiment, the anti-HER2 antibody or antibody fragment comprises the amino acid sequence of at least one of SEQ ID NOs: 1-13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises the amino acid sequence of at least two of SEQ ID NOs: 1-13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises a) SEQ ID NOs: 1 or 2; and b) any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises a) a heavy chain of SEQ ID NOs: 1 or 2; and b) a light chain of any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises a) SEQ ID NO: 1; and b) any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises a) SEQ ID NO: 2; and b) any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 3. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 4. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 5. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 6. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 7. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 8. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 9. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 10. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 11. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 12. In another embodiment, the anti-HER2 antibody or antibody fragment comprises SEQ ID NO: 2 and SEQ ID NO: 13. In another embodiment, the invention provides an anti-HER2 antibody or antibody fragment wherein the non-naturally encoded amino acid is site specifically incorporated at position 114 according to Kabat numbering.

[0060] 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 20 2026205050   29 Jun 2026 comprising 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 in the antibody or antibody fragment, the TC further comprising a chemotherapeutic or immunotherapeutic agent. In another embodiment, the present invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist selected from any one of the compounds of Tables 3-7, wherein the TLR agonist is conjugated to the antibody or antibody fragment via a linker covalently bonded to one or more non-naturally encoded amino acids incorporated in the antibody or antibody fragment. In another embodiment, the present invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist selected from any one of the compounds of Tables 3-7, wherein the TLR agonist is conjugated to the antibody or antibody fragment via a linker covalently bonded to one or more non-naturally encoded amino acids incorporated in the antibody or antibody fragment, the TC further comprising a chemotherapeutic or immunotherapeutic agent.

[0061] In another embodiment, the present invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist comprising a 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 in the antibody or antibody fragment, the TC further comprising an drug conjugate. In other embodiments the drug conjugate is an antibody drug conjugate. In another embodiment, the present invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist selected from any one of the compounds of Tables 3-7, wherein the TLR agonist is conjugated to the antibody or antibody fragment via a linker covalently bonded to one or more non-naturally encoded amino acids incorporated in the antibody or antibody fragment. In another embodiment, the present invention provides a TLR agonist conjugate (TC) comprising an anti-HER2 antibody or antibody fragment conjugated to a TLR agonist selected from any one of the compounds of Tables 3-7, wherein the TLR agonist is conjugated to the antibody or antibody fragment via a linker covalently bonded to one or more non-naturally encoded amino acids incorporated in the antibody or antibody fragment the TC further comprising an drug conjugate. In other embodiments the drug conjugate is an antibody drug conjugate. In other embodiments the TC further comprises a cytokine or cytotoxin. 2026205050   29 Jun 2026

[0062] In another embodiment, the present invention provides a method of treating a subject or patient having cancer or a disease or condition or indication or disorder comprising administering to the subject or patient a therapeutically-effective amount of a composition or TC of the invention. In certain embodiments, the tumor or cancer is a HER2 positive tumor or cancer. In certain embodiments, the tumor, cancer, indication, disease, disorder or condition is a HER2 positive tumor, cancer, indication, disease, disorder or condition. In certain embodiments, the tumor or cancer is selected from the group consisting of colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, glioblastoma, prostate cancer, bladder cancer, cervical cancer, pancreatic cancer, renal cancer, esophageal cancer, vaginal cancer, stomach cancer, and leukemia.

[0063] In another embodiment, the present invention provides a method of treating a subject or patient having cancer or a disease or condition comprising administering to the subject or patient a therapeutically-effective amount of a composition or TC of the invention., further comprising a chemotherapeutic or immunotherapeutic agent. In certain embodiments, the TC is co-administered with at least one chemotherapeutic agent. The chemotherapeutic agent can be selected from the group consisting of temozolomide, gemictabine, doxorubicin, cyclophosphamide, paclitaxel, cisplatin, fluoropyrimidine, taxane, anthracycline, lapatinib, capecitabine, letrozole, pertuzumab, docetaxel, IFN-a. In another embodiment of the present invention, TC is coadministered with at least one chemotherapeutic agent.

[0064] In another embodiment, the present invention provides a method of treating a subject or patient having cancer or a disease or condition comprising administering to the subject or patient a therapeutically-effective amount of a composition or TC of the invention, further comprising an antibody drug conjugate, a cytotoxic agent, or a checkpoint inhibitor.

[0065] In another embodiment, the present invention provides a method of killing a cell comprising contacting a cell with a TC of the invention. In other embodiments, the cell is a tumor or cancer cell. In certain embodiments, the tumor or cancer cell is a colon, ovarian, breast, melanoma, lung, glioblastoma, prostate, bladder, cervical, pancreatic, renal, esophageal, vaginal, stomach, or leukemia cancer cell. In certain embodiments, the tumor or cancer is a HER2 positive tumor or cancer. In certain embodiments, the tumor, cancer, indication, disease, disorder or condition to be treated is a HER2 positive tumor, cancer, indication, disease, disorder or condition.

[0066] The present invention provides methods of inhibiting or reducing growth of a tumor or cancer comprising contacting the tumor with an effective amount of TC of the present 22 2026205050   29 Jun 2026 invention to stimulate the immune system of the patient in proximity to the tumor. The present invention provides methods of inhibiting or reducing growth of a tumor or cancer comprising contacting the tumor with an effective amount of a PEGylated TC, or stable dimer or multimer of the TC, of the present invention. In one embodiment, the TC is non-pegylated or monopegylated. In one embodiment, the TC is dipegylated. In one embodiment, the TC has more than one and / or different TLR agonist molecules attached to it. Another embodiment of the present invention provides methods of using TCs of the present invention to modulate the immune response to tumor cells.

[0067] In some embodiments, the present invention provides methods of using a TC to treat cancer. In some embodiments, TCs of the present invention can be used in treating or preventing cancer-related diseases, disorders and conditions including conditions that are associated, directly or indirectly, with cancer, for example, angiogenesis and precancerous conditions such as dysplasia. In some embodiments, the tumor is a liquid or solid tumor. In some embodiments the condition to be treated is a cancer. The cancer may be, but is non-limited to, a breast cancer, a brain cancer, a pancreatic cancer, a skin cancer, a lung cancer, a liver cancer, a gall bladder cancer, a colon cancer, an ovarian cancer, a prostate cancer, a uterine cancer, a bone cancer, and a blood cancer (leukemic) cancer or a cancer or disease or conditions related to any of these cancers. Carcinomas are cancers that begin in the epithelial cells, which are cells that cover the surface of the body, produce hormones, and make up glands. By way of non-limiting example, carcinomas include breast cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, rectal cancer, kidney cancer, bladder cancer, stomach cancer, prostate cancer, liver cancer, ovarian cancer, brain cancer, vaginal cancer, vulvar cancer, uterine cancer, oral cancer, penile cancer, testicular cancer, esophageal cancer, skin cancer, cancer of the fallopian tubes, head and neck cancer, gastrointestinal stromal cancer, adenocarcinoma, cutaneous or intraocular melanoma, cancer of the anal region, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, cancer of the urethra, cancer of the renal pelvis, cancer of the ureter, cancer of the endometrium, cancer of the cervix, cancer of the pituitary gland, neoplasms of the central nervous system (CNS), primary CNS lymphoma, brain stem glioma, and spinal axis tumors. In some instances, the cancer is a skin cancer, such as a basal cell carcinoma, squamous, melanoma, nonmelanoma, or actinic (solar) keratosis. In some embodiments, the invention also relates to a method for treating an acute leukemia in a mammal, comprising administering a therapeutically effective amount of a TC of the present invention to said mammal. The invention also provides a method 23 2026205050   29 Jun 2026 for inhibiting proliferation of acute leukemia blast cells comprising administering a therapeutically effective dose of a TC of the present invention to a mammal suffering from an acute leukemia.

[0068] In another embodiment, the TCs disclosed herein may be used to modulate an immune response. Modulation of an immune response may comprise stimulating, activating, increasing, enhancing, or up-regulating an immune response. Modulation of an immune response may comprise suppressing, inhibiting, preventing, reducing, or downregulating an immune response.

[0069] In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a composition or TC of the invention and a pharmaceutically acceptable carrier or excipient.

[0070] In another embodiment, the present invention provides a use of the composition of the invention in the manufacture of a medicament.

[0071] In another embodiment, the present invention provides an immune stimulating antibody conjugate (ISAC) comprisisng a TLR-agonist according to any one of the structures of Figure 1. In another embodiment, the present invention provides an immune stimulating antibody conjugate (ISAC) comprisisng a TLR-agonist according to any one of the compounds of Tables 3, 4, 5, 6, 7. In another embodiment, the present invention provides ISACs wherein the TLR agonist comprises a compound selected from the group of: AXC-862, AXC-863, AXC-867, AXC-868, AXC-869, AXC-874, AXC-875, AXC-876, AXC-879, AXC-880, AXC-882, AXC-893, AXC-896, AXC-897, AXC-901, AXC-907, and AXC-910 compounds.

[0072] In another embodiment, the present invention provides a salt of any one of the compounds having a structure according to Figure 1. In another embodiment, the present invention provides a salt of any one of the compounds of Tables 3, 4, 5, 6, 7. In another embodiment, the present invention provides a pharmaceutical composition or salt thereof according to compositions, compounds and TC of the invention disclosure. In other embodiments, the pharmaceutical composition or salt further comprises a pharmaceutically acceptable excipient.

[0073] BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 depicts the general structure of TLR agonists suitable for use in the present invention.

[0075] Figure 2 depicts the structure of various TC conjugates. 24 2026205050   29 Jun 2026

[0076] Figure 3 depicts the structure of additional TC conjugates.

[0077] Figure 4 depicts the biological activity of selected TC conjugates in a cell proliferation assay.

[0078] Figures 5A and 5B depict TLR7 activities of various TLR7 agonists.

[0079] Figure 6 depicts TLR7 activities of various TLR7 agonists attached to a linker.

[0080] Figure 7 depicts TLR7 activities of additional TLR7 agonists and TLR7 agonists attached to a linker.

[0081] Figure 8 depicts TLR7 activities of additional TLR7 agonists and TLR7 agonists attached to a linker.

[0082] Figure 9 depicts TLR7 activities of different TLR7 agonist attached to a linker, (drug linker or DL), compared to a non-natural amino acid, pAF, (DL-pAF).

[0083] Figures 10A -10C depict HPLC chromatograms of unconjugated anti-HER2 antibody with a non-natural amino acid at amino acid position HA114 (Figures 10A), and anti-HER2 antibody conjugated at amino acid position HA114 with TLR agonist AXC-875 (Figure 10B) and AXC-880 (Figure 10C).

[0084] Figures 11A-11C compare tumor dependent ISAC activities of various payload linkers conjugated to anti-HER2 antibody in SKOV3 HER2 high expressing tumor cell line (Figure 11A); JIMT-1 HER2 medium / low expressing tumor cell line (Figure 11B); and A431 HER2 low expressing tumor cell line (Figure 11C).

[0085] Figures 12A and 12B compare tumor dependent ISAC activities of additional payload linkers conjugated to anti-HER2 antibody in SKBR3 HER2 high expressing tumor cell line, (Figure 12A), and HCC1806 HER2 very low expressing tumor cell line (Figure 12B).

[0086] Figures 13A and 13B compare tumor dependent ISAC activities of additional payload linkers conjugated to anti-HER2 antibody in SKBR3 HER2 high expressing tumor cell line, (Figure 13A), and HCC1806 HER2 very low expressing tumor cell line, (Figure 13B).

[0087] Figures 14A and 14B compare tumor-dependent ISAC activities of additional payload linkers conjugated to anti-HER2 antibody in SKBR3 HER2 high expressing tumor cell line (Figure 14A), and HCC1806 HER2 very low expressing tumor cell line, (Figure 14B).

[0088] Figures 15A and 15B compare tumor-dependent ISAC activities of three (3) payload linkers conjugated to anti-HER2 antibody in SKBR3 HER2 high expressing tumor cell line (Figure 15A), and HCC1806 HER2 very low expressing tumor cell line, (Figure 15B) showing HER2-AXC-879 has the best ISAC activity. 2026205050   29 Jun 2026 DETAILED DESCRIPTION OF THE INVENTION

[0089] Disclosed herein are TCs comprising a targeting moiety such as an antibody and one or more TLR agonists. The TLR agonist may further comprise one or more linker(s). The TCs of the present invention may comprise TLR agonists linked to non-natural amino acids in the targeting moiety. Also included are methods for making such TCs comprising non-natural amino acids incorporated into the targeting moiety polypeptides.

[0090] In certain embodiments, a pharmaceutical composition is provided comprising any of the compounds described and a pharmaceutically acceptable carrier, excipient, or binder.

[0091] In further or alternative embodiments are methods for detecting the presence of a polypeptide in a patient, the method comprising administering a polypeptide comprising at least one heterocycle-containing non-natural amino acid and the resulting heterocycle-containing nonnatural amino acid polypeptide modulates the immunogenicity of the polypeptide relative to the homologous naturally-occurring amino acid polypeptide.

[0092] It is to be understood that the methods and compositions described herein are not limited to the particular methodology, protocols, cell lines, constructs, and reagents described herein and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the methods and compositions described herein, which will be limited only by the appended claims.

[0093] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly indicates otherwise.

[0094] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the inventions described herein belong. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the inventions described herein, the preferred methods, devices and materials are now described.

[0095] All publications and patents mentioned herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the constructs and methodologies that are described in the publications, which might be used in connection with the presently described inventions. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as 2026205050   29 Jun 2026 an admission that the inventors described herein are not entitled to antedate such disclosure by virtue of prior invention or for any other reason.

[0096] The terms “aldol-based linkage” or “mixed aldol-based linkage” refers to the acid- or base-catalyzed condensation of one carbonyl compound with the enolate / enol of another carbonyl compound, which may or may not be the same, to generate a P-hydroxy carbonyl compound—an aldol.

[0097] The term “affinity label,” as used herein, refers to a label which reversibly or irreversibly binds another molecule, either to modify it, destroy it, or form a compound with it. By way of example, affinity labels include enzymes and their substrates, or antibodies and their antigens.

[0098] The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) are used in their conventional sense and refer to those alkyl groups linked to molecules via an oxygen atom, an amino group, or a sulfur atom, respectively.

[0099] The term “alkyl,” by itself or as part of another molecule means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e. C1-C10 means one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term “alkyl,” unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail herein, such as “heteroalkyl”, “haloalkyl” and “homoalkyl”.

[00100] The term “alkylene” by itself or as part of another molecule means a divalent radical derived from an alkane, as exemplified, by (-CH2-)n, wherein n may be 1 to about 24. By way of example only, such groups include, but are not limited to, groups having 10 or fewer carbon atoms such as the structures -CH2CH2- and -CH2CH2CH2CH2-. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. The term “alkylene,” unless otherwise noted, is also meant to include those groups described herein as “heteroalkylene.” 2026205050   29 Jun 2026

[00101] The term “amino acid” refers to naturally occurring and non-natural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the 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) and pyrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, by way of example only, an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs may have modified R groups (by way of example, norleucine) or may have modified peptide backbones while still retaining the same basic chemical structure as a naturally occurring amino acid. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.

[00102] Amino acids may be referred to herein by either their name, their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Additionally, nucleotides, may be referred to by their commonly accepted single-letter codes.

[00103] An “amino terminus modification group” refers to any molecule that can be attached to a terminal amine group. By way of example, such terminal amine groups may be at the end of polymeric molecules, wherein such polymeric molecules include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. Terminus modification groups include but are not limited to, various water soluble polymers, peptides or proteins. By way of example only, terminus modification groups include polyethylene glycol or serum albumin. Terminus modification groups may be used to modify therapeutic characteristics of the polymeric molecule, including but not limited to increasing the serum half-life of peptides.

[00104] By "antibody" herein is meant a protein consisting of one or more polypeptides substantially encoded by all or part of the antibody genes. The immunoglobulin genes include, but are not limited to, the kappa, lambda, alpha, gamma (IgG1, IgG2, IgG3, and IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Antibody herein is meant to include full-length antibodies and antibody fragments and include antibodies that exist naturally in any organism or are engineered (e.g. are variants).

[00105] By “antibody fragment” is meant any form of an antibody other than the full-length form. Antibody fragments herein include antibodies that are smaller components that exist within full-length antibodies, and antibodies that have been engineered. Antibody fragments 28 2026205050   29 Jun 2026 include but are not limited to Fv, Fc, Fab, and (Fab')2, single chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDR’s, variable regions, framework regions, constant regions, heavy chains, light chains, and variable regions, and alternative scaffold non-antibody molecules, bispecific antibodies, and the like (Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9:395-402). Another functional substructure is a single chain Fv (scFv), comprised of the variable regions of the immunoglobulin heavy and light chain, covalently connected by a peptide linker (S-z Hu et al., 1996, Cancer Research, 56, 3055-3061). These small (Mr 25,000) proteins generally retain specificity and affinity for antigen in a single polypeptide and can provide a convenient building block for larger, antigen-specific molecules. Unless specifically noted otherwise, statements and claims that use the term “antibody” or “antibodies” specifically includes “antibody fragment” and “antibody fragments”.

[00106] By “antibody-drug conjugate, or “ADC”, as used herein, refers to an antibody molecule, or fragment thereof, that is covalently bonded to one or more biologically active molecule(s). The biologically active molecule may be conjugated to the antibody through a linker, polymer, or other covalent bond.

[00107] The term “aromatic” or “aryl”, as used herein, refers to a closed ring structure which has at least one ring having a conjugated pi electron system and includes both carbocyclic aryl and heterocyclic aryl (or “heteroaryl” or “heteroaromatic”) groups. The carbocyclic or heterocyclic aromatic group may contain from 5 to 20 ring atoms. The term includes monocyclic rings linked covalently or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups. An aromatic group can be unsubstituted or substituted. Non-limiting examples of “aromatic” or “aryl”, groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, anthracenyl, and phenanthracenyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described herein.

[00108] For brevity, the term “aromatic” or “aryl” when used in combination with other terms (including but not limited to, aryloxy, arylthioxy, aralkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term “aralkyl” or “alkaryl” is meant to include those radicals in which an aryl group is attached to an alkyl group (including but not limited to, benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (including but not limited to, a methylene group) has been replaced by a heteroatom, by way of example only, by an oxygen atom. Examples of such aryl groups include, but are not limited to, phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like. 2026205050   29 Jun 2026

[00109] The term “arylene”, as used herein, refers to a divalent aryl radical. Non-limiting examples of “arylene” include phenylene, pyridinylene, pyrimidinylene and thiophenylene. Substituents for arylene groups are selected from the group of acceptable substituents described herein.

[00110] A “bifunctional polymer”, also referred to as a “bifunctional linker”, refers to a polymer comprising two functional groups that are capable of reacting specifically with other moieties to form covalent or non-covalent linkages. Such moieties may include, but are not limited to, the side groups on natural or non-natural amino acids or peptides which contain such natural or non-natural amino acids. The other moieties that may be linked to the bifunctional linker or bifunctional polymer may be the same or different moieties. By way of example only, a bifunctional linker may have a functional group reactive with a group on a first peptide, and another functional group which is reactive with a group on a second peptide, whereby forming a conjugate that includes the first peptide, the bifunctional linker and the second peptide. Many procedures and linker molecules for attachment of various compounds to peptides are known. See, e.g., European Patent Application No. 188,256; U.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; and 4,569,789 which are incorporated by reference herein in their entirety. A “multi-functional polymer” also referred to as a “multi-functional linker”, refers to a polymer comprising two or more functional groups that are capable of reacting with other moieties. Such moieties may include, but are not limited to, the side groups on natural or nonnatural amino acids or peptides which contain such natural or non-natural amino acids. (including but not limited to, amino acid side groups) to form covalent or non-covalent linkages. A bi-functional polymer or multi-functional polymer may be any desired length or molecular weight, and may be selected to provide a particular desired spacing or conformation between one or more molecules linked to a compound and molecules it binds to or the compound.

[00111] The term “bioavailability,” as used herein, refers to the rate and extent to which a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. Increases in bioavailability refers to increasing the rate and extent a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. By way of example, an increase in bioavailability may be indicated as an increase in concentration of the substance or its active moiety in the blood when compared to other substances or active moieties. Methods to evaluate increases in bioavailability are known in the art and may be used for evaluating the bioavailability of any polypeptide. 2026205050   29 Jun 2026

[00112] The term “biologically active molecule”, “biologically active moiety” or “biologically active agent” when used herein means any substance which can affect any physical or biochemical properties of a biological system, pathway, molecule, or interaction relating to an organism, including but not limited to, viruses, bacteria, bacteriophage, transposon, prion, insects, fungi, plants, animals, and humans. In particular, as used herein, biologically active molecules include but are not limited to any substance intended for diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental well-being of humans or animals. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, prodrugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles and micelles. Classes of biologically active agents that are suitable for use with the methods and compositions described herein include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, anti-viral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroidal agents, microbially derived toxins, and the like.

[00113] By “modulating biological activity” is meant increasing or decreasing the reactivity of a polypeptide, altering the selectivity of the polypeptide, enhancing or decreasing the substrate selectivity of the polypeptide. Analysis of modified biological activity can be performed by comparing the biological activity of the non-natural polypeptide to that of the natural polypeptide.

[00114] The term “biomaterial,” as used herein, refers to a biologically-derived material, including but not limited to material obtained from bioreactors and / or from recombinant methods and techniques.

[00115] The term “biophysical probe,” as used herein, refers to probes which can detect or monitor structural changes in molecules. Such molecules include, but are not limited to, proteins and the “biophysical probe” may be used to detect or monitor interaction of proteins with other macromolecules. Examples of biophysical probes include, but are not limited to, spin-labels, a fluorophores, and photoactivatible groups.

[00116] The term “biosynthetically,” as used herein, refers to any method utilizing a translation system (cellular or non-cellular), including use of at least one of the following components: a polynucleotide, a codon, a tRNA, and a ribosome. By way of example, nonnatural amino acids may be “biosynthetically incorporated” into non-natural amino acid 31 2026205050   29 Jun 2026 polypeptides using the methods and techniques described in WO 2002 / 085923, incorporated herein by reference in its entirety. Additionally, the methods for the selection of useful nonnatural amino acids which may be “biosynthetically incorporated” into non-natural amino acid polypeptides are described in WO 2002 / 085923, incorporated herein by reference in its entirety.

[00117] The term “biotin analogue,” or also referred to as “biotin mimic”, as used herein, is any molecule, other than biotin, which bind with high affinity to avidin and / or streptavidin.

[00118] The term “carbonyl” as used herein refers to a group containing at a moiety selecting from the group consisting of -C(O)-, -S(O)-, -S(O)2-, and -C(S)-, including, but not limited to, groups containing a least one ketone group, and / or at least one aldehyde groups, and / or at least one ester group, and / or at least one carboxylic acid group, and / or at least one thioester group. Such carbonyl groups include ketones, aldehydes, carboxylic acids, esters, and thioesters. In addition, such groups may be part of linear, branched, or cyclic molecules.

[00119] The term “carboxy terminus modification group” refers to any molecule that can be attached to a terminal carboxy group. By way of example, such terminal carboxy groups may be at the end of polymeric molecules, wherein such polymeric molecules include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. Terminus modification groups include but are not limited to, various water soluble polymers, peptides or proteins. By way of example only, terminus modification groups include polyethylene glycol or serum albumin. Terminus modification groups may be used to modify therapeutic characteristics of the polymeric molecule, including but not limited to increasing the serum half-life of peptides.

[00120] The term “chemically cleavable group,” also referred to as “chemically labile”, as used herein, refers to a group which breaks or cleaves upon exposure to acid, base, oxidizing agents, reducing agents, chemical inititiators, or radical initiators.

[00121] “Cofolding,” as used herein, refers to refolding processes, reactions, or methods which employ at least two molecules which interact with each other and result in the transformation of unfolded or improperly folded molecules to properly folded molecules. By way of example only, “cofolding,” employ at least two polypeptides which interact with each other and result in the transformation of unfolded or improperly folded polypeptides to native, properly folded polypeptides. Such polypeptides may contain natural amino acids and / or at least one non-natural amino acid.

[00122] "Conjugate", as used herein, refers to a polypeptide that is linked, e.g., covalently linked, either directly or through a linker to a compound or compound-linker described herein, e.g., a compound or salt of any one of structures according to Figure 1, or any one of structures 32 2026205050   29 Jun 2026 of Tables 3-7. The "targeting moiety" refers to a structure that has a selective affinity for a target molecule relative to other non-target molecules. A targeting moiety of the invention binds to a target molecule. A targeting moiety may include, for example, an antibody, a peptide, a ligand, a receptor, or a binding portion thereof. A target biological molecule may be a biological receptor or other structure of a cell such as a tumor antigen. As used herein, the term “conjugate of the invention,” “targeting moiety conjugate” “targeting conjugate,” “targeting moiety-active molecule conjugate" or “TC” refers to a targeting polypeptide or a portion, analog or derivative thereof that binds to a target present on a cell or subunit thereof conjugated to a biologically active molecule, a portion thereof or an analog thereof, including but not limited to a TLR7 and / or a TLR8 agonist. As used herein, the term “tumor-targeting moiety conjugate” "tumortargeting moiety-biologically active molecule conjugate" or “BTC” refers to a tumor targeting polypeptide or a portion, analog or derivative thereof that binds to a target present on tumor cells or subunit thereof conjugated to a biologically active molecule, a portion thereof or an analog thereof, including but not limited to a TLR7 and / or a TLR8 agonist. Unless otherwise indicated, the terms "compound of the invention" and "composition of the invention" are used as alternatives for the term "conjugate of the invention."

[00123] The term “conservatively modified variants” applies to both natural and non-natural amino acid and natural and non-natural nucleic acid sequences, and combinations thereof. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those natural and non-natural nucleic acids which encode identical or essentially identical natural and non-natural amino acid sequences, or where the natural and non-natural nucleic acid does not encode a natural and non-natural amino acid sequence, to essentially identical sequences. By way of example, because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Thus, by way of example, every natural or nonnatural nucleic acid sequence herein which encodes a natural or non-natural polypeptide also describes every possible silent variation of the natural or non-natural nucleic acid. One of ordinary skill in the art will recognize that each codon in a natural or non-natural nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. 33 2026205050   29 Jun 2026 Accordingly, each silent variation of a natural and non-natural nucleic acid which encodes a natural and non-natural polypeptide is implicit in each described sequence.

[00124] As to amino acid sequences, individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single natural and non-natural amino acid or a small percentage of natural and non-natural amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the deletion of an amino acid, addition of an amino acid, or substitution of a natural and non-natural amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar natural amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the methods and compositions described herein.

[00125] Conservative substitution tables providing functionally similar amino acids are known to those of ordinary skill in the art. The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M). (See, e.g., Creighton, Proteins:Structures and Molecular Properties (W H Freeman & Co.; 2nd edition (December 1993).

[00126] The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl”, respectively. Thus, a cycloalkyl or heterocycloalkyl include saturated, partially unsaturated and fully unsaturated ring linkages. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. 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, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. Additionally, the term encompasses multicyclic structures, including but not limited to, bicyclic and tricyclic ring structures. Similarly, the term “heterocycloalkylene” by itself or as part of another molecule means a divalent radical derived 2026205050   29 Jun 2026 from heterocycloalkyl, and the term “cycloalkylene” by itself or as part of another molecule means a divalent radical derived from cycloalkyl.

[00127] The term “cyclodextrin,” as used herein, refers to cyclic carbohydrates consisting of at least six to eight glucose molecules in a ring formation. The outer part of the ring contains water soluble groups; at the center of the ring is a relatively nonpolar cavity able to accommodate small molecules.

[00128] The term “cytotoxic,” as used herein, refers to a compound which harms cells.

[00129] “Denaturing agent” or “denaturant,” as used herein, refers to any compound or material which will cause a reversible unfolding of a polymer. By way of example only, “denaturing agent” or “denaturants,” may cause a reversible unfolding of a protein. The strength of a denaturing agent or denaturant will be determined both by the properties and the concentration of the particular denaturing agent or denaturant. By way of example, denaturing agents or denaturants include, but are not limited to, chaotropes, detergents, organic, water miscible solvents, phospholipids, or a combination thereof. Non-limiting examples of chaotropes include, but are not limited to, urea, guanidine, and sodium thiocyanate. Non-limiting examples of detergents may include, but are not limited to, strong detergents such as sodium dodecyl sulfate, or polyoxyethylene ethers (e.g. Tween or Triton detergents), Sarkosyl, mild non-ionic detergents (e.g., digitonin), mild cationic detergents such as N-›2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents (e.g. sodium cholate or sodium deoxycholate) or zwitterionic detergents including, but not limited to, sulfobetaines (Zwittergent), 3-(3-chlolamidopropyl)dimethylammonio-1-propane     sulfate     (CHAPS),     and     3-(3- chlolamidopropyl)dimethylammonio-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 alkandiols (C2 -C4 alkandiols such as ethylene-glycol) may be used as denaturants. Non-limiting examples of phospholipids include, but are not limited to, naturally occurring phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol or synthetic phospholipid derivatives or variants such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

[00130] The term “diamine,”as used herein, refers to groups / molecules comprising at least two amine functional groups, including, but not limited to, a hydrazine group, an amidine group, an imine group, a 1,1-diamine group, a 1,2-diamine group, a 1,3-diamine group, and a 1,4-diamine group. In addition, such groups may be part of linear, branched, or cyclic molecules. 35 2026205050   29 Jun 2026

[00131] The term “detectable label,” as used herein, refers to a label which may be observable using analytical techniques including, but not limited to, fluorescence, chemiluminescence, electron-spin resonance, ultraviolet / visible absorbance spectroscopy, mass spectrometry, nuclear magnetic resonance, magnetic resonance, and electrochemical methods.

[00132] The term “dicarbonyl” as used herein refers to a group containing at least two moieties selected from the group consisting of -C(O)-, -S(O)-, -S(O)2-, and -C(S)-, including, but not limited to, 1,2-dicarbonyl groups, a 1,3-dicarbonyl groups, and 1,4-dicarbonyl groups, and groups containing a least one ketone group, and / or at least one aldehyde groups, and / or at least one ester group, and / or at least one carboxylic acid group, and / or at least one thioester group. Such dicarbonyl groups include diketones, ketoaldehydes, ketoacids, ketoesters, and ketothioesters. In addition, such groups may be part of linear, branched, or cyclic molecules. The two moieties in the dicarbonyl group may be the same or different, and may include substituents that would produce, by way of example only, an ester, a ketone, an aldehyde, a thioester, or an amide, at either of the two moieties.

[00133] The term “drug,” as used herein, refers to any substance used in the prevention, diagnosis, alleviation, treatment, or cure of a disease or condition.

[00134] The term “effective amount,” as used herein, refers to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. By way of example, an agent or a compound being administered includes, but is not limited to, a natural amino acid polypeptide, non-natural amino acid polypeptide, modified natural amino acid polypeptide, or modified non-amino acid polypeptide. Compositions containing such natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides can be administered for prophylactic, enhancing, and / or therapeutic treatments. An appropriate “effective” amount in any individual case may be determined using techniques, such as a dose escalation study.

[00135] The terms “enhance” or “enhancing” means to increase or prolong either in potency or duration a desired effect. By way of example, “enhancing” the effect of therapeutic agents refers to the ability to increase or prolong, either in potency or duration, the effect of therapeutic agents on during treatment of a disease, disorder or condition. An “enhancing-effective amount,” as used herein, refers to an amount adequate to enhance the effect of a therapeutic agent in the 36 2026205050   29 Jun 2026 treatment of a disease, disorder or condition. When used in a patient, amounts effective for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.

[00136] As used herein, the term “eukaryote” refers to organisms belonging to the phylogenetic domain Eucarya, including but not limited to animals (including but not limited to, mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to, monocots, dicots, and algae), fungi, yeasts, flagellates, microsporidia, and protists.

[00137] The term “fatty acid,” as used herein, refers to carboxylic acids with about C6 or longer hydrocarbon side chain.

[00138] The term “fluorophore,” as used herein, refers to a molecule which upon excitation emits photons and is thereby fluorescent.

[00139] The terms “functional group”, “active moiety”, “activating group”, “leaving group”, “reactive site”, “chemically reactive group” and “chemically reactive moiety,” as used herein, refer to portions or units of a molecule at which chemical reactions occur. The terms are somewhat synonymous in the chemical arts and are used herein to indicate the portions of molecules that perform some function or activity and are reactive with other molecules.

[00140] The term “halogen” includes fluorine, chlorine, iodine, and bromine.

[00141] The term “haloacyl,” as used herein, refers to acyl groups which contain halogen moieties, including, but not limited to, -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like.

[00142] The term “haloalkyl,” as used herein, refers to alkyl groups which contain halogen moieties, including, but not limited to, -CF3 and -CH2CF3 and the like.

[00143] The term “heteroalkyl,” as used herein, refers to straight or branched chain, or cyclic hydrocarbon radicals, or combinations thereof, consisting of an alkyl group and at least one heteroatom selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3,  -CH2-S-CH2-CH3,  -CH2-CH2,-S(O)-CH3,  -CH2-CH2-S(O)2-CH3,  - CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. In addition, up to two heteroatoms may be consecutive, such as, by way of example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. 2026205050   29 Jun 2026

[00144] The terms “heterocyclic-based linkage” or “heterocycle linkage” refers to a moiety formed from the reaction of a dicarbonyl group with a diamine group. The resulting reaction product is a heterocycle, including a heteroaryl group or a heterocycloalkyl group. The resulting heterocycle group serves as a chemical link between a non-natural amino acid or non-natural amino acid polypeptide and another functional group. In one embodiment, the heterocycle linkage includes a nitrogen-containing heterocycle linkage, including by way of example only a pyrazole linkage, a pyrrole linkage, an indole linkage, a benzodiazepine linkage, and a pyrazalone linkage.

[00145] Similarly, the term “heteroalkylene” refers to a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, the same or different heteroatoms can also occupy either or both of the chain termini (including but not limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. By way of example, the formula -C(O)2R’-represents both -C(O)2R’- and -R’C(O)2-.

[00146] The term “heteroaryl” or “heteroaromatic,” as used herein, refers to aryl groups which contain at least one heteroatom selected from N, O, and S; wherein the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen atom(s) may be optionally quaternized. Heteroaryl groups may be substituted or unsubstituted. A heteroaryl group may be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.

[00147] The term “homoalkyl,” as used herein refers to alkyl groups which are hydrocarbon groups.

[00148] The term “identical,” as used herein, refers to two or more sequences or subsequences which are the same. In addition, the term “substantially identical,” as used herein, refers to two or more sequences which have a percentage of sequential units which are the same when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using comparison algorithms or by manual alignment and visual inspection. 38 2026205050   29 Jun 2026 By way of example only, two or more sequences may be “substantially identical” if the sequential units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. Such percentages to describe the “percent identity” of two or more sequences. The identity of a sequence can exists over a region that is at least about 75-100 sequential units in length, over a region that is about 50 sequential units in length, or, where not specified, across the entire sequence. This definition also refers to the complement of a test sequence. By way of example only, two or more polypeptide sequences are identical when the amino acid residues are the same, while two or more polypeptide sequences are “substantially identical” if the amino acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. The identity can exist over a region that is at least about 75 to about 100 amino acids in length, over a region that is about 50 amino acids in length, or, where not specified, across the entire sequence of a polypeptide sequence. In addition, by way of example only, two or more polynucleotide sequences are identical when the nucleic acid residues are the same, while two or more polynucleotide sequences are “substantially identical” if the nucleic acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. The identity can exist over a region that is at least about 75 to about 100 nucleic acids in length, over a region that is about 50 nucleic acids in length, or, where not specified, across the entire sequence of a polynucleotide sequence.

[00149] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[00150] The term “immunogenicity,” as used herein, refers to an antibody response to administration of a therapeutic drug. The immunogenicity toward therapeutic non-natural amino acid polypeptides can be obtained using quantitative and qualitative assays for detection of anti-non-natural amino acid polypeptides antibodies in biological fluids. Such assays include, but are not limited to, Radioimmunoassay (RIA), Enzyme-linked immunosorbent assay (ELISA), 39 2026205050   29 Jun 2026 luminescent immunoassay (LIA), and fluorescent immunoassay (FIA). Analysis of immunogenicity toward therapeutic non-natural amino acid polypeptides involves comparing the antibody response upon administration of therapeutic non-natural amino acid polypeptides to the antibody response upon administration of therapeutic natural amino acid polypeptides.

[00151] The term “isolated,” as used herein, refers to separating and removing a component of interest from components not of interest. Isolated substances can be in either a dry or semi-dry state, or in solution, including but not limited to an aqueous solution. The isolated component can be in a homogeneous state or the isolated component can be a part of a pharmaceutical composition that comprises 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. In addition, when a component of interest is isolated and is the predominant species present in a preparation, the component is described herein as substantially purified. The term “purified,” as used herein, may refer to a component of interest which is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or greater pure. By way of example only, nucleic acids or proteins are “isolated” when such nucleic acids or proteins are free of at least some of the cellular components with which it is associated in the natural state, or that the nucleic acid or protein has been concentrated to a level greater than the concentration of its in vivo or in vitro production. Also, by way of example, a gene is isolated when separated from open reading frames which flank the gene and encode a protein other than the gene of interest.

[00152] The term “label,” as used herein, refers to a substance which is incorporated into a compound and is readily detected, whereby its physical distribution may be detected and / or monitored.

[00153] The term “linkage” or “linker” as used herein to refer to bonds or chemical moiety formed from a chemical reaction between the functional group of a linker and another molecule. Such bonds may include, but are not limited to, covalent linkages and non-covalent bonds, while such chemical moieties may include, but are not limited to, esters, carbonates, imines phosphate esters, hydrazones, acetals, orthoesters, peptide linkages, and oligonucleotide linkages. Hydrolytically stable linkages mean that the linkages are substantially stable in water and do not react with water at useful pH values, including but not limited to, under physiological conditions for an extended period of time, perhaps even indefinitely. Hydrolytically unstable or degradable linkages mean that the linkages are degradable in water or in aqueous solutions, including for example, blood. Enzymatically unstable or degradable linkages mean that the linkage can be 40 2026205050   29 Jun 2026 degraded by one or more enzymes. By way of example only, PEG and related polymers may include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. Such degradable linkages include, but are not limited to, ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent, wherein such ester groups generally hydrolyze under physiological conditions to release the biologically active agent. Other hydrolytically degradable linkages include but are not limited to carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which are reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; peptide linkages formed by an amine group, including but not limited to, at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polymer, and a 5' hydroxyl group of an oligonucleotide. Linkers include but are not limited to short linear, branched, multi-armed, or dendrimeric molecules such as polymers. In some embodiments of the 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. A number of different cleavable linkers are known to those of skill in the art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. The mechanisms for release of an agent from these linker groups include, for example, irradiation of a photolabile bond and acid-catalyzed hydrolysis. U.S. Pat. No. 4,671,958, for example, includes a description of immunoconjugates comprising linkers which are cleaved at the target site in vivo by the proteolytic enzymes of the patient's complement system. The length of the linker may be predetermined or selected depending upon a desired spatial relationship between the polypeptide and the molecule linked to it. In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and other agents to antibodies one skilled in the art will be able to determine a suitable method for attaching a given agent or molecule to a polypeptide.

[00154] The term “modified,” as used herein refers to the presence of a change to a natural amino acid, a non-natural amino acid, a natural amino acid polypeptide or a non-natural amino acid polypeptide. Such changes, or modifications, may be obtained by post synthesis modifications of natural amino acids, non-natural amino acids, natural amino acid polypeptides 41 2026205050   29 Jun 2026 or non-natural amino acid polypeptides, or by co-translational, or by post-translational modification of natural amino acids, non-natural amino acids, natural amino acid polypeptides or non-natural amino acid polypeptides. The form “modified or unmodified” means that the natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide being discussed are optionally modified, that is, he natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide under discussion can be modified or unmodified.

[00155] As used herein, the term “modulated serum half-life” refers to positive or negative changes in the circulating half-life of a modified biologically active molecule relative to its nonmodified form. By way of example, the modified biologically active molecules include, but are not limited to, natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide. By way of example, serum half-life is measured by taking blood samples at various time points after administration of the biologically active molecule or modified biologically active molecule, and determining the concentration of that molecule in each sample. Correlation of the serum concentration with time allows calculation of the serum half-life. By way of example, modulated serum half-life may be an increased in serum half-life, which may enable improved dosing regimens or avoid toxic effects. Such increases in serum may be at least about two fold, at least about three-fold, at least about five-fold, or at least about ten-fold. Methods to evaluate increases in serum half-life of any polypeptide are well know to the skilled artisan.

[00156] The term “modulated therapeutic half-life,” as used herein, refers to positive or negative change in the half-life of the therapeutically effective amount of a modified biologically active molecule, relative to its non-modified form. By way of example, the modified biologically active molecules include, but are not limited to, natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide. By way of example, therapeutic half-life is measured by measuring pharmacokinetic and / or pharmacodynamic properties of the molecule at various time points after administration. Increased therapeutic halflife may enable a particular beneficial dosing regimen, a particular beneficial total dose, or avoids an undesired effect. By way of example, the increased therapeutic half-life may result from increased potency, increased or decreased binding of the modified molecule to its target, an increase or decrease in another parameter or mechanism of action of the non-modified molecule, or an increased or decreased breakdown of the molecules by enzymes such as, by way of 2026205050   29 Jun 2026 example only, proteases. Methods to evaluate increases in therapeutic half-life of any polypeptide are well known to the skilled artisan.

[00157] A “non-natural amino acid” refers to an amino acid that is not one of the 20 common amino acids or pyrolysine or selenocysteine. Other terms that may be used synonymously with the term “non-natural amino acid” is “non-naturally encoded amino acid,” “unnatural amino acid,” “non-naturally-occurring amino acid,” and variously hyphenated and non-hyphenated versions thereof. The term “non-natural amino acid” includes, but is not limited to, amino acids which occur naturally by modification of a naturally encoded amino acid (including but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine) but are not themselves incorporated into a growing polypeptide chain by the translation complex. Examples of such amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine. Additionally, the term “non-natural amino acid” includes, but is not limited to, amino acids which do not occur naturally and may be obtained synthetically or may be obtained by modification of non-natural amino acids. In some embodiments, non-natural amino acids comprise a lysine analog, for example, N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, or allyloxycarbonyl lysine. In some embodiments, non-natural amino acids comprise a saccharide moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine,    N-acetyl-L-galactosaminyl-L-serine,    N-acetyl-L-glucosaminyl-L- threonine, N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine. Examples of such amino acids also include examples where the naturally-occurring N- or O- linkage between the amino acid and the saccharide is replaced by a covalent linkage not commonly found in nature - including but not limited to, an alkene, an oxime, a thioether, an amide and the like. Examples of such amino acids also include saccharides that are not commonly found in naturally-occurring proteins such as 2-deoxy-glucose, 2-deoxygalactose and the like. Specific examples of non-natural amino acids include, but are not limited to, a p-acetyl-L- phenylalanine, a p-propargyloxyphenylalanine, O-methyl-l-tyrosine, an l-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAcp-serine, an L-Dopa, a fluorinated phenylalanine, a isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, a L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, a p-amino-L-phenylalanine, a p-propargyloxy-L-phenylalanine, a 4-azido-L-phenylalanine, a para- 2026205050   29 Jun 2026 azidoethoxy phenylalanine, and a para-azidomethyl-phenylalanine, and the like. In some embodiments, the non-natural amino acid is selected from a group consisting of para-acetyl-phenylalanine, 4-azido-L-phenylalanine, para-azidoethoxy phenylalanine or para-azidomethyl-phenylalanine.

[00158] The term “nucleic acid,” as used herein, refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in either single-or double-stranded form. By way of example only, such nucleic acids and nucleic acid polymers include, but are not limited to, (i) analogues of natural nucleotides which have similar binding properties as a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides; (ii) oligonucleotide analogs including, but are not limited to, PNA (peptidonucleic acid), analogs of DNA used in antisense technology (phosphorothioates, phosphoroamidates, and the like); (iii) conservatively modified variants thereof (including but not limited to, degenerate codon substitutions) and complementary sequences and sequence explicitly indicated. By way of example, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[00159] The term “oxidizing agent,” as used herein, refers to a compound or material which is capable of removing an electron from a compound being oxidized. By way of example oxidizing agents include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythreitol, and oxygen. A wide variety of oxidizing agents are suitable for use in the methods and compositions described herein.

[00160] The term “pharmaceutically acceptable”, as used herein, refers to a material, including but not limited, to a salt, carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[00161] The term “polyalkylene glycol,” or “poly(alkene glycol)” as used herein, refers to linear or branched polymeric polyether polyols. Such polyalkylene glycols, including, but are not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof. Other exemplary embodiments are listed, for example, in commercial supplier catalogs, such as Shearwater Corporation's catalog “Polyethylene Glycol and Derivatives for Biomedical 44 2026205050   29 Jun 2026 Applications” (2001). By way of example only, such polymeric polyether polyols have average molecular weights between about 0.1 kDa to about 100 kDa. By way of example, such polymeric polyether polyols include, but are not limited to, between about 100 Da and about 100,000 Da or more. The molecular weight of the polymer may be between about 100 Da and about 100,000 Da, including but not limited to, about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da, about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, about 1,000 Da, about 900 Da, about 800 Da, about 700 Da, about 600 Da, about 500 Da, 400 Da, about 300 Da, about 200 Da, and about 100 Da. In some embodiments molecular weight of the polymer is between about 100 Da and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 2,000 to about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da. In some embodiments, the poly(ethylene glycol) molecule is a branched polymer. The molecular weight of the branched chain PEG may be between about 1,000 Da and about 100,000 Da, including but not limited to, about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da, about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, and about 1,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 50,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 20,000 Da. In other embodiments, the molecular weight of the branched chain PEG is between about 2,000 to about 50,000 Da. 2026205050   29 Jun 2026

[00162] The term “polymer,” as used herein, refers to a molecule composed of repeated subunits. Such molecules include, but are not limited to, polypeptides, polynucleotides, or polysaccharides or polyalkylene glycols.

[00163] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description directed to a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-natural amino acid. Additionally, such “polypeptides,” “peptides” and “proteins” include amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[00164] The term “post-translationally modified” refers to any modification of a natural or non-natural amino acid which occurs after such an amino acid has been translationally incorporated into a polypeptide chain. Such modifications include, but are not limited to, co-translational in vivo modifications, co-translational in vitro modifications (such as in a cell-free translation system), post-translational in vivo modifications, and post-translational in vitro modifications.

[00165] The terms “prodrug” or “pharmaceutically acceptable prodrug,” as used herein, refers to an agent that is converted into the parent drug in vivo or in vitro, wherein which does not abrogate the biological activity or properties of the drug, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. Prodrugs are generally drug precursors that, following administration to a subject and subsequent absorption, are converted to an active, or a more active species via some process, such as conversion by a metabolic pathway. Some prodrugs have a chemical group present on the prodrug that renders it less active and / or confers solubility or some other property to the drug. Once the chemical group has been cleaved and / or modified from the prodrug the active drug is generated. Prodrugs are converted into active drug within the body through enzymatic or non-enzymatic reactions. Prodrugs may provide improved physiochemical properties such as better solubility, enhanced delivery characteristics, such as specifically targeting a particular cell, tissue, organ or ligand, and improved therapeutic value of the drug. The benefits of such prodrugs include, but are not limited to, (i) ease of administration compared with the parent drug; (ii) the prodrug may be bioavailable by oral administration whereas the parent is not; and (iii) the prodrug may also have improved solubility in pharmaceutical compositions compared 46 2026205050   29 Jun 2026 with the parent drug. A pro-drug includes a pharmacologically inactive, or reduced-activity, derivative of an active drug. Prodrugs may be designed to modulate the amount of a drug or biologically active molecule that reaches a desired site of action through the manipulation of the properties of a drug, such as physiochemical, biopharmaceutical, or pharmacokinetic properties. An example, without limitation, of a prodrug would be a non-natural amino acid polypeptide which is administered as an ester (the “prodrug”) to facilitate transmittal across a cell membrane where water solubility is detrimental to mobility, but which then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water solubility is beneficial. Prodrugs may be designed as reversible drug derivatives, for use as modifiers to enhance drug transport to site-specific tissues.

[00166] The term “prophylactically effective amount,” as used herein, refers that amount of a composition containing at least one non-natural amino acid polypeptide or at least one modified non-natural amino acid polypeptide prophylactically applied to a patient which will relieve to some extent one or more of the symptoms of a disease, condition or disorder being treated. In such prophylactic applications, such amounts may depend on the patient's state of health, weight, and the like. It is considered well within the skill of the art for one to determine such prophylactically effective amounts by routine experimentation, including, but not limited to, a dose escalation clinical trial.

[00167] The term “protected,” as used herein, refers to the presence of a “protecting group” or moiety that prevents reaction of the chemically reactive functional group under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group being protected. By way of example only, (i) if the chemically reactive group is an amine or a hydrazide, the protecting group may be selected from tert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc); (ii) if the chemically reactive group is a thiol, the protecting group may be orthopyridyldisulfide; and (iii) if the chemically reactive group is a carboxylic acid, such as butanoic or propionic acid, or a hydroxyl group, the protecting group may be benzyl or an alkyl group such as methyl, ethyl, or tert-butyl.

[00168] By way of example only, blocking / protecting groups may be selected from: 2026205050   29 Jun 2026 „ C-lur H2 c-c H2 allyl H3C" Et / O (CH3)3C Boc (H3C)3C Cbz alloc H3CX zCH3 (H3C)3C-Si t-butyl            TBDMS pMBn (C6H5)3C — trityl acetyl H3C Me (H3C)3Si Teoc Fmoc

[00169] Additionally, protecting groups include, but are not limited to, including photolabile groups such as Nvoc and MeNvoc and other protecting groups known in the art. Other protecting groups are described in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, which is incorporated herein by reference in its entirety.

[00170] The term “recombinant host cell,” also referred to as “host cell,” refers to a cell which includes an exogenous polynucleotide, wherein the methods used to insert the exogenous polynucleotide into a cell include, but are not limited to, direct uptake, transduction, f-mating, or other methods known in the art to create recombinant host cells. By way of example only, such exogenous polynucleotide may be a nonintegrated vector, including but not limited to a plasmid, or may be integrated into the host genome.

[00171] The term “redox-active agent,” as used herein, refers to a molecule which oxidizes or reduces another molecule, whereby the redox active agent becomes reduced or oxidized. Examples of redox active agent include, but are not limited to, ferrocene, quinones, Ru2+ / 3+ complexes, Co2+ / 3+ complexes, and Os2+ / 3+ complexes.

[00172] The term “reducing agent,” as used herein, refers to a compound or material which is capable of adding an electron to a compound being reduced. By way of example reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2-aminoethanethiol), and reduced glutathione. Such reducing agents may be used, by way of example only, to maintain sulfhydryl groups in the reduced state and to reduce intra- or intermolecular disulfide bonds. 2026205050   29 Jun 2026

[00173] “Refolding,” as used herein describes any process, reaction or method which transforms an improperly folded or unfolded state to a native or properly folded conformation. By way of example only, refolding transforms disulfide bond containing polypeptides from an improperly folded or unfolded state to a native or properly folded conformation with respect to disulfide bonds. Such disulfide bond containing polypeptides may be natural amino acid polypeptides or non-natural amino acid polypeptides.

[00174] The term “safety” or “safety profile,” as used herein, refers to side effects that might be related to administration of a drug relative to the number of times the drug has been administered. By way of example, a drug which has been administered many times and produced only mild or no side effects is said to have an excellent safety profile. Methods used for evaluating the safety profile of any polypeptide are known in the art.

[00175] The phrase “selectively hybridizes to” or “specifically hybridizes to,” as used herein, refers to the binding, duplexing, or hybridizing of a molecule to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture including but not limited to, total cellular or library DNA or RNA.

[00176] The phrase “stringent hybridization conditions” refers to hybridization of sequences of DNA, RNA, PNA or other nucleic acid mimics, or combinations thereof, under conditions of low ionic strength and high temperature. By way of example, under stringent conditions a probe will hybridize to its target subsequence in a complex mixture of nucleic acid (including but not limited to, total cellular or library DNA or RNA) but does not hybridize to other sequences in the complex mixture. Stringent conditions are sequence-dependent and will be different in different circumstances. By way of example, longer sequences hybridize specifically at higher temperatures. Stringent hybridization conditions include, but are not limited to, (i) about 5-10 oC lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH; (ii) the salt concentration is about 0.01 M to about 1.0 M at about pH 7.0 to about pH 8.3 and the temperature is at least about 30 oC for short probes (including but not limited to, about 10 to about 50 nucleotides) and at least about 60 oC for long probes (including but not limited to, greater than 50 nucleotides); (iii) the addition of destabilizing agents including, but not limited to, formamide, (iv) 50% formamide, 5X SSC, and 1% SDS, incubating at 42 oC, or 5X SSC, about 1% SDS, incubating at 65 oC, with wash in 0.2X SSC, and about 0.1% SDS at 65 oC for between about 5 minutes to about 120 minutes. By way of example only, detection of selective or specific hybridization, includes, but is not limited to, a positive signal at least two times background. An extensive guide to the hybridization of nucleic acids is found in Tijssen, 49 2026205050   29 Jun 2026 Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993).

[00177] The term “subject” as used herein, refers to an animal which is the object of treatment, observation or experiment. By way of example only, a subject may be, but is not limited to, a mammal including, but not limited to, a human.

[00178] The term “substantially purified,” as used herein, refers to a component of interest that may be substantially or essentially free of other components which normally accompany or interact with the component of interest prior to purification. By way of example only, a component of interest may be “substantially purified” when the preparation of the component of interest contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about l% (by dry weight) of contaminating components. Thus, a “substantially purified” component of interest 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 greater. By way of example only, a natural amino acid polypeptide or a non-natural amino acid polypeptide may be purified from a native cell, or host cell in the case of recombinantly produced natural amino acid polypeptides or non-natural amino acid polypeptides. By way of example a preparation of a natural amino acid polypeptide or a non-natural amino acid polypeptide may be “substantially purified” when the preparation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about l% (by dry weight) of contaminating material. By way of example when a natural amino acid polypeptide or a non-natural amino acid polypeptide is recombinantly produced by host cells, the natural amino acid polypeptide or non-natural amino acid polypeptide may be present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cells. By way of example when a natural amino acid polypeptide or a non-natural amino acid polypeptide is recombinantly produced by host cells, the natural amino acid polypeptide or non-natural amino acid polypeptide may be present in the culture medium at about 5g / L, about 4g / L, about 3g / L, about 2g / L, about 1g / L, about 750mg / L, about 500mg / L, about 250mg / L, about 100mg / L, about 50mg / L, about 10mg / L, or about 1mg / L or less of the dry weight of the cells. By way of example, “substantially purified” natural amino acid polypeptides or non-natural amino acid polypeptides may have a purity level of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, 50 2026205050   29 Jun 2026 about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater as determined by appropriate methods, including, but not limited to, SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[00179] The term “substituents” also referred to as “non-interfering substituents” “refers to groups which may be used to replace another group on a molecule. Such groups include, but are not limited to, halo, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 alkoxy, C5-C12 aralkyl, C3-C12 cycloalkyl, C4-C12 cycloalkenyl, phenyl, substituted phenyl, toluolyl, xylenyl, biphenyl, C2-C12 alkoxyalkyl, C5-C12 alkoxyaryl, C5-C12 aryloxyalkyl, C7-C12 oxyaryl, C1-C6 alkylsulfinyl, C1-C10 alkylsulfonyl, -(CH2)m-O-(C1-C10 alkyl) wherein m is from 1 to 8, aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic radical, nitroalkyl, -NO2, -CN, -NRC(O)-(C1-C10 alkyl), -C(O)-(C1-C10 alkyl), C2-C10 alkthioalkyl, -C(O)O-(C1-C10 alkyl), -OH, -SO2, =S, -COOH, -NR2, carbonyl, -C(O)-(C1-C10 alkyl)-CF3, -C(O)-CF3, -C(O)NR2, -(C1-C10 aryl)-S-(C6-C10 aryl), -C(O)-(C6-C10 aryl), -(CH2)m-O-(CH2)m-O-(C1-C10 alkyl) wherein each m is from 1 to 8, -C(O)NR2, -C(S)NR2, -SO2NR2, -NRC(O)NR2, -NRC(S)NR2, salts thereof, and the like. Each R group in the preceding list includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl, or alkaryl. Where substituent groups are specified by their conventional chemical formulas, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left; for example, -CH2O- is equivalent to -OCH2-.

[00180] By way of example only, substituents for alkyl and heteroalkyl radicals (including those groups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) includes, but is 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 preceding list includes, but is not limited to, hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, including but not limited to, aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or aralkyl groups. When two R groups are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR2 is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl.

[00181] By way of example, substituents for aryl and heteroaryl groups include, but are not limited to, -OR, =O, =NR, =N-OR, -NR2, -SR, -halogen, -SiR3, -OC(O)R, -C(O)R, -CO2R, -CONR2, -OC(O)NR2, -NRC(O)R, -NRC(O)NR2, -NR(O)2R, -NR-C(NR2)=NR, -S(O)R, - 51 2026205050   29 Jun 2026 S(O)2R, -S(O)2NR2, -NRSO2R, -CN, -NO2, -R, -N3, -CH(Ph)2, fluoro(Ci-C4)alkoxy, and fluoro(C1-C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where each R group in the preceding list includes, but is not limited to, hydrogen, alkyl, heteroalkyl, aryl and heteroaryl.

[00182] The term “therapeutically effective amount,” as used herein, 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 already suffering from a disease, condition or disorder, sufficient to cure or at least partially arrest, or relieve to some extent one or more of the symptoms of the disease, disorder or condition being treated. The effectiveness of such compositions depends on conditions including, but not limited to, the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician. By way of example only, therapeutically effective amounts may be determined by routine experimentation, including but not limited to a dose escalation clinical trial.

[00183] The term “thioalkoxy,” as used herein, refers to sulfur containing alkyl groups linked to molecules via an oxygen atom.

[00184] The term “toxic moiety” or “toxic group” as used herein, refers to a compound which can cause harm, disturbances, or death. Toxic moieties include, but are not limited to, auristatin, DNA minor groove binding agent, DNA minor groove alkylating agent, enediyne, lexitropsin, duocarmycin, taxane, puromycin, TLR-agonist, maytansinoid, vinca alkaloid, AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin,    TLR-agonist-10, echinomycin, combretatstatin, chalicheamicin, maytansine, DM-1, netropsin, podophyllotoxin (e.g. etoposide, teniposide, etc.), baccatin and its derivatives, anti-tubulin agents, cryptophysin, combretastatin, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, epothilone A, epothilone B, nocodazole, colchicines, colcimid, estramustine, cemadotin, discodermolide, maytansine, eleutherobin, mechlorethamine, cyclophosphamide, melphalan, carmustine, lomustine, semustine, streptozocin, chlorozotocin, uracil mustard, chlormethine,     ifosfamide,     chlorambucil,     pipobroman,     triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide, ytarabine, cytosine arabinoside, fluorouracil, floxuridine, 6-thioguanine, 6-mercaptopurine, pentostatin, 5-fluorouracil, methotrexate, 10-propargyl-5,8-dideazafolate, 5,8-dideazatetrahydrofolic acid, leucovorin, fludarabine phosphate, pentostatine, gemcitabine, Ara-C, paclitaxel, docetaxel, 52 2026205050   29 Jun 2026 deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine, brequinar, antibiotics (e.g., anthracycline, gentamicin, cefalotin, 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 (e.g., abacavir, acyclovir, ampligen, cidofovir, delavirdine, didanosine, efavirenz, entecavir, fosfonet, ganciclovir, ibacitabine, imunovir, idoxuridine, inosine, lopinavir, methisazone, nexavir, nevirapine, oseltamivir, penciclovir, stavudine, trifluridine, truvada, valaciclovir, zanamivir, etc.), daunorubicin hydrochloride, daunomycin, rubidomycin, cerubidine, idarubicin, doxorubicin, epirubicin and morpholino derivatives, phenoxizone biscyclopeptides (e.g., dactinomycin), basic glycopeptides (e.g., bleomycin), anthraquinone glycosides (e.g., plicamycin, mithramycin), anthracenediones (e.g., mitoxantrone), azirinopyrrolo indolediones (e.g., mitomycin), macrocyclic immunosuppressants (e.g., cyclosporine, FK-506, tacrolimus, prograf, rapamycin etc.), navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, droloxafine, allocolchicine, Halichondrin B, colchicine, colchicine derivatives , maytansine, rhizoxin, paclitaxel, paclitaxel derivatives, docetaxel, thiocolchicine, trityl cysterin, vinblastine sulfate, vincristine sulfate, cisplatin, carboplatin, hydroxyurea, N-methylhydrazine, epidophyllotoxin, procarbazine, mitoxantrone, leucovorin, and tegafur. “Taxanes” include paclitaxel, as well as any active taxane derivative or pro-drug.

[00185] The terms “treat,” “treating” or “treatment”, as used herein, include alleviating, abating or ameliorating a disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition. The terms “treat,” “treating” or “treatment”, include, but are not limited to, prophylactic and / or therapeutic treatments.

[00186] 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, mono C1-C10 alkoxy or aryloxy derivatives thereof (described in U.S. Patent No. 5,252,714 which is incorporated by reference herein), monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinylether maleic anhydride, N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran 53 2026205050   29 Jun 2026 derivatives including dextran sulfate, polypropylene glycol, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives, including but not limited to methylcellulose and carboxymethyl cellulose, serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers, and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixtures thereof. By way of example only, coupling of such water soluble polymers to natural amino acid polypeptides or non-natural polypeptides may result in changes including, but not limited to, increased water solubility, increased or modulated serum half-life, increased or modulated therapeutic half-life relative to the unmodified form, increased bioavailability, modulated biological activity, extended circulation time, modulated immunogenicity, modulated physical association characteristics including, but not limited to, aggregation and multimer formation, altered receptor binding, activity modulator, or other targeting polypeptide binding, altered binding to one or more binding partners, and altered targeting polypeptide receptor dimerization or multimerization. In addition, such water soluble polymers may or may not have their own biological activity, and may be utilized as a linker for attaching targeting polypeptide to other substances, including but not limited to one or more targeting polypeptides, or one or more biologically active molecules.

[00187] Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art are employed.

[00188] Compounds, (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) presented herein include isotopically-labeled compounds, which are identical to those recited in the various formulas and structures presented herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the present compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as 2H, 3H, 13C, 14C, 15N, 18O, 17O, 35S, 18F, 36Cl, respectively. Certain isotopically-labeled compounds described herein, for example those into which radioactive isotopes such as 3H and 14C are incorporated, are useful in drug and / or substrate tissue distribution assays. Further, substitution with isotopes such as deuterium, i.e., 2H, 2026205050   29 Jun 2026 can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements.

[00189] Some of the compounds herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) have asymmetric carbon atoms and can therefore exist as enantiomers or diastereomers. Diasteromeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods known, for example, by chromatography and / or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. All such isomers, including diastereomers, enantiomers, and mixtures thereof are considered as part of the compositions described herein.

[00190] In additional or further embodiments, the compounds described herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified nonnatural amino acid polypeptides, and reagents for producing the aforementioned compounds) are used in the form of pro-drugs. In additional or further embodiments, the compounds described herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) are metabolized upon administration to an organism in need to produce a metabolite that is then used to produce a desired effect, including a desired therapeutic effect. In further or additional embodiments are active metabolites of non-natural amino acids and “modified or unmodified” non-natural amino acid polypeptides.

[00191] The methods and formulations described herein include the use of N-oxides, crystalline forms (also known as polymorphs), or pharmaceutically acceptable salts of nonnatural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides. In certain embodiments, non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides may exist as tautomers. All tautomers are included within the scope of the non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides presented herein. In addition, the non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of 55 2026205050   29 Jun 2026 the non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides presented herein are also considered to be disclosed herein.

[00192] Some of the compounds herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reagents for producing the aforementioned compounds) may exist in several tautomeric forms. All such tautomeric forms are considered as part of the compositions described herein. Also, for example all enol-keto forms of any compounds (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reagents for producing the aforementioned compounds) herein are considered as part of the compositions described herein.

[00193] Some of the compounds herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reagents for producing either of the aforementioned compounds) are acidic and may form a salt with a pharmaceutically acceptable cation. Some of the compounds herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified nonnatural amino acid polypeptides and reagents for producing the aforementioned compounds) can be basic and accordingly, may form a salt with a pharmaceutically acceptable anion. All such salts, including di-salts 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 the acidic and basic entities, in either an aqueous, non-aqueous or partially aqueous medium. The salts are recovered by using at least one of the following techniques: filtration, precipitation with a non-solvent followed by filtration, evaporation of the solvent, or, in the case of aqueous solutions, lyophilization.

[00194] Pharmaceutically acceptable salts of the non-natural amino acid polypeptides disclosed herein may be formed when an acidic proton present in the parent non-natural amino acid polypeptides either is replaced by a metal ion, by way of example an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. In addition, the salt forms of the disclosed non-natural amino acid polypeptides can be prepared using salts of the starting materials or intermediates. The non-natural amino acid polypeptides described herein may be prepared as a pharmaceutically acceptable acid addition salt (which is a type of a pharmaceutically acceptable salt) by reacting the free base form of non-natural amino acid polypeptides described herein with a pharmaceutically acceptable inorganic or organic acid. Alternatively, the non-natural amino acid polypeptides described herein may be prepared as 56 2026205050   29 Jun 2026 pharmaceutically acceptable base addition salts (which are a type of a pharmaceutically acceptable salt) by reacting the free acid form of non-natural amino acid polypeptides described herein with a pharmaceutically acceptable inorganic or organic base.

[00195] The type of pharmaceutical acceptable salts, include, but are not limited to: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed 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-ene-1-carboxylic acid, glucoheptonic acid, 4,4’-methylenebis-(3-hydroxy-2-ene-1 -carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.

[00196] The corresponding counterions of the non-natural amino acid polypeptide pharmaceutical acceptable salts may be analyzed and identified using various methods including, but not limited to, ion exchange chromatography, ion chromatography, capillary electrophoresis, inductively coupled plasma, atomic absorption spectroscopy, mass spectrometry, or any combination thereof. In addition, the therapeutic activity of such non-natural amino acid polypeptide pharmaceutical acceptable salts may be tested using the techniques and methods described in the examples.

[00197] It should be understood that a reference to a salt includes the solvent addition forms or crystal forms thereof, particularly solvates or polymorphs. Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent, and are often formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Polymorphs include the different crystal packing arrangements of the same 57 2026205050   29 Jun 2026 elemental composition of a compound. Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. Various factors such as the recrystallization solvent, rate of crystallization, and storage temperature may cause a single crystal form to dominate.

[00198] The screening and characterization of non-natural amino acid polypeptide pharmaceutical acceptable salts polymorphs and / or solvates may be accomplished using a variety of techniques including, but not limited to, thermal analysis, x-ray diffraction, spectroscopy, vapor sorption, and microscopy. Thermal analysis methods address thermo chemical degradation or thermo physical processes including, but not limited to, polymorphic transitions, and such methods are used to analyze the relationships between polymorphic forms, determine weight loss, to find the glass transition temperature, 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 Thermogravi-metric and Infrared analysis (TG / IR). X-ray diffraction methods include, but are not limited to, single crystal and powder diffractometers and synchrotron sources. The various spectroscopic techniques used include, but are not limited to, Raman, FTIR, UVIS, and NMR (liquid and solid state). The various microscopy techniques include, but are not limited to, polarized light microscopy, Scanning Electron Microscopy (SEM) with Energy Dispersive X-Ray Analysis (EDX), Environmental Scanning Electron Microscopy with EDX (in gas or water vapor atmosphere), IR microscopy, and Raman microscopy.

[00199] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur 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 may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. TLR-agonist Linker Derivatives

[00200] At one level, described herein are the tools (methods, compositions, techniques) for creating and using a targeting polypeptide of the TCs or analogs comprising at least one nonnatural amino acid or modified non-natural amino acid with a carbonyl, dicarbonyl, oxime or hydroxylamine group. Such targeting polypeptide of the TCs comprising non-natural amino 58 2026205050   29 Jun 2026 acids may contain further functionality, including but not limited to, a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; and any combination thereof. Note that the various aforementioned functionalities are not meant to imply that the members of one functionality cannot be classified as members of another functionality. Indeed, there will be overlap depending upon the particular circumstances. By way of example only, a water-soluble polymer overlaps in scope with a derivative of polyethylene glycol, however the overlap is not complete and thus both functionalities are cited above.

[00201] In one aspect are methods for selecting and designing a TLR-agonist linker derivative, and the targeting polypeptide, to be modified using the methods, compositions and techniques described herein. The new TLR-agonist linker derivative and the targeting polypeptide may be designed de novo, including by way of example only, as part of high-throughput screening process (in which case numerous polypeptides may be designed, synthesized, characterized and / or tested) or based on the interests of the researcher. The new TLR-agonist linker derivative and the targeting polypeptide may also be designed based on the structure of a known or partially characterized polypeptide. By way of example only, TLR-agonist has been the subject of intense study by the scientific community; a new compound may be designed based on the structure of TLR-agonist. The principles for selecting which amino acid(s) to substitute and / or modify are described separately herein. The choice of which modification to employ is also described herein and can be used to meet the need of the experimenter or end user. Such needs may include, but are not limited to, manipulating the therapeutic effectiveness of the polypeptide, improving the safety profile of the polypeptide, adjusting the pharmacokinetics, pharmacologics and / or pharmacodynamics of the polypeptide, such as, by way of example only, increasing water solubility, bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending the circulation time. In addition, such modifications include, by way of example only, providing additional functionality to the polypeptide, incorporating an antibody, and any combination of the aforementioned modifications.

[00202] Also described herein are TLR-agonist linker derivatives and the targeting polypeptide that have or can be modified to contain an oxime, carbonyl, dicarbonyl, or hydroxylamine group. Included with this aspect are methods for producing, purifying, characterizing and using such TLR-agonist linker derivatives and the targeting polypeptides. 2026205050   29 Jun 2026

[00203] The TLR-agonist linker derivative or the targeting polypeptide may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more of a carbonyl or dicarbonyl group, oxime group, hydroxylamine group, or protected forms thereof. The TLR-agonist linker derivative or the targeting polypeptide can be the same or different, for example, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different sites in the derivative that comprise 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.

[00204] As described herein, the present disclosures provide targeting polypeptides coupled to another molecule having the formula “targeting polypeptide-L-M”, wherein L is a linking group or a 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.

[00205] Targeting polypeptide and M can be linked together through L using standard linking agents and procedures known to those skilled in the art. In some aspects, targeting polypeptide and M are fused directly and L is a bond. In other aspects, targeting polypeptide and M are fused through a linking group L. For example, in some embodiments, targeting polypeptide and M are linked together via a peptide bond, optionally through a peptide or amino acid spacer. In some embodiments, targeting polypeptide and M are linked together through chemical conjugation, optionally through a linking group (L). In some embodiments, L is directly conjugated to each of targeting polypeptide and M.

[00206] Chemical conjugation can occur by reacting a nucleophilic reactive group of one compound to an electrophilic reactive group of another compound. In some embodiments when L is a bond, targeting polypeptide is conjugated to M either by reacting a nucleophilic reactive moiety on targeting polypeptide with an electrophilic reactive moiety on Y, or by reacting an electrophilic reactive moiety on targeting polypeptide with a nucleophilic reactive moiety on M. In embodiments when L is a group that links targeting polypeptide and M together, targeting polypeptide and / or M can be conjugated to L either by reacting a nucleophilic reactive moiety on targeting polypeptide and / or M with an electrophilic reactive moiety on L, or by reacting an electrophilic reactive moiety on targeting polypeptide and / or M with a nucleophilic reactive moiety on L. Nonlimiting examples of nucleophilic reactive groups include amino, thiol, and hydroxyl. Nonlimiting examples of electrophilic reactive groups include carboxyl, acyl chloride, anhydride, ester, succinimide ester, alkyl halide, sulfonate ester, maleimido, haloacetyl, and isocyanate. In embodiments where targeting polypeptide and M are conjugated together by 60 2026205050   29 Jun 2026 reacting a carboxylic acid with an amine, an activating agent can be used to form an activated ester of the carboxylic acid.

[00207] The activated ester of the carboxylic acid can be, for example, N-hydroxysuccinimide (NHS), tosylate (Tos), mesylate, triflate, a carbodiimide, or a hexafluorophosphate. In some embodiments, the carbodiimide is 1,3-dicyclohexylcarbodiimide (DCC), 1 ,1'-carbonyldiimidazole (CDI), l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), or 1,3-diisopropylcarbodiimide (DICD). In some embodiments, the hexafluorophosphate is selected from a group consisting of hexafluorophosphate benzotriazol-l-yl-oxy-tris(dimethylamino)phosphonium     hexafluorophosphate     (BOP),     benzotriazol-l-yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 2-(lH-7-azabenzotriazol-l-yl)-1,1 ,3,3-tetramethyl uronium hexafluorophosphate (HATU), and o-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate (HBTU).

[00208] In some embodiments, targeting polypeptide comprises a nucleophilic reactive group (e.g. the amino group, thiol group, or hydroxyl group of the side chain of lysine, cysteine or serine) that is capable of conjugating to an electrophilic reactive group on M or L. In some embodiments, targeting polypeptide comprises an electrophilic reactive group (e.g. the carboxylate group of the side chain of Asp or Glu) that is capable of conjugating to a nucleophilic reactive group on M or L. In some embodiments, targeting polypeptide is chemically modified to comprise a reactive group that is capable of conjugating directly to M or to L. In some embodiments, targeting polypeptide is modified at the N-terminus or C-terminus to comprise a natural or non-natural amino acid with a nucleophilic side chain. In exemplary embodiments, the N-terminus or C-terminus amino acid of targeting polypeptide is selected from the group consisting of lysine, ornithine, serine, cysteine, and homocysteine. For example, the N-terminus or C-terminus amino acid of targeting polypeptide can be modified to comprise a lysine residue. In some embodiments, targeting polypeptide is modified at the N-terminus or C-terminus amino acid to comprise a natural or non-natural amino acid with an electrophilic side chain such as, for example, Asp and Glu. In some embodiments, an internal amino acid of targeting polypeptide is substituted with a natural or non-natural amino acid having a nucleophilic side chain, as previously described herein. In exemplary embodiments, the internal amino acid of targeting polypeptide that is substituted is selected from the group consisting of lysine, ornithine, serine, cysteine, and homocysteine. For example, an internal amino acid of targeting polypeptide can be substituted with a lysine residue. In some embodiments, an internal 2026205050   29 Jun 2026 amino acid of targeting polypeptide is substituted with a natural or non-natural amino acid with an electrophilic side chain, such as, for example, Asp and Glu.

[00209] In some embodiments, M comprises a reactive group that is capable of conjugating directly to targeting polypeptide or to L. In some embodiments, M comprises a nucleophilic reactive group (e.g. amine, thiol, hydroxyl) that is capable of conjugating to an electrophilic reactive group on targeting polypeptide or L. In some embodiments, M comprises electrophilic reactive group (e.g. carboxyl group, activated form of a carboxyl group, compound with a leaving group) that is capable of conjugating to a nucleophilic reactive group on targeting polypeptide or L. In some embodiments, M is chemically modified to comprise either a nucleophilic reactive group that is capable of conjugating to an electrophilic reactive group on targeting polypeptide or L. In some embodiments, M is chemically modified to comprise an electrophilic reactive group that is capable of conjugating to a nucleophilic reactive group on targeting polypeptide or L.

[00210] In some embodiments, conjugation can be carried out through organosilanes, for example, aminosilane treated with glutaraldehyde; carbonyldiimidazole (CDI) activation of silanol groups; or utilization of dendrimers. A variety of dendrimers are known in the art and include poly (amidoamine) (PAMAM) dendrimers, which are synthesized by the divergent method starting from ammonia or ethylenediamine initiator core reagents; a sub-class of PAMAM dendrimers based on a tris-aminoethylene-imine core; radially layered poly(amidoamine-organosilicon) dendrimers (PAMAMOS), which are inverted unimolecular micelles that consist of hydrophilic, nucleophilic polyamidoamine (PAMAM) interiors and hydrophobic organosilicon (OS) exteriors; Poly (Propylene Imine) (PPI) dendrimers, which are generally poly-alkyl amines having primary amines as end groups, while the dendrimer interior consists of numerous of tertiary tris-propylene amines; Poly (Propylene Amine) (POPAM) dendrimers; Diaminobutane (DAB) dendrimers; amphiphilic dendrimers; micellar dendrimers which are unimolecular micelles of water soluble hyper branched polyphenylenes; polylysine dendrimers; and dendrimers based on poly-benzyl ether hyper branched skeleton.

[00211] In some embodiments, conjugation can be carried out through olefin metathesis. In some embodiments, M and targeting polypeptide, M and L, or targeting polypeptide and L both comprise an alkene or alkyne moiety that is capable of undergoing metathesis. In some embodiments a suitable catalyst (e.g. copper, ruthenium) is used to accelerate the metathesis reaction. Suitable methods of performing olefin metathesis reactions are described in the art. See, for example, Schafmeister et al., J. Am. Chem. Soc. 122: 5891-5892 (2000), Walensky et al., 62 2026205050   29 Jun 2026 Science 305: 1466-1470 (2004), and Blackwell et al., Angew, Chem., Int. Ed. 37: 3281-3284 (1998).

[00212] In some embodiments, conjugation can be carried out using click chemistry. A "click reaction" is wide in scope and easy to perform, uses only readily available reagents, and is insensitive to oxygen and water. In some embodiments, the click reaction is a cycloaddition reaction between an alkynyl group and an azido group to form a triazolyl group. In some embodiments, the click reaction uses a copper or ruthenium catalyst. Suitable methods of performing click reactions are described in the art. See, for example, Kolb et al., Drug Discovery Today 8: 1128 (2003); Kolb et al., Angew. Chem. Int. Ed. 40:2004 (2001); Rostovtsev et al., Angew. Chem. Int. Ed. 41 :2596 (2002); Tornoe et al., J. Org. Chem. 67:3057 (2002); Manetsch et al., J. Am. Chem. Soc. 126: 12809 (2004); Lewis et al., Angew. Chem. Int. Ed. 41: 1053 (2002); Speers, J. Am. Chem. Soc. 125:4686 (2003); Chan et al. Org. Lett. 6:2853 (2004); Zhang et al., J. Am. Chem. Soc. 127: 15998 (2005); and Waser et al., J. Am. Chem. Soc. 127:8294 (2005).

[00213] Indirect conjugation via high affinity specific binding partners, e.g. streptavidin / biotin or avidin / biotin or lectin / carbohydrate is also contemplated.

[00214] In some embodiments, targeting polypeptide and / or M are functionalized to comprise a nucleophilic reactive group or an electrophilic reactive group with an organic derivatizing agent. This derivatizing agent is capable of reacting with selected side chains or the N- or C-terminal residues of targeted amino acids on targeting polypeptide and functional groups on M. Reactive groups on targeting polypeptide and / or M include, e.g., aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group. Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride or other agents known in the art. Alternatively, targeting polypeptide and / or M can be linked to each other indirectly through intermediate carriers, such as polysaccharide or polypeptide carriers. Examples of polysaccharide carriers include aminodextran. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixed polymers of these amino acids and others, e.g., serines, to confer desirable solubility properties on the resultant loaded carrier.

[00215] Cysteinyl residues most commonly are reacted with a-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with 63 2026205050   29 Jun 2026 bromotrifluoroacetone, alpha-bromo-P-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-l,3-diazole.

[00216] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

[00217] Lysinyl and amino-terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reaction with glyoxylate.

[00218] Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1 ,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

[00219] The specific modification of tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.

[00220] Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R-N=C=N-R'), where R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or l-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[00221] Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), deamidation of asparagine or glutamine, acetylation of the N-terminal amine, and / or amidation or esterification of the C-terminal carboxylic acid group. 2026205050   29 Jun 2026

[00222] Another type of covalent modification involves chemically or enzymatically coupling glycosides to the peptide. Sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO1987 / 05330, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[00223] In some embodiments, L is a bond. In these embodiments, targeting polypeptide and M are conjugated together by reacting a nucleophilic reactive moiety on targeting polypeptide with and electrophilic reactive moiety on M. In alternative embodiments, targeting polypeptide and M are conjugated together by reacting an electrophilic reactive moiety on targeting polypeptide with a nucleophilic moiety on M. In exemplary embodiments, L is an amide bond that forms upon reaction of an amine on targeting polypeptide (e.g. an s-amine of a lysine residue) with a carboxyl group on M. In alternative embodiments, targeting polypeptide and or M is derivatized with a derivatizing agent before conjugation.

[00224] In some embodiments, L is a linking group. In some embodiments, L is a bifunctional linker and comprises only two reactive groups before conjugation to targeting polypeptide and M. In embodiments where both targeting polypeptide and M have electrophilic reactive groups, L comprises two of the same or two different nucleophilic groups (e.g. amine, hydroxyl, thiol) before conjugation to targeting polypeptide and M. In embodiments where both targeting polypeptide and M have nucleophilic reactive groups, L comprises two of the same or two different electrophilic groups (e.g. carboxyl group, activated form of a carboxyl group, compound with a leaving group) before conjugation to targeting polypeptide and M. In embodiments where one of targeting polypeptide or M has a nucleophilic reactive group and the other of targeting polypeptide or M has an electrophilic reactive group, L comprises one nucleophilic reactive group and one electrophilic group before conjugation to targeting polypeptide and M.

[00225] L can be any molecule with at least two reactive groups (before conjugation to targeting polypeptide and M) capable of reacting with each of targeting polypeptide and M. In some embodiments L has only two reactive groups and is bifunctional. L (before conjugation to the peptides) can be represented by Formula VI: Linking Group 2026205050   29 Jun 2026 (L) wherein A and B are independently nucleophilic or electrophihc reactive groups. In some embodiments A and B are either both nucleophilic groups or both electrophihc groups. In some embodiments one of A or B is a nucleophilic group and the other of A or B is an electrophihc group. Nonlimiting combinations of A and B are shown below in Table 1. Table 1: Nonlimiting combinations of Nucleophilic and Electrophilic Groups Both Nucleophilic Both Electrophilic Nucleophilic / Electrophilic A B A B A B Amino Amino Carboxyl Carboxyl Amino Carboxyl Amino Thiol Carboxyl Acyl chloride Amino Acyl chloride Amino Hydroxyl Carboxyl Anhydride Amino Anhydride Thiol Amino Carboxyl Ester Amino Ester Thiol Thiol Carboxyl NHS Amino NHS Thiol Hydroxyl Carboxyl Halogen Amino Halogen Hydroxyl Amino Carboxyl Sulfonate ester Amino Sulfonate ester Hydroxyl Thiol Carboxyl Maleimido Amino Maleimido Hydroxyl Hydroxyl Carboxyl Haloacetyl Amino Haloacetyl Carboxyl Isocyanate Amino Isocyanate Acyl chloride Carboxyl Thiol Carboxyl Acyl chloride Acyl chloride Thiol Acyl chloride Acyl chloride Anhydride Thiol Anhydride 2026205050   29 Jun 2026 Both Nucleophilic Both Electrophilic Nucleophilic / Electrophilic Acyl chloride Ester Thiol Ester Acyl chloride NHS Thiol NHS Acyl chloride Halogen Thiol Halogen Acyl chloride Sulfonate ester Thiol Sulfonate ester Acyl chloride Maleimido Thiol Maleimido Acyl chloride Haloacetyl Thiol Haloacetyl Acyl chloride Isocyanate Thiol Isocyanate Anhydride Carboxyl Hydroxyl Carboxyl Anhydride Acyl chloride Hydroxyl Acyl chloride Anhydride Anhydride Hydroxyl Anhydride Anhydride Ester Hydroxyl Ester Anhydride NHS Hydroxyl NHS Anhydride Halogen Hydroxyl Halogen Anhydride Sulfonate ester Hydroxyl Sulfonate ester Anhydride Maleimido Hydroxyl Maleimido 2026205050   29 Jun 2026 Both Nucleophilic Both Electrophilic Nucleophilic / Electrophilic Anhydride Haloacetyl Hydroxyl Haloacetyl Anhydride Isocyanate Hydroxyl Isocyanate Ester Carboxyl Ester Acyl chloride Ester Anhydride Ester Ester Ester NHS Ester Halogen Ester Sulfonate ester Ester Maleimido Ester Haloacetyl Ester Isocyanate NHS Carboxyl NHS Acyl chloride NHS Anhydride NHS Ester NHS NHS NHS Halogen NHS Sulfonate ester NHS Maleimido 2026205050   29 Jun 2026 Both Nucleophilic Both Electrophilic Nucleophilic / Electrophilic NHS Haloacetyl NHS Isocyanate Halogen Carboxyl Halogen Acyl chloride Halogen Anhydride Halogen Ester Halogen NHS Halogen Halogen Halogen Sulfonate ester Halogen Maleimido Halogen Haloacetyl Halogen Isocyanate Sulfonate ester Carboxyl Sulfonate ester Acyl chloride Sulfonate ester Anhydride Sulfonate ester Ester Sulfonate ester NHS Sulfonate ester Halogen 2026205050   29 Jun 2026 Both Nucleophilic Both Electrophilic Nucleophilic / Electrophilic Sulfonate ester Sulfonate ester Sulfonate ester Maleimido Sulfonate ester Haloacetyl Sulfonate ester Isocyanate Maleimido Carboxyl Maleimido Acyl chloride Maleimido Anhydride Maleimido Ester Maleimido NHS Maleimido Halogen Maleimido Sulfonate ester Maleimido Maleimido Maleimido Haloacetyl Maleimido Isocyanate Haloacetyl Carboxyl Haloacetyl Acyl chloride Haloacetyl Anhydride Haloacetyl Ester 2026205050   29 Jun 2026 Both Nucleophilic Both Electrophilic Nucleophilic / Electrophilic Haloacetyl NHS Haloacetyl Halogen Haloacetyl Sulfonate ester Haloacetyl Maleimido Haloacetyl Haloacetyl Haloacetyl Isocyanate Isocyanate Carboxyl Isocyanate Acyl chloride Isocyanate Anhydride Isocyanate Ester Isocyanate NHS Isocyanate Halogen Isocyanate Sulfonate ester Isocyanate Maleimido Isocyanate Haloacetyl Isocyanate Isocyanate

[00226] In some embodiments, A and B may include alkene and / or alkyne functional groups that are suitable for olefin metathesis reactions. In some embodiments, A and B include moieties that are suitable for click chemistry (e.g. alkene, alkynes, nitriles, azides). Other nonlimiting examples of reactive groups (A and B) include pyridyldithiol, aryl azide, diazirine, carbodiimide, and hydrazide. 2026205050   29 Jun 2026

[00227] In some embodiments, L is hydrophobic. Hydrophobic linkers are known in the art. See, e.g., Bioconjugate Techniques, G. T. Hermanson (Academic Press, San Diego, CA, 1996), which is incorporated by reference in its entirety. Suitable hydrophobic linking groups known in the art include, for example, 8 -hydroxy octanoic acid and 8-mercaptooctanoic acid. Before conjugation to the peptides of the composition, the hydrophobic linking group comprises at least two reactive groups (A and B), as described herein and as shown below: Hydrophobic Linking Group

[00228] In some embodiments, the hydrophobic linking group comprises either a maleimido or an iodoacetyl group and either a carboxylic acid or an activated carboxylic acid (e.g. NHS ester) as the reactive groups. In these embodiments, the maleimido or iodoacetyl group can be coupled to a thiol moiety on targeting polypeptide or M and the carboxylic acid or activated carboxylic acid can be coupled to an amine on targeting polypeptide or M with or without the use of a coupling reagent. Any coupling agent known to one skilled in the art can be used to couple the carboxylic acid with the free amine such as, for example, DCC, DIC, HATU, HBTU, TBTU, and other activating agents described herein. In specific embodiments, the hydrophilic linking group comprises an aliphatic chain of 2 to 100 methylene groups wherein A and B are carboxyl groups or derivatives thereof (e.g. succinic acid). In other specific embodiments the L is iodoacetic acid. succinic acid            iodoacetic acid

[00229] In some embodiments, the linking group is hydrophilic such as, for example, polyalkylene glycol. Before conjugation to the peptides of the composition, the hydrophilic linking group comprises at least two reactive groups (A and B), as described herein and as shown below: Hydrophilic Linking 2026205050   29 Jun 2026 Group

[00230] In specific embodiments, the linking group is polyethylene glycol (PEG). The PEG in certain embodiments has a molecular weight of about 100 Daltons to about 10,000 Daltons, e.g. about 500 Daltons to about 5000 Daltons. The PEG in some embodiments has a molecular weight of about 10,000 Daltons to about 40,000 Daltons.

[00231] In some embodiments, the hydrophilic linking group comprises either a maleimido or an iodoacetyl group and either a carboxylic acid or an activated carboxylic acid (e.g. NHS ester) as the reactive groups. In these embodiments, the maleimido or iodoacetyl group can be coupled to a thiol moiety on targeting polypeptide or M and the carboxylic acid or activated carboxylic acid can be coupled to an amine on targeting polypeptide or M with or without the use of a coupling reagent. Any appropriate coupling agent known to one skilled in the art can be used to couple the carboxylic acid with the amine such as, for example, DCC, DIC, HATU, HBTU, TBTU, and other activating agents described herein. In some embodiments, the linking group is maleimido-polymer(20-40 kDa)-COOH, iodoacetyl-polymer(20-40 kDa)-COOH, maleimido-polymer(20-40 kDa)-NHS, or iodoacetyl-polymer(20-40 kDa)-NHS.

[00232] In some embodiments, the linking group is comprised of an amino acid, a dipeptide, a tripeptide, or a polypeptide, wherein the amino acid, dipeptide, tripeptide, or polypeptide comprises at least two activating groups, as described herein. In some embodiments, the linking group (L) comprises a moiety selected from the group consisting of: amino, ether, thioether, maleimido, disulfide, amide, ester, thioester, alkene, cycloalkene, alkyne, trizoyl, carbamate, carbonate, cathepsin B-cleavable, and hydrazone.

[00233] In some embodiments, L comprises a chain of atoms from 1 to about 60, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms long. In some embodiments, the chain atoms are all carbon atoms. In some embodiments, the chain atoms in the backbone of the linker are selected from the group consisting of C, O, N, and S. Chain atoms and linkers may be selected according to their expected solubility (hydrophilicity) so as to provide a more soluble conjugate. In some embodiments, L provides a functional group that is subject to cleavage by an enzyme or other catalyst or hydrolytic conditions found in the target tissue or organ or cell. In some embodiments, the length of L is long enough to reduce the potential for steric hindrance. 2026205050   29 Jun 2026

[00234] In some embodiments, L is stable in biological fluids such as blood or blood fractions. In some embodiments, L is stable in blood serum for at least 5 minutes, e.g. less than 25%, 20%, 15%, 10% or 5% of the conjugate is cleaved when incubated in serum for a period of 5 minutes. In other embodiments, L is stable in blood serum for at least 10, or 20, or 25, or 30, or 60, or 90, or 120 minutes, or 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18 or 24 hours. In these embodiments, L does not comprise a functional group that is capable of undergoing hydrolysis in vivo. In some exemplary embodiments, L is stable in blood serum for at least about 72 hours. Nonlimiting examples of functional groups that are not capable of undergoing significant hydrolysis in vivo include amides, ethers, and thioethers. For example, the following compound does not undergoing significant hydrolysis in vivo: 0

[00235] In some embodiments, L is hydrolyzable in vivo. In these embodiments, L comprises a functional group that is capable of undergoing hydrolysis in vivo. Nonlimiting examples of functional groups that are capable of undergoing hydrolysis in vivo include esters, anhydrides, and thioesters. For example the following compound is capable of undergoing hydrolysis in vivo because it comprises an ester group:

[00236] In some exemplary embodiments L is labile and undergoes substantial hydrolysis within 3 hours in blood plasma at 37°C, with complete hydrolysis within 6 hours. In some exemplary embodiments, L is not labile.

[00237] In some embodiments, L is metastable in vivo. In these embodiments, L comprises a functional group that is capable of being chemically or enzymatically cleaved in vivo (e.g., an acid-labile, reduction-labile, or enzyme-labile functional group), optionally over a period of time. In these embodiments, L can comprise, for example, a hydrazone moiety, a disulfide moiety, or a cathepsin-cleavable moiety. When L is metastable, and without intending to be bound by any particular theory, the targeting polypeptide-L-M conjugate is stable in an extracellular environment, e.g., stable in blood serum for the time periods described above, but labile in the intracellular environment or conditions that mimic the intracellular environment, so that it cleaves upon entry into a cell. In some embodiments when L is metastable, L is stable in 2026205050   29 Jun 2026 blood serum for at least about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, or 48 hours, for example, at least about 48, 54, 60, 66, or 72 hours, or about 24-48, 48-72, 24-60, 36-48, 3672, or 48-72 hours.

[00238] In another embodiment, the polymer derivatives of the invention comprise a polymer backbone having the structure: X—CH2CH2O--(CH2CH2O)n --CH2CH2 - O-(CH2)m-W-N=N=N wherein: W is an aliphatic or aromatic linker moiety comprising between 1-10 carbon atoms; n is 1 to about 4000; and X is a functional group as described above; m is between 1 and 10.

[00239] The azide-containing polymer derivatives of the invention can be prepared by a variety of methods known in the art and / or disclosed herein. In one method, shown below, a water soluble polymer backbone having an average molecular weight from about 800 Da to about 100,000 Da, the polymer backbone having a first terminus bonded to a first functional group and a second terminus bonded to a suitable leaving group, is reacted with an azide anion (which may be paired with any of a number of suitable counter-ions, including sodium, potassium, tert-butylammonium and so forth). The leaving group undergoes a nucleophilic displacement and is replaced by the azide moiety, affording the desired azide-containing polymer polymer; X-polymer-LY + Nf^ X-polymer-L N3

[00240] As illustrated, a suitable polymer backbone for use in the present invention has the formula X-polymer-LY, wherein polymer is poly(ethylene glycol) and X is a functional group which does not react with azide groups and Y is a suitable leaving group. Examples of suitable functional groups include, but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl, amine, aminooxy, protected amine, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine, and vinylpyridine, and ketone. Examples of suitable leaving groups include, but are not limited to, chloride, bromide, iodide, mesylate, tresylate, and tosylate.

[00241] In another method for preparation of the azide-containing polymer derivatives of the present invention, a linking agent bearing an azide functionality is contacted with a water soluble polymer backbone having an average molecular weight from about 800 Da to about 100,000 Da, wherein the linking agent bears a chemical functionality that will react selectively with a chemical functionality on the polymer to form an azide-containing polymer derivative product wherein the azide is separated from the polymer backbone by a linking group.

[00242] An exemplary reaction scheme is shown below: 75 2026205050   29 Jun 2026 X-polymer-Y + N-linker-N=N=N -> PG-X-polymer-linker-N=N=N wherein: polymer is poly(ethylene glycol) and X is a capping group such as alkoxy or a functional group as described above; and Y is a functional group that is not reactive with the azide functionality but that will react efficiently and selectively with the N functional group.

[00243] Examples of suitable functional groups include, but are not limited to, Y being a carboxylic acid, carbonate or active ester if N is an amine; Y being a ketone if N is a hydrazide or aminooxy moiety; Y being a leaving group if N is a nucleophile. Purification of the crude product may be accomplished by known methods including, but are not limited to, precipitation of the product followed by chromatography, if necessary.

[00244] A more specific example is shown below in the case of polymer diamine, in which one of the amines is protected by a protecting group moiety such as tert-butyl-Boc and the resulting mono-protected polymer diamine is reacted with a linking moiety that bears the azide functionality: BocHN-polymer-NH2 + HO2C-(CH2)3-N=N=N

[00245] In this instance, the amine group can be coupled to the carboxylic acid group using a variety of activating agents such as thionyl chloride or carbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazole to create an amide bond between the monoamine polymer derivative and the azide-bearing linker moiety. After successful formation of the amide bond, the resulting N-tert-butyl-Boc-protected azide-containing derivative can be used directly to modify bioactive molecules or it can be further elaborated to install other useful functional groups. For instance, the N-t-Boc group can be hydrolyzed by treatment with strong acid to generate an omega-amino-polymer-azide. The resulting amine can be used as a synthetic handle to install other useful functionality such as maleimide groups, activated disulfides, activated esters and so forth for the creation of valuable heterobifunctional reagents.

[00246] Heterobifunctional derivatives are particularly useful when it is desired to attach different molecules to each terminus of the polymer. For example, the omega-N-amino-N-azido polymer would allow the attachment of a molecule having an activated electrophilic group, such as an aldehyde, ketone, activated ester, activated carbonate and so forth, to one terminus of the polymer and a molecule having an acetylene group to the other terminus of the polymer.

[00247] In another embodiment of the present invention, A is an aliphatic linker of between 110 carbon atoms or a substituted aryl ring of between 6-14 carbon atoms. X is a functional group which does not react with azide groups and Y is a suitable leaving group.

[00248] Multiple targeting polypeptides may be joined by a linker polypeptide, wherein the linker polypeptide optionally is 6-14, 7-13, 8-12, 7-11, 9-11, or 9 amino acids in length. Other 76 2026205050   29 Jun 2026 linkers include but are not limited to small polymers such as PEG, which may be multi-armed allowing for multiple targeting polypeptide molecules to be linked together. Multiple targeting polypeptides and modified targeting polypeptides may be linked to each other via their N-termini in a head-to-head configuration through the use of such a linker or by direct chemical bonding between the respective N-terminus of each polypeptide. For example, two targeting polypeptides may be linked to form a dimer by chemical bonding between their N-terminal amino groups or modified N-terminal amino groups, Also, a linking molecule that is designed to comprise multiple chemical functional groups for bonding with the N-terminus of each targeting polypeptide may be used to join multiple targeting polypeptides each at their respective N-terminus. In addition, multiple targeting polypeptides may be linked through bonding between amino acids other than the N-terminal amino acid or C-terminal amino acid. An example of covalent bonds that may be utilized to form the dimmers and multimers of targeting polypeptide that are described herein include, but are not limited to disulphide or sulfhydral or thiol bonds. In addition, certain enzymes, such as sortase, may be used to form covalent bonds between the targeting polypeptides and the linker, including at the N-termini of the targeting polypeptides.

[00249] The linker may have a wide range of molecular weight or molecular length. Larger or smaller molecular weight linkers may be used to provide a desired spatial relationship or conformation between targeting polypeptide and the linked entity or between the linked entity and its binding partner, if any. Linkers having longer or shorter molecular length may also be used to provide a desired space or flexibility between targeting polypeptide and the linked entity, or between the linked entity and its binding partner.

[00250] In some embodiments, the invention provides water-soluble bifunctional linkers that have a dumbbell structure that includes: a) an azide, an alkyne, a hydrazine, a hydrazide, a hydroxylamine, or a carbonyl-containing moiety on at least a first end of a polymer backbone; and b) at least a second functional group on a second end of the polymer backbone. The second functional group can be the same or different as the first functional group. The second functional group, in some embodiments, is not reactive with the first functional group. The invention provides, in some embodiments, water-soluble compounds that comprise at least one arm of a branched molecular structure. For example, the branched molecular structure can be dendritic.

[00251] In exemplary embodiments, the polymer is linked to the targeting polypeptide or modified targeting polypeptide through a linker. For example, the linker can comprise one or two amino acids which at one end bind to the polymer - such as an albumin binding moiety - and at the other end bind to any available position on the polypeptide backbone. Additional 77 2026205050   29 Jun 2026 exemplary linkers include a hydrophilic linker such as a chemical moiety which comprises at least 5 non-hydrogen atoms where 30-50% of these are either N or O. Additional exemplary linkers which may link a polymer to a targeting polypeptide or modified targeting polypeptide are disclosed in U.S. 2012 / 0295847 and WO / 2012 / 168430, each of which is hereby incorporated by reference in its entirety.

[00252] Optionally, multiple targeting polypeptide or modified targeting polypeptide molecules may be joined by a linker polypeptide, wherein said linker polypeptide optionally is 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12 amino acids in length, and longer in length, wherein optionally the N-terminus of one targeting polypeptide is fused to the C-terminus of the linker polypeptide and the N-terminus of the linker polypeptide is fused to the N-terminus of another targeting polypeptide. Further exemplary linker polypeptides which may be utilized are disclosed in WO / 2013 / 004607, which is hereby incorporated by reference in its entirety.

[00253] The terms "electrophilic group", "electrophile" and the like as used herein refers to an atom or group of atoms that can accept an electron pair to form a covalent bond. The "electrophilic group" used herein includes but is not limited to halide, carbonyl and epoxide containing compounds. Common electrophiles may be halides such as thiophosgene, glycerin dichlorohydrin, phthaloyl chloride, succinyl chloride, chloroacetyl chloride, chlorosucciriyl chloride, etc.; ketones such as chloroacctone, bromoacetone, etc.; aldehydes such as glyoxal, etc.; isocyanates such as hexamethylene diisocyanate, tolylene diisocyanate, meta-xylylene diisocyanate, cyclohexylmethane-4,4-diisocyanate, etc and derivatives of these compounds.

[00254] The terms "nucleophilic group", "nucleophile" and the like as used herein refers to an atom or group of atoms that have an electron pair capable of forming a covalent bond. Groups of this type may be iohizable groups that react as anionic groups. The "nucleophilic group" used herein includes but is not limited to hydroxyl, primary amines, secondary amines, tertiary amines and thiols.

[00255] Table 2 provides various starting electrophiles and nucleophiles which may be combined to create a desired functional group. The information provided is meant to be illustrative and not limiting to the synthetic techniques described herein. Table 2: Examples of Covalent Linkages and Precursors Thereof Covalent Linkage Product Electrophile Nucleophile Carboxamides Activated esters amines / anilines Carboxamides acyl azides amines / anilines 2026205050   29 Jun 2026 Carboxamides acyl halides amines / anilines Esters acyl halides alcohols / phenols Esters acyl nitriles alcohols / phenols Carboxamides acyl nitriles amines / anilines Imines Aldehydes amines / anilines Hydrazones aldehydes or ketones Hydrazines Oximes aldehydes or ketones Hydroxylamines Alkyl amines alkyl halides amines / anilines Esters alkyl halides carboxylic acids Thioethers alkyl halides Thiols Ethers alkyl halides alcohols / phenols Thioethers alkyl sulfonates Thiols Esters alkyl sulfonates carboxylic acids Ethers alkyl sulfonates alcohols / phenols Esters Anhydrides alcohols / phenols Carboxamides Anhydrides amines / anilines Thiophenols aryl halides Thiols Aryl amines aryl halides Amines Thioethers Azindines Thiols Boronate esters Boronates Glycols Carboxamides carboxylic acids amines / anilines Esters carboxylic acids Alcohols hydrazines Hydrazides carboxylic acids N- acylureas or Anhydrides Carbodiimides carboxylic acids Esters Diazoalkanes carboxylic acids Thioethers Epoxides Thiols Thioethers Haloacetamides Thiols Ammotriazines Halotriazines amines / anilines Triazinyl ethers Halotriazines alcohols / phenols Amidines imido esters amines / anilines Ureas Isocyanates amines / anilines 2026205050   29 Jun 2026 Urethanes Isocyanates alcohols / phenols Thioureas Isothiocyanates amines / anilines Thioethers Maleimides Thiols Phosphite esters Phosphoramidites Alcohols Silyl ethers silyl halides Alcohols Alkyl amines sulfonate esters amines / anilines Thioethers sulfonate esters Thiols Esters sulfonate esters carboxylic acids Ethers sulfonate esters Alcohols Sulfonamides sulfonyl halides amines / anilines Sulfonate esters sulfonyl halides phenols / alcohols

[00256] In general, carbon electrophiles are susceptible to attack by complementary nucleophiles, including carbon nucleophiles, wherein an attacking nucleophile brings an electron pair to the carbon electrophile in order to form a new bond between the nucleophile and the carbon electrophile.

[00257] Non-limiting examples of carbon nucleophiles include, but are not limited to alkyl, alkenyl, aryl and alkynyl Grignard, organolithium, organozinc, alkyl-, alkenyl , aryl- and alkynyl-tin reagents (organostannanes), alkyl-, alkenyl-, aryl- and alkynyl-borane reagents (organoboranes and organoboronates); these carbon nucleophiles have the advantage of being kinetically stable in water or polar organic solvents. Other non-limiting examples of carbon nucleophiles include phosphorus ylids, enol and enolate reagents; these carbon nucleophiles have the advantage of being relatively easy to generate from precursors well known to those skilled in the art of synthetic organic chemistry. Carbon nucleophiles, when used in conjunction with carbon electrophiles, engender new carbon-carbon bonds between the carbon nucleophile and carbon electrophile.

[00258] Non-limiting examples of non-carbon nucleophiles suitable for coupling to carbon electrophiles include but are not limited to primary and secondary amines, thiols, thiolates, and thioethers, alcohols, alkoxides, azides, semicarbazides, and the like. These non-carbon nucleophiles, when used in conjunction with carbon electrophiles, typically generate heteroatom linkages (C-X-C), wherein X is a hetereoatom, including, but not limited to, oxygen, sulfur, or nitrogen. 2026205050   29 Jun 2026

[00259] In some cases, a polymer used in the invention terminates on one end with hydroxy or methoxy, i.e., X is H or CH3 ("methoxy PEG"). Alternatively, the polymer can terminate with a reactive group, thereby forming a bifunctional polymer. Typical reactive groups can include those reactive groups that are commonly used to react with the functional groups found in the 20 common amino acids (including but not limited to, maleimide groups, activated carbonates (including but not limited to, p-nitrophenyl ester), activated esters (including but not limited to, N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well as functional groups that are inert to the 20 common amino acids but that react specifically with complementary functional groups (including but not limited to, azide groups, alkyne groups). It is noted that the other end of the polymer, which is shown in the above formula by Y, will attach either directly or indirectly to a targeting polypeptide via a naturally-occurring or non-naturally encoded amino acid. For instance, Y may be an amide, carbamate or urea linkage to an amine group (including but not limited to, the epsilon amine of lysine or the N-terminus) of the polypeptide. Alternatively, Y may be a maleimide linkage to a thiol group (including but not limited to, the thiol group of cysteine). Alternatively, Y may be a linkage to a residue not commonly accessible via the 20 common amino acids. For example, an azide group on the polymer can be reacted with an alkyne group on the targeting polypeptide to form a Huisgen [3+2] cycloaddition product. Alternatively, an alkyne group on the polymer can be reacted with an azide group present in a targeting polypeptide to form a similar product. In some embodiments, a strong nucleophile (including but not limited to, hydrazine, hydrazide, hydroxylamine, semicarbazide) can be reacted with an aldehyde or ketone group present in a targeting polypeptide to form a hydrazone, oxime or semicarbazone, as applicable, which in some cases can be further reduced by treatment with an appropriate reducing agent. Alternatively, the strong nucleophile can be incorporated into the targeting polypeptide via a non-naturally encoded amino acid and used to react preferentially with a ketone or aldehyde group present in the water soluble polymer.

[00260] Any molecular mass for a polymer can be used as practically desired, including but not limited to, from about 100 Daltons (Da) to 100,000 Da or more as desired (including but not limited to, sometimes 0.1-50 kDa or 10-40 kDa). The molecular weight of polymer may be of a wide range, including but not limited to, between about 100 Da and about 100,000 Da or more. polymer may be between about 100 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 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, 81 2026205050   29 Jun 2026 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, polymer is between about 100 Da and about 50,000 Da. Branched chain polymers, including but not limited to, polymer molecules with each chain having a molecular weight ranging from 1-100 kDa (including but not limited to, 1-50 kDa or 520 kDa) can also be used. The molecular weight of each chain of the branched chain polymer may be, including but not limited to, between about 1,000 Da and about 100,000 Da or more. The molecular weight of each chain of the branched chain polymer may be between about 1,000 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 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 chain polymer is between about 1,000 Da and about 50,000 Da. In some embodiments, the molecular weight of each chain of the branched chain polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of each chain of the branched chain polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of each chain of the branched chain polymer is between about 5,000 Da and about 20,000 Da. A wide range of polymer molecules are described in, including but not limited to, the Shearwater Polymers, Inc. catalog, Nektar Therapeutics catalog, incorporated herein by reference.

[00261] The invention provides in some embodiments azide- and acetylene-containing polymer derivatives comprising a water soluble polymer backbone having an average molecular weight from about 800 Da to about 100,000 Da. The polymer backbone of the water-soluble polymer can be poly(ethylene glycol). However, it should be understood that a wide variety of water soluble polymers including but not limited to poly(ethylene)glycol and other related polymers, including poly(dextran) and poly(propylene glycol), are also suitable for use in the practice of this invention and that the use of the term PEG or poly(ethylene glycol) is intended to encompass and include all such molecules. The term PEG includes, but is not limited to, poly(ethylene glycol) in any of its forms, including bifunctional PEG, multiarmed PEG, derivatized PEG, forked PEG, branched PEG, pendent PEG (i.e. PEG or related polymers having one or more functional groups pendent to the polymer backbone), or PEG with degradable linkages therein. 2026205050   29 Jun 2026

[00262] In addition to these forms of polymer, the polymer can also be prepared with weak or degradable linkages in the backbone. For example, polymer can be prepared with ester linkages in the polymer backbone that are subject to hydrolysis. As shown below, this hydrolysis results in cleavage of the polymer into fragments of lower molecular weight: -polymer-CO2-polymer-+H2O ^polymer-CO2H+HO-polymer-

[00263] Many polymers are also suitable for use in the present invention. In some embodiments, polymer backbones that are water-soluble, with from 2 to about 300 termini, are particularly useful in the invention. Examples of suitable polymers include, but are not limited to, other poly(alkylene glycols), such as poly(propylene glycol) (“PPG”), copolymers thereof (including but not limited to copolymers of ethylene glycol and propylene glycol), terpolymers thereof, mixtures thereof, and the like. Although the molecular weight of each chain of the polymer backbone can vary, it is typically in the range of from about 800 Da to about 100,000 Da, often from about 6,000 Da to about 80,000 Da. The molecular weight of each chain of the polymer backbone may be between about 100 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 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.

[00264] In one feature of this embodiment of the invention, the intact polymer-conjugate, prior to hydrolysis, is minimally degraded upon administration, such that hydrolysis of the cleavable bond is effective to govern the slow rate of release of active targeting polypeptide into the bloodstream, as opposed to enzymatic degradation of targeting polypeptide prior to its release into the systemic circulation.

[00265] Appropriate physiologically cleavable linkages include but are not limited to ester, carbonate ester, carbamate, sulfate, phosphate, acyloxyalkyl ether, acetal, and ketal. Such 83 2026205050   29 Jun 2026 conjugates should possess a physiologically cleavable bond that is stable upon storage and upon administration. For instance, a targeting polypeptide or modified targeting polypeptide linked to a polymer should maintain its integrity upon manufacturing of the final pharmaceutical composition, upon dissolution in an appropriate delivery vehicle, if employed, and upon administration irrespective of route.

[00266] The present invention also includes phosphate-based linkers with tunable stability for intracellular delivery of drug conjugates disclosed in US 2017 / 0182181, incorporated by reference herein. The phosphate-based linkers comprise a monophosphate, diphosphate, triphosphate, or tetraphosphate group (phosphate group) covalently linked to the distal end of a linker arm comprising from the distal to the proximal direction a tuning element, optionally a spacer element, and a reactive functional group. The phosphate group of the phosphate-based linker is capable of being conjugated to a payload and the reactive functional group is capable of being conjugated to a cell-specific targeting ligand such as an antibody. The general structure of the phosphate-based linkers is: Phosphate group-Tuning element-Optional spacer elementFunctional reactive group A phosphate-based linker conjugated to a payload has the general structure:  Payload-Phosphate group-Tuning element-Optional spacer element-Functional reactive group and when conjugated to a targeting ligand has the general structure Payload-Phosphate group-Tuning element-Optional spacer element-Targeting ligand. These phosphatebased linkers have a differentiated and tunable stability in blood vs. an intracellular environment (e.g. lysosomal compartment). The rate at which the phosphate group is cleaved in the intracellular environment to release the payload in its native or active form may be affected by the structure of the tuning element with further effects mediated by substitutions of the phosphate group as well as whether the phosphate group is a monophosphate, diphosphate, triphosphate, or tetraphosphate. Further, these phosphate-based linkers provide the ability to construct conjugates such as antibody-drug conjugates in which the propensity of the conjugate to form aggregates is reduced compared to conjugates in which the same payload is conjugated to the antibody or targeting ligand using a linker that is not a phosphate-based linker as disclosed herein. Structure and Synthesis of TLR-agonist Linker Derivatives: Electrophilic and Nucleophilic Groups

[00267] TLR-agonist derivatives with linkers containing a hydroxylamine (also called an aminooxy) group allow for reaction with a variety of electrophilic groups to form conjugates 2026205050   29 Jun 2026 (including but not limited to, with PEG or other water soluble polymers). Like hydrazines, hydrazides and semicarbazides, the enhanced nucleophilicity of the aminooxy group permits it to react efficiently and selectively with a variety of molecules that contain carbonyl- or dicarbonylgroups, including but not limited to, ketones, aldehydes or other functional groups with similar chemical reactivity. See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34(9): 727-736 (2001). Whereas the result of reaction with a hydrazine group is the corresponding hydrazone, however, an oxime results generally from the reaction of an aminooxy group with a carbonyl- or dicarbonyl-containing group such as, by way of example, a ketones, aldehydes or other functional groups with similar chemical reactivity. In some embodiments, TLR-agonist derivatives with linkers comprising an azide, alkyne or cycloalkyne allow for linking of molecules via cycloaddition reactions (e.g., 1,3-dipolar cycloadditions, azide-alkyne Huisgen cycloaddition, etc.). (Described in U.S. Patent No. 7,807,619 which is incorporated by reference herein to the extent relative to the reaction).

[00268] Thus, in certain embodiments described herein are TLR-agonist derivatives with linkers comprising a hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, keto-amine, keto-alkyne, and ene-dione hydroxylamine group, a hydroxylamine-like group (which has reactivity similar to a hydroxylamine group and is structurally similar to a hydroxylamine group), a masked hydroxylamine group (which can be readily converted into a hydroxylamine group), or a protected hydroxylamine group (which has reactivity similar to a hydroxylamine group upon deprotection). In some embodiments, the TLR-agonist derivatives with linkers comprise azides, alkynes or cycloalkynes.

[00269] Such TLR-agonist linker derivatives or the targeting polypeptide may be in the form of a salt or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00270] In certain embodiments, compounds of Formula (I)-(VII) are stable in aqueous solution for at least 1 month under mildly acidic conditions. In certain embodiments, compounds of Formula (I)-(VII) are stable for at least 2 weeks under mildly acidic conditions. In certain embodiments, compound of Formula (I)-(VII) are stable for at least 5 days under mildly acidic conditions. In certain embodiments, such acidic conditions are pH 2 to 8.

[00271] The methods and compositions provided and described herein include polypeptides comprising non-natural amino acids having at least one carbonyl or dicarbonyl group, oxime group, hydroxylamine group, or protected or masked forms thereof. Introduction of at least one 85 2026205050   29 Jun 2026 reactive group into a TLR-agonist linker derivative or the targeting polypeptide can allow for the application of conjugation chemistries that involve specific chemical reactions, including, but not limited to, with one or more targeting polypeptide(s) while not reacting with the commonly occurring amino acids. Once incorporated, the targeting polypeptide of the TC side chains can also be modified by utilizing chemistry methodologies described herein or suitable for the particular functional groups or substituents present in the TLR-agonist linker derivative or the targeting polypeptide.

[00272] The TLR-agonist linker derivative and the targeting polypeptide methods and compositions described herein provide conjugates of substances having a wide variety of functional groups, substituents or moieties, with other substances including but not limited to a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; and any combination thereof.

[00273] In certain embodiments, the TLR-agonist linker derivatives, the targeting polypeptide, TCs, linkers and reagents described herein, including compounds of Formulas (I)-(VII) are stable in aqueous solution under mildly acidic conditions (including but not limited to pH 2 to 8). In other embodiments, such compounds are stable for at least one month under mildly acidic conditions. In other embodiments, such compounds are stable for at least 2 weeks under mildly acidic conditions. In other embodiments, such compounds are stable for at least 5 days under mildly acidic conditions.

[00274] In another aspect of the compositions, methods, techniques and strategies described herein are methods for studying or using any of the aforementioned “modified or unmodified” non-natural amino acid targeting polypeptide. Included within this aspect, by way of example only, are therapeutic, diagnostic, assay-based, industrial, cosmetic, plant biology, environmental, energy-production, consumer-products, and / or military uses which would benefit from a targeting polypeptide comprising a “modified or unmodified” non-natural amino acid polypeptide or protein.

[00275] TC molecules comprising at least one non-natural amino acid are provided in the invention. In certain embodiments of the invention, the TC with at least one non-natural amino acid includes at least one post-translational modification. In one embodiment, the at least one post-translational modification comprises attachment of a molecule including but not limited to, a label, a dye, a linker, another TC polypeptide, a polymer, a water-soluble polymer, a derivative of polyethylene glycol, a photocrosslinker, a radionuclide, a cytotoxic compound, a drug, an 86 2026205050   29 Jun 2026 affinity label, a photoaffinity label, a reactive compound, a resin, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, an antisense polynucleotide, a saccharide, a cyclodextrin, an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocaged moiety, an actinic radiation excitable moiety, a photoisomerizable moiety, biotin, a derivative of biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, an elongated side chain, a carbon-linked sugar, a redox-active agent, an amino thioacid, a toxic moiety, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chemiluminescent group, an electron dense group, a magnetic group, an intercalating group, a chromophore, an energy transfer agent, a biologically active agent, a detectable label, a small molecule, a quantum dot, a nanotransmitter, a radionucleotide, a radiotransmitter, a neutron-capture agent, or any combination of the above or any other desirable compound or substance, comprising a second reactive group to at least one non-natural amino acid comprising a first reactive group utilizing chemistry methodology that is known to one of ordinary skill in the art to be suitable for the particular reactive groups. For example, the first reactive group is an alkynyl moiety (including but not limited to, in the non-natural amino acid p-propargyloxyphenylalanine, where the propargyl group is also sometimes referred to as an acetylene moiety) and the second reactive group is an azido moiety, and [3+2] cycloaddition chemistry methodologies are utilized. In another example, the first reactive group is the azido moiety (including but not limited to, in the non-natural amino acid p-azido-L-phenylalanine or pAZ as it is sometimes referred to within this specification) and the second reactive group is the alkynyl moiety. In certain embodiments of the modified TC of the present invention, at least one non-natural amino acid (including but not limited to, non-natural amino acid containing a keto functional group) comprising at least one post-translational modification, is used where the at least one post-translational modification comprises a saccharide moiety. In certain embodiments, the post-translational modification is made in vivo in a eukaryotic cell or in a non-eukaryotic cell. A linker, polymer, water soluble polymer, or other molecule may attach the molecule to the polypeptide. In an additional embodiment the linker attached to the TC is long enough to permit formation of a dimer. The molecule may also be linked directly to the polypeptide. 2026205050   29 Jun 2026

[00276] In certain embodiments, the TC protein includes at least one post-translational modification that is made in vivo by one host cell, where the post-translational modification is not normally made by another host cell type. In certain embodiments, the protein includes at least one post-translational modification that is made in vivo by a eukaryotic cell, where the post-translational modification is not normally made by a non-eukaryotic cell. Examples of post-translational modifications include, but are not limited to, glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linkage modification, and the like.

[00277] In some embodiments, the TC comprise one or more non-naturally encoded amino acids for glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, or glycolipid-linkage modification of the polypeptide. In some embodiments, the TC comprise one or more non-naturally encoded amino acids for glycosylation of the polypeptide. In some embodiments, the TC comprise one or more naturally encoded amino acids for glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, or glycolipid-linkage modification of the polypeptide. In some embodiments, the TC, comprise one or more naturally encoded amino acids for glycosylation of the polypeptide.

[00278] In some embodiments, the TC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation of the polypeptide. In some embodiments, the TC comprises one or more deletions that enhance glycosylation of the polypeptide. In some embodiments, the TC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation at a different amino acid in the polypeptide. In some embodiments, the TC comprises one or more deletions that enhance glycosylation at a different amino acid in the polypeptide. In some embodiments, the TC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation at a non-naturally encoded amino acid in the polypeptide. In some embodiments, the TC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation at a naturally encoded amino acid in the polypeptide. In some embodiments, the TC comprises one or more naturally encoded amino acid additions and / or substitutions that enhance glycosylation at a different amino acid in the polypeptide. In some embodiments, the TC comprises one or more non-naturally encoded amino acid additions and / or substitutions that enhance glycosylation at a naturally encoded amino acid in the polypeptide. In some embodiments, the TC comprises one or more non-naturally encoded amino 88 2026205050   29 Jun 2026 acid additions and / or substitutions that enhance glycosylation at a non-naturally encoded amino acid in the polypeptide.

[00279] In one embodiment, the post-translational modification comprises attachment of an oligosaccharide to an asparagine by a GlcNAc-asparagine linkage (including but not limited to, where the oligosaccharide comprises (GlcNAc-Man)2-Man-GlcNAc-GlcNAc, and the like). In another embodiment, the post-translational modification comprises attachment of an oligosaccharide (including but not limited to, Gal-GalNAc, Gal-GlcNAc, etc.) to a serine or threonine by a GalNAc-serine, a GalNAc-threonine, a GlcNAc-serine, or a GlcNAc-threonine linkage. In certain embodiments, a protein or polypeptide of the invention can comprise a secretion or localization sequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, and / or the like. Examples of secretion signal sequences include, but are not limited to, a prokaryotic secretion signal sequence, a eukaryotic secretion signal sequence, a eukaryotic secretion signal sequence 5’-optimized for bacterial expression, a novel secretion signal sequence, pectate lyase secretion signal sequence, Omp A secretion signal sequence, and a phage secretion signal sequence. Examples of secretion signal sequences include, but are not limited to, STII (prokaryotic), Fd GIII and M13 (phage), Bgl2 (yeast), and the signal sequence bla derived from a transposon. Any such sequence may be modified to provide a desired result with the polypeptide, including but not limited to, substituting one signal sequence with a different signal sequence, substituting a leader sequence with a different leader sequence, etc.

[00280] The protein or polypeptide of interest can contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more non-natural amino acids. The non-natural amino acids can be the same or different, for example, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different non-natural amino acids. In certain embodiments, at least one, but fewer than all, of a particular amino acid present in a naturally occurring version of the protein is substituted with an non-natural amino acid.

[00281] The present invention provides methods and compositions based on TC comprising at least one non-naturally encoded amino acid. Introduction of at least one non-naturally encoded amino acid into TC can allow for the application of conjugation chemistries that involve specific chemical reactions, including, but not limited to, with one or more non-naturally encoded amino acids while not reacting with the commonly occurring 20 amino acids. In some embodiments, TC comprising 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 89 2026205050   29 Jun 2026 acid. This invention provides a highly efficient method for the selective modification of proteins with PEG derivatives or TLR-linker derivatives, which involves the selective incorporation of non-genetically encoded amino acids, including but not limited to, those amino acids containing functional groups or substituents not found in the 20 naturally incorporated amino acids, including but not limited to a ketone, an azide or acetylene moiety, into proteins in response to a selector codon and the subsequent modification of those amino acids with a suitably reactive PEG derivative. Once incorporated, the amino acid side chains can then be modified by utilizing chemistry methodologies known to those of ordinary skill in the art to be suitable for the particular functional groups or substituents present in the non-naturally encoded amino acid. Known chemistry methodologies of a wide variety are suitable for use in the present invention to incorporate a water soluble polymer into the protein. Such methodologies include but are not limited to a Huisgen [3+2] cycloaddition reaction (see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991) Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1109; and, Huisgen, R. in 1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, New York, p. 1-176) with, including but not limited to, acetylene or azide derivatives, respectively.

[00282] Because the Huisgen [3+2] cycloaddition method involves a cycloaddition rather than a nucleophilic substitution reaction, proteins can be modified with extremely high selectivity. The reaction can be carried out at room temperature in aqueous conditions with excellent regioselectivity (1,4 > 1,5) by the addition of catalytic amounts of Cu(I) salts to the reaction mixture. See, e.g., Tornoe, et al., (2002) J. Org. Chem. 67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599; and WO 03 / 101972. A molecule that can be added to a protein of the invention through a [3+2] cycloaddition includes virtually any molecule with a suitable functional group or substituent including but not limited to an azido or acetylene derivative. These molecules can be added to an non-natural amino acid with an acetylene group, including but not limited to, p-propargyloxyphenylalanine, or azido group, including but not limited to p-azido-phenylalanine, respectively.

[00283] The five-membered ring that results from the Huisgen [3+2] cycloaddition is not generally reversible in reducing environments and is stable against hydrolysis for extended periods in aqueous environments. Consequently, the physical and chemical characteristics of a wide variety of substances can be modified under demanding aqueous conditions with the active PEG derivatives or TLR-linker derivatives of the present invention. Even more importantly, because the azide and acetylene moieties are specific for one another (and do not, for example, 2026205050   29 Jun 2026 react with any of the 20 common, genetically-encoded amino acids), proteins can be modified in one or more specific sites with extremely high selectivity.

[00284] The 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. The PEG polymer derivatives that contain acetylene moieties are highly selective for coupling with azide moieties that have been introduced selectively into proteins in response to a selector codon. Similarly, PEG polymer derivatives that contain azide moieties are highly selective for coupling with acetylene moieties that have been introduced selectively into proteins in response to a selector codon. More specifically, the azide moieties comprise, but are not limited to, alkyl azides, aryl azides and derivatives of these azides. The derivatives of the alkyl and aryl azides can include other substituents so long as the acetylenespecific reactivity is maintained. The acetylene moieties comprise alkyl and aryl acetylenes and derivatives of each. The derivatives of the alkyl and aryl acetylenes can include other substituents so long as the azide-specific reactivity is maintained.

[00285] The present invention provides conjugates of substances having a wide variety of functional groups, substituents or moieties, with other substances including but not limited to a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a radionuclide; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide; a water-soluble dendrimer; a cyclodextrin; an inhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing moiety; a radioactive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; an actinic radiation excitable moiety; a photoisomerizable moiety; biotin; a derivative of biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent; an amino thioacid; a toxic moiety; an isotopically labeled moiety; a biophysical probe; a phosphorescent group; a chemiluminescent group; an electron dense group; a magnetic group; an intercalating group; a chromophore; an energy transfer agent; a biologically active agent; a detectable label; a small molecule; a quantum dot; a nanotransmitter; a radionucleotide; a radiotransmitter; a neutron-capture agent; or any combination of the above, or any other desirable compound or substance. The present invention also includes conjugates of substances 91 2026205050   29 Jun 2026 having azide or acetylene moieties with PEG polymer derivatives having the corresponding acetylene or azide moieties. For example, a PEG polymer containing an azide moiety can be coupled to a biologically active molecule at a position in the protein that contains a non-genetically encoded amino acid bearing an acetylene functionality. The linkage by which the PEG and the biologically active molecule are coupled includes but is not limited to the Huisgen [3+2] cycloaddition product.

[00286] It is well established in the art that PEG can be used to modify the surfaces of biomaterials (see, e.g., U.S. Patent 6,610,281; Mehvar, R., J. Pharm Pharm Sci., 3(1):125-136 (2000) which are incorporated by reference herein). 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 coupled to the surface via the Huisgen [3+2] cycloaddition linkage. Biomaterials and other substances can also be coupled to the azide-or acetylene-activated polymer derivatives through a linkage other than the azide or acetylene linkage, such as through a linkage comprising a carboxylic acid, amine, alcohol or thiol moiety, to leave the azide or acetylene moiety available for subsequent reactions.

[00287] The invention includes a method of synthesizing the azide- and acetylene- containing polymers of the invention. In the case of the azide-containing PEG derivative, the azide can be bonded directly to a carbon atom of the polymer. Alternatively, the azide-containing PEG derivative can be prepared by attaching a linking agent that has the azide moiety at one terminus to a conventional activated polymer so that the resulting polymer has the azide moiety at its terminus. In the case of the acetylene-containing PEG derivative, the acetylene can be bonded directly to a carbon atom of the polymer. Alternatively, the acetylene-containing PEG derivative can be prepared by attaching a linking agent that has the acetylene moiety at one terminus to a conventional activated polymer so that the resulting polymer has the acetylene moiety at its terminus.

[00288] More specifically, in the case of the azide-containing PEG derivative, a water soluble polymer having at least one active hydroxyl moiety undergoes a reaction to produce a substituted polymer having a more reactive moiety, such as a mesylate, tresylate, tosylate or halogen leaving group, thereon. The preparation and use of PEG derivatives or TLR-linker derivatives containing sulfonyl acid halides, halogen atoms and other leaving groups are known to those of ordinary skill in the art. The resulting substituted polymer then undergoes a reaction to substitute for the more reactive moiety an azide moiety at the terminus of the polymer. Alternatively, a water soluble polymer having at least one active nucleophilic or electrophilic 92 2026205050   29 Jun 2026 moiety undergoes a reaction with a linking agent that has an azide at one terminus so that a covalent bond is formed between the PEG polymer and the linking agent and the azide moiety is positioned at the terminus of the polymer. Nucleophilic and electrophilic moieties, including amines, thiols, hydrazides, hydrazines, alcohols, carboxylates, aldehydes, ketones, thioesters and the like, are known to those of ordinary skill.

[00289] More specifically, in the case of the acetylene-containing PEG derivative, a water soluble polymer having at least one active hydroxyl moiety undergoes a reaction to displace a halogen or other activated leaving group from a precursor that contains an acetylene moiety. Alternatively, a water soluble polymer having at least one active nucleophilic or electrophilic moiety undergoes a reaction with a linking agent that has an acetylene at one terminus so that a covalent bond is formed between the PEG polymer and the linking agent and the acetylene moiety is positioned at the terminus of the polymer. The use of halogen moieties, activated leaving group, nucleophilic and electrophilic moieties in the context of organic synthesis and the preparation and use of PEG derivatives or TLR-linker derivatives is well established to practitioners in the art.

[00290] The invention also provides a method for the selective modification of proteins to add other substances to the modified protein, including but not limited to water soluble polymers such as PEG and PEG derivatives or TLR-linker derivatives, linkers, or another TC polypeptide, containing an azide or acetylene moiety. The azide- and acetylene-containing PEG derivatives or TLR-linker derivatives can be used to modify the properties of surfaces and molecules where biocompatibility, stability, solubility and lack of immunogenicity are important, while at the same time providing a more selective means of attaching the PEG derivatives or TLR-linker derivatives to proteins than was previously known in the art. General Recombinant Nucleic Acid Methods For Use With The Invention

[00291] In numerous embodiments of the present invention, nucleic acids encoding a targeting polypeptide of the TC of interest will be isolated, cloned and often altered using recombinant methods. Such embodiments are used, including but not limited to, for protein expression or during the generation of variants, derivatives, expression cassettes, or other sequences derived from a targeting polypeptide of the TC. In some embodiments, the sequences encoding the polypeptides of the invention are operably linked to a heterologous promoter.

[00292] A nucleotide sequence encoding a targeting polypeptide of the TC comprising a non-naturally encoded amino acid may be synthesized on the basis of the amino acid sequence of the parent polypeptide, and then changing the nucleotide sequence so as to effect introduction (i.e., 93 2026205050   29 Jun 2026 incorporation or substitution) or removal (i.e., deletion or substitution) of the relevant amino acid residue(s). The nucleotide sequence may be conveniently modified by site-directed mutagenesis in accordance with conventional methods. Alternatively, the nucleotide sequence may be prepared by chemical synthesis, including but not limited to, by using an oligonucleotide synthesizer, wherein oligonucleotides are designed based on the amino acid sequence of the desired polypeptide, and preferably selecting those codons that are favored in the host cell in which the recombinant polypeptide will be produced.   For example, several small oligonucleotides coding for portions of the desired polypeptide may be synthesized and assembled by PCR, ligation or ligation chain reaction. See, e.g., Barany, et al., Proc. Natl. Acad. Sci. 88: 189-193 (1991); U.S. Patent 6,521,427 which are incorporated by reference herein.

[00293] This invention utilizes routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).

[00294] The invention also relates to eukaryotic host cells, non-eukaryotic host cells, and organisms for the in vivo incorporation of a non-natural amino acid via orthogonal tRNA / RS pairs. Host cells are genetically engineered (including but not limited to, transformed, transduced or transfected) with the polynucleotides of the invention or constructs which include a polynucleotide of the invention, including but not limited to, a vector of the invention, which can be, for example, a cloning vector or an expression vector.

[00295] Several well-known methods of introducing target nucleic acids into cells are available, any of which can be used in the invention. These include: fusion of the recipient cells with bacterial protoplasts containing the DNA, electroporation, projectile bombardment, and infection with viral vectors (discussed further, below), etc. Bacterial cells can be used to amplify the number of plasmids containing DNA constructs of this invention. The bacteria are grown to log phase and the plasmids within the bacteria can be isolated by a variety of methods known in the art (see, for instance, Sambrook). In addition, kits are commercially available for the purification of plasmids from bacteria, (see, e.g., EasyPrep™, FlexiPrep™, both from Pharmacia Biotech; StrataClean™ from Stratagene; and, QIAprep™ from Qiagen). The isolated and purified plasmids are then further manipulated to produce other plasmids, used to transfect cells or incorporated into related vectors to infect organisms. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters 94 2026205050   29 Jun 2026 useful for regulation of the expression of the particular target nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (including but not limited to, shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or both. See, Gillam & Smith, Gene 8:81 (1979); Roberts, et al., Nature, 328:731 (1987); Schneider, E., et al., Protein Expr. Purif. 6(1):10-14 (1995); Ausubel, Sambrook, Berger (all supra). A catalogue of bacteria and bacteriophages useful for cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria and Bacteriophage (1992) Gherna et al. (eds) published by the ATCC. Additional basic procedures for sequencing, cloning and other aspects of molecular biology and underlying theoretical considerations are also found in Watson et al. (1992) Recombinant DNA Second Edition Scientific American Books, NY. In addition, essentially any nucleic acid (and virtually any labeled nucleic acid, whether standard or nonstandard) can be custom or standard ordered from any of a variety of commercial sources, such as the Midland Certified Reagent Company (Midland, TX available on the World Wide Web at mcrc.com), The Great American Gene Company (Ramona, CA available on the World Wide Web at genco.com), ExpressGen Inc. (Chicago, IL available on the World Wide Web at expressgen.com), Operon Technologies Inc. (Alameda, CA) and many others. Selector Codons

[00296] Selector codons of the invention expand the genetic codon framework of protein biosynthetic machinery. For example, a selector codon includes, but is not limited to, a unique three base codon, a nonsense codon, such as a stop codon, including but not limited to, an amber codon (UAG), an ochre codon, or an opal codon (UGA), an unnatural codon, a four or more base codon, a rare codon, or the like. It is readily apparent to those of ordinary skill in the art that there is a wide range in the number of selector codons that can be introduced into a desired gene or polynucleotide, including but not limited to, one or more, two or more, three or more, 4, 5, 6, 7, 8, 9, 10 or more in a single polynucleotide encoding at least a portion of the TC.

[00297] In one embodiment, the methods involve the use of a selector codon that is a stop codon for the incorporation of one or more non-natural amino acids in vivo. For example, an O-tRNA is produced that recognizes the stop codon, including but not limited to, UAG, and is aminoacylated by an O-RS with a desired non-natural amino acid. This O-tRNA is not recognized by the naturally occurring host’s aminoacyl-tRNA synthetases. Conventional site-directed mutagenesis can be used to introduce the stop codon, including but not limited to, TAG, 95 2026205050   29 Jun 2026 at the site of interest in a polypeptide of interest. See, e.g., Sayers, J.R., 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 the nucleic acid that encodes the polypeptide of interest are combined in vivo, the non-natural amino acid is incorporated in response to the UAG codon to give a polypeptide containing the non-natural amino acid at the specified position.

[00298] The incorporation of non-natural amino acids in vivo can be done without significant perturbation of the eukaryotic host cell. For example, because the suppression efficiency for the UAG codon depends upon the competition between the O-tRNA, including but not limited to, the amber suppressor tRNA, and a eukaryotic release factor (including but not limited to, eRF) (which binds to a stop codon and initiates release of the growing peptide from the ribosome), the suppression efficiency can be modulated by, including but not limited to, increasing the expression level of O-tRNA, and / or the suppressor tRNA.

[00299] Non-natural amino acids can also be encoded with rare codons. For example, when the arginine concentration in an in vitro protein synthesis reaction is reduced, the rare arginine codon, AGG, has proven to be efficient for insertion of Ala by a synthetic tRNA acylated with alanine. See, e.g., Ma et al., Biochemistry, 32:7939 (1993). In this case, the synthetic tRNA competes with the naturally occurring tRNAArg, which exists as a minor species in Escherichia coli. Some organisms do not use all triplet codons. An unassigned codon AGA in Micrococcus luteus has been utilized for insertion of amino acids in an in vitro transcription / translation extract. See, e.g., Kowal and Oliver, Nucl. Acid. Res., 25:4685 (1997). Components of the present invention can be generated to use these rare codons in vivo.

[00300] Selector codons also comprise extended codons, including but not limited to, four or more base codons, such as, four, five, six or more base codons. Examples of four base codons include, but are not limited to, AGGA, CUAG, UAGA, CCCU and the like. Examples of five base codons include, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC and the like. A feature of the invention includes using extended codons based on frameshift suppression. Four or more base codons can insert, including but not limited to, one or multiple non-natural amino acids into the same protein. For example, in the presence of mutated O-tRNAs, including but not limited to, a special frameshift suppressor tRNAs, with anticodon loops, for example, with at least 8-10 nt anticodon loops, the four or more base codon is read as single amino acid. In other embodiments, the anticodon loops can decode, including but not limited to, at least a four-base codon, at least a five-base codon, or at least a six-base codon or more. Since there are 256 possible four-base codons, multiple non-natural amino acids can be 96 2026205050   29 Jun 2026 encoded in the same cell using a four or more base codon. See, Anderson et al., (2002) Exploring the Limits of Codon and Anticodon Size, Chemistry and Biology, 9:237-244; Magliery, (2001) Expanding the Genetic Code: Selection of Efficient Suppressors of Four-base Codons and Identification of “Shifty” Four-base Codons with a Library Approach in Escherichia coli, J. Mol. Biol. 307: 755-769.

[00301] For example, four-base codons have been used to incorporate non-natural amino acids into proteins using in vitro biosynthetic methods. See, e.g., Ma et al., (1993) Biochemistry, 32:7939; and Hohsaka et al., (1999) J. Am. Chem. Soc., 121:34. CGGG and AGGU were used to simultaneously incorporate 2-naphthylalanine and an NBD derivative of lysine into streptavidin in vitro with two chemically acylated frameshift suppressor tRNAs. See, e.g., Hohsaka et al., (1999) J. Am. Chem. Soc., 121:12194. In an in vivo study, Moore et al. examined the ability of tRNALeu derivatives with NCUA anticodons to suppress UAGN codons (N can be U, A, G, or C), and found that the quadruplet UAGA can be decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13 to 26% with little decoding in the 0 or -1 frame. See, Moore et al., (2000) J. Mol. Biol., 298:195. In one embodiment, extended codons based on rare codons or nonsense codons can be used in the present invention, which can reduce missense readthrough and frameshift suppression at other unwanted sites.

[00302] For a given system, a selector codon can also include one of the natural three base codons, where the endogenous system does not use (or rarely uses) the natural base codon. For example, this includes a system that is lacking a tRNA that recognizes the natural three base codon, and / or a system where the three base codon is a rare codon.

[00303] Selector codons optionally include unnatural base pairs. These unnatural base pairs further expand the existing genetic alphabet. One extra base pair increases the number of triplet codons from 64 to 125. Properties of third base pairs include stable and selective base pairing, efficient enzymatic incorporation into DNA with high fidelity by a polymerase, and the efficient continued primer extension after synthesis of the nascent unnatural base pair. Descriptions of unnatural base pairs which can be adapted for methods and compositions include, e.g., Hirao, et al., (2002) An unnatural base pair for incorporating amino acid analogues into protein, Nature Biotechnology, 20:177-182. See, also, Wu, Y., et al., (2002) J. Am. Chem. Soc. 124:1462614630. Other relevant publications are listed below.

[00304] For in vivo usage, the unnatural nucleoside is membrane permeable and is phosphorylated to form the corresponding triphosphate. In addition, the increased genetic information is stable and not destroyed by cellular enzymes. Previous efforts by Benner and 97 2026205050   29 Jun 2026 others took advantage of hydrogen bonding patterns that are different from those in canonical Watson-Crick pairs, the most noteworthy example of which is the iso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc., 111:8322; and Piccirilli et al., (1990) Nature, 343:33; Kool, (2000) Curr. Opin. Chem. Biol., 4:602. These bases in general mispair to some degree with natural bases and cannot be enzymatically replicated. Kool and co-workers demonstrated that hydrophobic packing interactions between bases can replace hydrogen bonding to drive the formation of base pair. See, Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and Guckian and Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort to develop an unnatural base pair satisfying all the above requirements, Schultz, Romesberg and co-workers have systematically synthesized and studied a series of unnatural hydrophobic bases. A PICS:PICS self-pair is found to be more stable than natural base pairs and can be efficiently incorporated into DNA by Klenow fragment of Escherichia coli DNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem. Soc., 121:11585-6; and Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274. A 3MN:3MN self-pair can be synthesized by KF with efficiency and selectivity sufficient for biological function. See, e.g., Ogawa et al., (2000) J. Am. Chem. Soc., 122:8803. However, both bases act as a chain terminator for further replication. A mutant DNA polymerase has been recently evolved that can be used to replicate the PICS self pair. In addition, a 7AI self pair can be replicated. See, e.g., Tae et al., (2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py, has also been developed, which forms a stable pair upon binding Cu(II). See, Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Because extended codons and unnatural codons are intrinsically orthogonal to natural codons, the methods of the invention can take advantage of this property to generate orthogonal tRNAs for them.

[00305] A translational bypassing system can also be used to incorporate a non-natural amino acid in a desired polypeptide. In a translational bypassing system, a large sequence is incorporated into a gene but is not translated into protein. The sequence contains a structure that serves as a cue to induce the ribosome to hop over the sequence and resume translation downstream of the insertion.

[00306] Nucleic acid molecules encoding a protein of interest such as a targeting polypeptide of the TC may be readily mutated to introduce a cysteine at any desired position of the polypeptide. Cysteine is widely used to introduce reactive molecules, water soluble polymers, proteins, or a wide variety of other molecules, onto a protein of interest. Methods suitable for the incorporation of cysteine into a desired position of a polypeptide are known to those of 98 2026205050   29 Jun 2026 ordinary skill in the art, such as those described in U.S. Patent No. 6,608,183, which is incorporated by reference herein, and standard mutagenesis techniques. III.         Non-Naturally Encoded Amino Acids

[00307] A very wide variety of non-naturally encoded amino acids are suitable for use in the present invention. Any number of non-naturally encoded amino acids can be introduced into a TC. In general, the introduced non-naturally encoded amino acids are substantially chemically inert toward the 20 common, genetically-encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). In some embodiments, the non-naturally encoded amino acids include side chain functional groups that react efficiently and selectively with functional groups not found in the 20 common amino acids (including but not limited to, azido, ketone, aldehyde and aminooxy groups) to form stable conjugates. For example, a targeting polypeptide of the TC that includes a non-naturally encoded amino acid containing an azido functional group can be reacted with a polymer (including but not limited to, poly(ethylene glycol) or, alternatively, a second polypeptide or linker containing an alkyne moiety) to form a stable conjugate resulting from the selective reaction of the azide and the alkyne functional groups to form a Huisgen [3+2] cycloaddition product.

[00308] The generic structure of an alpha-amino acid is illustrated as follows (Formula I): I H2N R COOH

[00309] A non-naturally encoded amino acid is typically any structure having the above-listed formula wherein the R group is any substituent other than one used in the twenty natural amino acids, and may be suitable for use in the present invention. Because the non-naturally encoded amino acids of the invention typically differ from the natural amino acids only in the structure of the side chain, the non-naturally encoded amino acids form amide bonds with other amino acids, including but not limited to, natural or non-naturally encoded, in the same manner in which they are formed in naturally occurring polypeptides. However, the non-naturally encoded amino acids have side chain groups that distinguish them from the natural amino acids. For example, R optionally comprises an alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono, 2026205050   29 Jun 2026 phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino group, or the like or any combination thereof. Other non-naturally occurring amino acids of interest that may be suitable for use in the present invention include, but are not limited to, amino acids comprising a photoactivatable cross-linker, spin-labeled amino acids, fluorescent amino acids, metal binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, photocaged and / or photoisomerizable amino acids, amino acids comprising biotin or a biotin analogue, glycosylated amino acids such as a sugar substituted serine, other carbohydrate modified amino acids, keto-containing amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids with an elongated side chains as compared to natural amino acids, including but not limited to, polyethers or long chain hydrocarbons, including but not limited to, greater than about 5 or greater than about 10 carbons, carbon-linked sugar-containing amino acids, redox-active amino acids, amino thioacid containing amino acids, and amino acids comprising one or more toxic moiety.

[00310] Exemplary non-naturally encoded amino acids that may be suitable for use in the present invention and that are useful for reactions with water soluble polymers include, but are not limited to, those with carbonyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactive groups. In some embodiments, non-naturally encoded amino acids comprise a saccharide moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,      N-acetyl-L-glucosaminyl-L-threonine,      N-acetyl-L- glucosaminyl-L-asparagine and O-mannosaminyl-L-serine. Examples of such amino acids also include examples where the naturally-occuring N- or O- linkage between the amino acid and the saccharide is replaced by a covalent linkage not commonly found in nature - including but not limited to, an alkene, an oxime, a thioether, an amide and the like. Examples of such amino acids also include saccharides that are not commonly found in naturally-occuring proteins such as 2-deoxy-glucose, 2-deoxygalactose and the like.

[00311] Many of the non-naturally encoded amino acids provided herein are commercially available, e.g., from Sigma-Aldrich (St. Louis, MO, USA), Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), or Peptech (Burlington, MA, USA). Those that are not commercially available are optionally synthesized as provided herein or using standard methods known to those of ordinary skill in the art. For organic synthesis techniques, see, e.g., Organic Chemistry by Fessendon and Fessendon, (1982, Second Edition, Willard Grant Press, Boston 100 2026205050   29 Jun 2026 Mass.); Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistry by Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York). See, also, U.S. Patent Nos. 7,045,337 and 7,083,970, which are incorporated by reference herein. In addition to non-natural amino acids that contain novel side chains, non-natural amino acids that may be suitable for use in the present invention also optionally comprise modified backbone structures, including but not limited to, as illustrated by the structures of Formula II and III: II R Z'^^'C--YH II X III R    R' H2N^^^^Co2H wherein Z typically comprises OH, NH2, SH, NH-R’, or S-R'; X and Y, which can be the same or different, typically comprise S or O, and R and R', which are optionally the same or different, are typically selected from the same list of constituents for the R group described above for the nonnatural amino acids having Formula I as well as hydrogen. For example, nonnatural amino acids of the invention optionally comprise substitutions in the amino or carboxyl group as illustrated by Formulas II and III. nonnatural amino acids of this type include, but are not limited to, a-hydroxy acids, a-thioacids, a-aminothiocarboxylates, including but not limited to, with side chains corresponding to the common twenty natural amino acids or unnatural side chains. In addition, substitutions at the a-carbon optionally include, but are not limited to, L, D, or a-a-disubstituted amino acids such as D-glutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Other structural alternatives include cyclic amino acids, such as proline analogues as well as 3, 4 ,6, 7, 8, and 9 membered ring proline analogues, p and y amino acids such as substituted p-alanine and Y-amino butyric acid.

[00312] Many nonnatural amino acids are based on natural amino acids, such as tyrosine, glutamine, phenylalanine, and the like, and are suitable for use in the present invention. 2026205050   29 Jun 2026 Tyrosine analogs include, but are not limited to, para-substituted tyrosines, ortho-substituted tyrosines, and meta substituted tyrosines, where the substituted tyrosine comprises, including but not limited to, a keto group (including but not limited to, an acetyl group), a benzoyl group, an amino group, a hydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropyl group, a methyl group, a C6 - C20 straight chain or branched hydrocarbon, a saturated or unsaturated hydrocarbon, an O-methyl group, a polyether group, a nitro group, an alkynyl group or the like. In addition, multiply substituted aryl rings are also contemplated. Glutamine analogs that may be suitable for use in the present invention include, but are not limited to, a-hydroxy derivatives, Y—substituted derivatives, cyclic derivatives, and amide substituted glutamine derivatives. Example phenylalanine analogs that may be suitable for use in the present invention include, but are not limited to, para-substituted phenylalanines, ortho-substituted phenyalanines, and metasubstituted phenylalanines, where the substituent comprises, including but not limited to, a hydroxy group, a methoxy group, a methyl group, an allyl group, an aldehyde, an azido, an iodo, a bromo, a keto group (including but not limited to, an acetyl group), a benzoyl, an alkynyl group, or the like. Specific examples of non-natural amino acids that may be suitable for use in the present invention include, but are not limited to, a p-acetyl-L- phenylalanine, an O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GleNAcP-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, a p-amino-L-phenylalanine, an isopropyl-L-phenylalanine, and a p-propargyloxy-phenylalanine, and the like. Examples of structures of a variety of nonnatural amino acids that may be suitable for use in the present invention are provided in, for example, WO 2002 / 085923 entitled “In vivo incorporation of unnatural amino acids.” See also Kiick et al., (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation, PNAS 99:19-24, which is incorporated by reference herein, for additional methionine analogs. International Application No. PCT / US06 / 47822 entitled “Compositions Containing, Methods Involving, and Uses of Nonnatural Amino Acids and Polypeptides,” which is incorporated by reference herein, describes reductive alkylation of an aromatic amine moieties, including but not limited to, p-amino-phenylalanine and reductive amination. 2026205050   29 Jun 2026

[00313] In another embodiment of the present invention, the TC polypeptides with one or more non-naturally encoded amino acids are covalently modified. Selective chemical reactions that are orthogonal to the diverse functionality of biological systems are recognized as important tools in chemical biology. As relative newcomers to the repertoire of synthetic chemistry, these bioorthogonal reactions have inspired new strategies for compound library synthesis, protein engineering, functional proteomics, and chemical remodeling of cell surfaces. The azide has secured a prominent role as a unique chemical handle for bioconjugation. The Staudinger ligation has been used with phosphines to tag azidosugars metabolically introduced into cellular glycoconjugates. The Staudinger ligation can be performed in living animals without physiological harm; nevertheless, the Staudinger reaction is not without liabilities. The requisite phosphines are susceptible to air oxidation and their optimization for improved water solubility and increased reaction rate has proven to be synthetically challenging.

[00314] The azide group has an alternative mode of bioorthogonal reactivity: the [3+2] cycloaddition with alkynes described by Huisgen. In its classic form, this reaction has limited applicability in biological systems due to the requirement of elevated temperatures (or pressures) for reasonable reaction rates. Sharpless and coworkers surmounted this obstacle with the development of a copper(I)-catalyzed version, termed "click chemistry," that proceeds readily at physiological temperatures and in richly functionalized biological environs. This discovery has enabled the selective modification of virus particles, nucleic acids, and proteins from complex tissue lysates. Unfortunately, the mandatory copper catalyst is toxic to both bacterial and mammalian cells, thus precluding applications wherein the 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 reaction with biological nucleophiles.

[00315] In one embodiment, compositions of a targeting polypeptide of the TC that include a non-natural amino acid (such as p-(propargyloxy)-phenyalanine) are provided. Various compositions comprising p-(propargyloxy)-phenyalanine and, including but not limited to, proteins and / or cells, are also provided. In one aspect, a composition that includes the p-(propargyloxy)-phenyalanine non-natural amino acid, further includes an orthogonal tRNA. The non-natural amino acid can be bonded (including but not limited to, covalently) to the orthogonal tRNA, including but not limited to, covalently bonded to the orthogonal tRNA though an aminoacyl bond, covalently bonded to a 3’OH or a 2’OH of a terminal ribose sugar of the orthogonal tRNA, etc. 2026205050   29 Jun 2026

[00316] The chemical moieties via nonnatural amino acids that can be incorporated into proteins offer a variety of advantages and manipulations of the protein. For example, the unique reactivity of a keto functional group allows selective modification of proteins with any of a number of hydrazine- or hydroxylamine-containing reagents in vitro and in vivo. A heavy atom nonnatural amino acid, for example, can be useful for phasing X-ray structure data. The sitespecific introduction of heavy atoms using nonnatural amino acids also provides selectivity and flexibility in choosing positions for heavy atoms. Photoreactive nonnatural amino acids (including but not limited to, amino acids with benzophenone and arylazides (including but not limited to, phenylazide) side chains), for example, allow for efficient in vivo and in vitro photocrosslinking of protein. Examples of photoreactive nonnatural amino acids include, but are not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine. The protein with the photoreactive nonnatural amino acids can then be crosslinked at will by excitation of the photoreactive group-providing temporal control. In one example, the methyl group of an nonnatural amino can be substituted with an isotopically labeled, including but not limited to, methyl group, as a probe of local structure and dynamics, including but not limited to, with the use of nuclear magnetic resonance and vibrational spectroscopy. Alkynyl or azido functional groups, for example, allow the selective modification of proteins with molecules through a [3+2] cycloaddition reaction.

[00317] A nonnatural amino acid incorporated into a polypeptide at the amino terminus can be composed of an R group that is any substituent other than one used in the twenty natural amino acids and a 2nd reactive group different from the NH2 group normally present in alphaamino acids. A similar nonnatural amino acid can be incorporated at the C-terminus with a 2nd reactive group different from the COOH group normally present in alpha-amino acids.

[00318] The nonnatural amino acids of the invention may be selected or designed to provide additional characteristics unavailable in the twenty natural amino acids. For example, nonnatural amino acid may be optionally designed or selected to modify the biological properties of a protein, e.g., into which they are incorporated. For example, the following properties may be optionally modified by inclusion of an nonnatural amino acid into a protein: toxicity, biodistribution, solubility, stability, e.g., thermal, hydrolytic, oxidative, resistance to enzymatic degradation, and the like, facility of purification and processing, structural properties, spectroscopic properties, chemical and / or photochemical properties, catalytic activity, redox potential, half-life, ability to react with other molecules, e.g., covalently or noncovalently, and the like. 2026205050   29 Jun 2026

[00319] In some embodiments the present invention provides TC linked to a water soluble polymer, e.g., a PEG, by an oxime bond. Many types of non-naturally encoded amino acids are suitable for formation of oxime bonds. These include, but are not limited to, non-naturally encoded amino acids containing a carbonyl, dicarbonyl, or hydroxylamine group. Such amino acids are described in U.S. Patent Publication Nos. 2006 / 0194256, 2006 / 0217532, and 2006 / 0217289 and 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. Patent No. 7,083,970 and U.S. Patent No. 7,045,337, which are incorporated by reference herein in their entirety.

[00320] Some embodiments of the invention utilize TC polypeptides that are substituted at one or more positions with a para-acetylphenylalanine amino acid. The synthesis of p-acetyl-(+ / -)-phenylalanine and m-acetyl-(+ / -)-phenylalanine are described in Zhang, Z., et al., Biochemistry 42: 6735-6746 (2003), incorporated by reference. Other carbonyl- or dicarbonylcontaining amino acids can be similarly prepared by one of ordinary skill in the art. Further, non-limiting examplary syntheses of non-natural amino acid that are included herein are presented in U.S. Patent No. 7,083,970, which is incorporated by reference herein in its entirety.

[00321] Amino acids with an electrophilic reactive group allow for a variety of reactions to link molecules via nucleophilic addition reactions among others. Such electrophilic reactive groups include a carbonyl group (including a keto group and a dicarbonyl group), a carbonyl-like group (which has reactivity similar to a carbonyl group (including a keto group and a dicarbonyl group) and is structurally similar to a carbonyl group), a masked carbonyl group (which can be readily converted into a carbonyl group (including a keto group and a dicarbonyl group)), or a protected carbonyl group (which has reactivity similar to a carbonyl group (including a keto group and a dicarbonyl group) upon deprotection). Such amino acids include amino acids having the structure of Formula (IV): wherein: (IV), 2026205050   29 Jun 2026 A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-,   -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-,  -C(R’)=N-N=,  -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; J is O , , R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; each R” is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R” group is present, two R” optionally form a heterocycloalkyl; R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; or the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group; 2026205050   29 Jun 2026 or the -J-R group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group; with a proviso that when A is phenylene and each R3 is H, B is present; and that when A is -(CH2)4- and each R3 is H, B is not -NHC(O)(CH2CH2)-; and that when A and B are absent and each R3 is H, R is not methyl.

[00322] In addition, having the structure of Formula (V) are included: O R2 (V) wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-,   -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-,  -C(R’)=N-N=,  -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and 2026205050   29 Jun 2026 R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; with a proviso that when A is phenylene, B is present; and that when A is -(CH2)4-, B is not -NHC(O)(CH2CH2)-; and that when A and B are absent, R is not methyl.

[00323] In addition, amino acids having the structure of Formula (VI) are included: Ra wherein: BR R2 (VI) B is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each Ra is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O)kR’ where k is 1, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is independently H, alkyl, or substituted alkyl.

[00324] In addition, the following amino acids are included: 2026205050   29 Jun 2026 compounds are optionally amino protected group, carboxyl protected or a salt thereof. In addition, any of the following non-natural amino acids may be incorporated into a non-natural amino acid polypeptide.

[00325] In addition, the following amino acids having the structure of Formula (VII) are included: O / (CRa)nX wherein (VII) B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-,   -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-,  -C(R’)=N-N=,  -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and 2026205050   29 Jun 2026 R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each Ra is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O)kR’ where k is 1, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is independently H, alkyl, or substituted alkyl; and n is 0 to 8; with a proviso that when A is -(CH2)4-, B is not -NHC(O)(CH2CH2)-.

[00326] In addition, the following amino acids are included: wherein such compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof. In addition, these non-natural amino acids and any of the following non-natural amino acids may be incorporated into a non-natural amino acid polypeptide.

[00327] In addition, the following amino acids having the structure of Formula (VIII) are included: wherein A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, 2026205050   29 Jun 2026 substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-,   -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-,  -C(R’)=N-N=,  -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide.

[00328] In addition, the following amino acids having the structure of Formula (IX) are included: Ra Ra B^^O, Ra R2 Ra (IX) B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-,   -S(O)kN(R’)-, 2026205050   29 Jun 2026 -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N- N(R’)-,  -C(R’)=N-N=,  -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; wherein each Ra is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O)kR’ where k is 1, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is independently H, alkyl, or substituted alkyl.

[00329] In addition, the following amino acids are included: wherein such compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof. In addition, these non-natural amino acids and any of the following non-natural amino acids may be incorporated into a non-natural amino acid polypeptide.

[00330] In addition, the following amino acids having the structure of Formula (X) are included: wherein B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, 2026205050   29 Jun 2026 -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-,   -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N- N(R’)-,  -C(R’)=N-N=,  -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each Ra is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O)kR’ where k is 1, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is independently H, alkyl, or substituted alkyl; and n is 0 to 8.

[00331] In addition, the following amino acids are included: , and H2N O , wherein such compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof. In addition, these non-natural amino acids and any of the following non-natural amino acids may be incorporated into a non-natural amino acid polypeptide.

[00332] In addition to monocarbonyl structures, the non-natural amino acids described herein may include groups such as dicarbonyl, dicarbonyl like, masked dicarbonyl and protected dicarbonyl groups.

[00333] For example, the following amino acids having the structure of Formula (XI) are included: 2026205050   29 Jun 2026 O O            (XI), wherein A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-,   -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-,  -C(R’)=N-N=,  -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide.

[00334] included: In addition, the following amino acids having the structure of Formula (XII) are R 2026205050   29 Jun 2026 B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-,   -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N- N(R’)-,  -C(R’)=N-N=,  -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; wherein each Ra is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O)kR’ where k is 1, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is independently H, alkyl, or substituted alkyl.

[00335] In addition, the following amino acids are included: and wherein such compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof. In addition, these non-natural amino acids and any of the following non-natural amino acids may be incorporated into a non-natural amino acid polypeptide.

[00336] In addition, the following amino acids having the structure of Formula (XIII) are included: O O                    (XIII), 2026205050   29 Jun 2026 wherein B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-,   -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N- N(R’)-,  -C(R’)=N-N=,  -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each Ra is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O)kR’ where k is 1, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is independently H, alkyl, or substituted alkyl; and n is 0 to 8.

[00337] In addition, the following amino acids are included: O H2N , wherein such compounds are optionally amino protected, 2026205050   29 Jun 2026 optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof. In addition, these non-natural amino acids and any of the following non-natural amino acids may be incorporated into a non-natural amino acid polypeptide.

[00338] In addition, the following amino acids having the structure of Formula (XIV) are included: wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralky...

Claims

1. A composition comprising one or more targeting polypeptides having one or morenonnaturally encoded amino acids incorporated, wherein at least one of said 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 polypeptide is a same ordifferent targeting polypeptide.

3. The composition of claim 1, wherein the one or more targeting polypeptide binds to a cellsurface target or tumor cell target.

4. The composition of claim 1, wherein the one or more targeting polypeptide is amonospecific, bispecific, or multi-specific targeting polypeptide.

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

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

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

8. The composition of claim 6, wherein the targeting polypeptide is an antibody or antibodyfragment that binds to a target selected from the group consisting of HER2, HER3, PD-1, PDL1, EGFR, TROP2, PSMA, VEGFR, CTLA-4, EpCAM, MUC1, MUC16, c-met, GPC3, ENPP3, TIM-1, FOLR1, STEAPI, Mesothelin, 5T4, CEA, CA9, Cadherin 6, RORI, SLC34A2, SLC39A6, SLC44A4, LY6E, DLL3, ePhA2, GPNMB, SLITRI(6, 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 orantibody fragment that binds to HER2.

10. The composition of claim 9, wherein the targeting polypeptide is trastuzumab.

11. The composition of claim 6, wherein the antibody or antibody fragment comprises an 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 (seFv) mutations.

13. The composition of claim 6, wherein the antibody or antibody fragment comprises one or more Fc mutations.2026205050   29 Jun 202614. The composition of claim 6, wherein the antibody or antibody fragment comprises one to six Fe mutations.

15. The composition of claim 6, wherein the antibody or antibody fragment comprises one or more non-naturally encoded amino acid incorporated in the heavy chain, light chain, or both the heavy and light chains.

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 of para-acetyl phenylalanine,      p-nitrophenylalanine,      p-sulfotyrosine,      p-carboxyphenylalanine,onitrophenylalanine, m-nitrophenylalanine, p-boronyl phenylalanine, o-boronylphenylalanine, mboronylphenylalanine, p-aminophenylalanine, o-aininophenylalanine, m-aminophenylalanine, oacylphenylalanine, m-acylphenylalanine, p-OMe phenylalanine, o-OMe phenylalanine, m-OMe phenylalanine, p-sulfophenylalanine, o-sulfophenylalanine, m-sulfophenylalanine, 5-nitro His, 3nitro Tyr, 2-nitro Tyr, nitro substituted Leu, nitro substituted His, nitro substituted De, nitro substituted Trp, 2-nitro Trp, 4-nitro Trp, 5-nitro Trp, 6-nitro Trp, 7-nitro Trp, 3-aminotyrosine, 2-aminotyrosine,     0-sulfotyrosine, 2-sulfooxyphenylalanine,     3-sulfooxyphenylalanine,ocarboxyphenylalanine,      m-carboxyphenylalanine,      p-acetyl-L-phenylalanine,      p-propargylphenylalanine, 0-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, 0-4allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAc-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenytalanine, p-acyl-L-phenylalanine, pbenzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propargyloxy-Lphenylalanine, 4-azido-L-phenylalanine, para-azidoethoxy phenylalanine, and paraazidomethylphenylalanine.

18. The composition of claim 17, wherein the non-natural amino acid is selected from a group consisting of para-acetyl-phenylalanine, 4-azido-L-phenylalanine, para-azidoethoxy phenylalanineorpara-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 polypeptide.

20. The composition of claim 1, wherein the TLR agonist is a TLR7 agonist, a TLRS agonist, or a TLR7 / TLR8 dual agonist.2026205050   29 Jun 202621. The composition of claim 1, wherein the TLR agonist is a TLR agonist comprising a molecule structure according to any one of structure 1, 2, 3, 4 or 5 of Figure 1.

22. The composition of claim 1, wherein the TLR agonist is any one of 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 linker, polymer, or biologically active molecule.

24. The composition of claim 23, wherein the one or more linker is a cleavable or noncleavable linker.

25. The composition of claim 23, wherein the one or more linker is O.OlkDa to 50kDa26.    The composition of claim 25, wherein the one or more linker is 0.0 lkDa to 1kDa.

27. The composition of claim 23, wherein the one or more linker or polymer is linear, branched, multimeric, or dendrimeric.

28. The composition of claim 23, wherein the one or more linker or polymer is a bifunctional or multifunctional linker or a bifunctional or multifunctional polymer.

29. The composition of claim 23, wherein the one or more polymer is a water soluble polymer.

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

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.lkDa and 100kDa.

33. The composition of claim 32, wherein the PEG has a molecular weight between 0.lkDa and 50kDa.

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

35. The composition of claim 1, wherein the targeting polypeptide comprises one or moreamino acid substitution, addition or deletion that increases the stability or solubility of the composition.

36. The composition of claim 1, wherein the composition comprises one or more amino acid substitution, addition or deletion that increases the expression of the targeting polypeptide in a recombinant host cell or synthesized in vitro.2026205050   29 Jun 202637. The composition of claim 1, wherein the non-naturally encoded amino acid is reactive toward a linker, polymer, or biologically active molecule that is otherwise unreactive toward any of the 20 common amino acids in the polypeptide.

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 aTLR agonist comprising 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 in the antibody or antibody fragment.

42. The TC of 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 theTLR 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 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, AXC668, 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,AXC742, AXC-747, AXC-748, AXC-749, AXC-750, AXC-751, AXC-752, AXC-754, AXC-755, AXC-756, AXC-757, AXC-758, AXC-759, AXC-760, AXC-761, AXC-762, AXC-764, AXC771, 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-807,2026205050   29 Jun 2026AXC808, 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 acid incorporated in the heavy chain, light chain, or both the heavy and light chains.

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

47. The TC of claim 41, wherein the one or more non-naturally encoded amino acids is selectedfrom  the group of para-acetyl phenylalanine, p-nitrophenylalanine, p-sulfotyrosine,pcarboxyphenylalanine, oanitrophenylalanine, m-nitrophenylalanine, p-boronyl phenylalanine, oboronylphenylalanine, m-boronylphenylalanine, p-aminophenylalanine, o-aminophenylalanine, m-aminophenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-OMe phenylalanine, oOMe phenylalanine, m-OMe phenylalanine, p-sulfophenylalanine, o-sulfophenylalanine, msulfophenylalanine, 5-nitro His, 3-nitro Tyr, 2-nitro Tyr, nitro substituted Leu, nitro substituted His, nitro substituted De, nitro substituted Trp, 2-nitro Trp, 4-nitro Trp, 5-nitro Trp, 6-nitro Trp, 7-nitro Trp, 3-aminotyrosine, 2-aminotyrosine, 0-sulfotyrosine, 2-sulfooxyphenylalanine, 3sulfooxyphenylalanine, o-carboxyphenylalanine, m-carboxyphenylalanine, p-acetyl-Lphenylalanine, p-propargyl-phenylalanine, 0-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri--acetyl-GlcNAcp-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acylL-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, ppropargyloxy-L-phenylalanine, 4-azido-L-phenylalanine, para-azidoethoxy phenylalanine, and para-azidomethyl-phenylalanine.

48. The TC of any of claims 47, wherein the non-natural amino acid is paraacetylphenylalanine, 4-azido-L-phenylalanine, para-azidomethyl-phenylalanine, or paraazidoethoxy phenylalanine.

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

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

51. The TC of claim 41, wherein the TLR agonist comprises a structure according to structure2 of Figure 1.

52. The TC of 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 1.2026205050   29 Jun 202654. The TC of claim 53, wherein the TLR agonist comprises a structure according to structure 3 is 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 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 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, AXC882, 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, AXC903, 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.

59. The TC of any 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 of any 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 of any of claims 41-60, wherein the anti-HER2 antibody or antibody fragment comprises a) SEQ ID NOs: 1 or 2; and b) any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13.

62. The TC of claim 41, further comprising a chemotherapeutic or immunotherapeutic agent.

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

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

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

66. The method of claim 64, further comprising an antibody drug conjugate, a cytotoxic agent, or a checkpoint inhibitor.2026205050   29 Jun 202667. A pharmaceutical composition comprising a therapeutically-effective amount of a composition or TC of any of the preceding claims, and a pharmaceutically acceptable carrier or excipient.

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

69. The TC of claim 44, wherein the TLR agonist comprising a structure according to structure1 is selected from the group 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, AXC675, AXC-681, AXC-687, AXC-688, AXC-689, AXC-690, AXC-691, AXC-697, AXC-699, AXC-700, AXC-701, AXC-702, AXC-709, AXC-710, AXC-711, AXC-713, AXC-714, AXC717, 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, AXC740, 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, AXC762, 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, AXC807, AXC-808, AXC-809, AXC-810, AXC-831 and AXC-910 compounds.

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

71. The TC of claim 54, wherein the TLR agonist comprises a structure of 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 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,AXC897, AXC-901, AXC-907, AXC-909, AXC-913, and AXC-914 compounds.

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

74. An Immune stimulating 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 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, AXC907, and AXC-910 compounds.

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

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

78. A method of killing a cell comprising contacting a cell with a TC according to claim 41.2026205050   29 Jun 202679. The method of claim 78, wherein the cell is a tumor or cancer cell.

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

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

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