Bridging Antibodies

Abstract

Provided herein are compositions and methods for crosslinking single domain antibodies with conjugates to targets.Further provided herein are conjugates comprising a targeting domain, the targeting domain comprising a non-natural amino acid.Further provided herein are methods for treating disease by crosslinking single domain and multispecific antibodies.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 293,025, filed December 22, 2021, U.S. Provisional Patent Application No. 63 / 346,799, filed May 27, 2022, and U.S. Provisional Patent Application No. 63 / 388,072, filed July 11, 2022, each of which is incorporated by reference in its entirety. [Background technology]

[0002] Proteins primarily use non-covalent interactions within or between proteins, since the amino acid side chains of proteins cannot normally form covalent bonds with each other, except for cysteine, which creates a relatively weak disulfide bond that is reversible. Thus, potential bioreactive unnatural amino acids (UAAs) that can specifically react with multiple natural amino acid residues on target proteins expand the diversity of proteins susceptible to covalent conjugation in vivo, which may enable existing protein properties to be enhanced or new functions to evolve by utilizing novel covalent bonds. Furthermore, covalent conjugation between proteins allows irreversible capture of protein-protein interactions in vivo, which may be useful for protein identification, drug discovery, irreversible antagonist and payload delivery. Summary of the Invention

[0003] Provided herein is a conjugate comprising a targeting domain and a payload, wherein the targeting domain comprises at least one unnatural amino acid (UAA) residue, wherein the targeting domain is configured to bind to a target, wherein the UAA residue is in sufficient proximity to form a covalent bond with the target when the targeting domain is bound. Further provided herein is a conjugate wherein the payload is attached to an amino acid at n+x positions from the UAA residue, where n is an amino acid position and x is at least 1. Further provided herein is a conjugate wherein the payload is attached to an amino acid at nx positions from the UAA residue, where n is an amino acid position and x is at least 1. Further provided herein is a conjugate wherein the UAA residue is within 5-20 angstroms of the target when the targeting domain is bound to the target. Further provided herein is a conjugate wherein the targeting domain binds to a cell surface molecule. Further provided herein are conjugates in which the UAA residue is configured to form a covalent bond with a target histidine, lysine, or tyrosine residue. Further provided herein are conjugates in which the UAA residue comprises a fluorosulfate moiety. Further provided herein are conjugates in which the UAA residue comprises an aryl-fluorosulfate moiety. Further provided herein are conjugates in which the UAA residue comprises a moiety represented by Formula I:

[0004] [ka] A conjugate comprising: Further provided herein is a UAA residue, UAA, having the structure:

[0005] [ka] A conjugate having the formula: Further provided herein is a UAA residue, UAA, having the structure:

[0006] [ka] A conjugate having the formula: Further herein, the UAA residue is represented by formula II:

[0007] [ka] A conjugate comprising: Further provided herein is a UAA residue, UAA, having the structure:

[0008] [ka] A conjugate having the formula: Further herein, the UAA residue UAA has the structure:

[0009] [ka] A conjugate having the formula: Further herein, the UAA residue is represented by formula III:

[0010] [ka] A conjugate comprising: Further provided herein is a UAA residue, UAA, having the structure:

[0011] [ka] A conjugate having the formula: Further provided herein is a UAA residue, UAA, having the structure:

[0012] [ka] A conjugate having the formula:

[0013] Further provided herein is a compound in which the UAA residue UAA has the structure of formula (IA):

[0014] [ka] A conjugate is provided having the formula: During the ceremony, Each X is independently O or NR'; Y is a bond, -O-, -NR-, or -N=; A is a bond or -(CH2) n -, m is 1 or 2, and n is an integer from 1 to 4; each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, L is -(CH2) P - or -C(O)NH-(CH2) P -, p is an integer from 1 to 6, and wherein m is 1 when Y is -O- or -NR-, and m is 2 when Y is -N=.

[0015] Further provided herein is a compound represented by the formula (IA) having the structure (IA-a):

[0016] [ka] A conjugate having the formula: Further provided herein is a UAA of formula (IA) having the structure of formula (IA-b):

[0017] [ka] A conjugate having the formula: Further provided herein is a compound having the structure of formula (IB):

[0018] [ka] A conjugate having the formula: Further provided herein is a compound having the structure of formula (IC):

[0019] [ka] A conjugate having the formula:

[0020] Further provided herein is a UAA of formula (IA) having the structure of formula (ID):

[0021] [ka] A conjugate is provided having the formula: During the ceremony, Each X is independently O or NR; Y is a bond, -O-, -NR-, or -N=; A is a bond or -(CH2) n -, m is 1 or 2, and n is an integer from 1 to 4; each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, and wherein m is 1 when Y is a bond, -O- or -NR-, and m is 2 when Y is -N=.

[0022] Further provided herein is a UAA of formula (IA) having the structure of formula (IE):

[0023] [ka] A conjugate is provided having the formula: During the ceremony, Each X is independently O or NR'; Y is a bond, -O-, -NR-, or -N=; A is a bond or -(CH2) n -, m is 1 or 2, and n is an integer from 1 to 4; each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, and wherein m is 1 when Y is a bond, -O- or -NR-, and m is 2 when Y is -N=.

[0024] Further provided herein is a UAA of formula (IA) having the structure of formula (IIA):

[0025] [ka] A conjugate is provided having the formula: During the ceremony, X is independently O or NR, and R', if present, is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.

[0026] Further provided herein is a UAA of formula (IA) having the structure of formula (IIB):

[0027] [ka] A conjugate is provided having the formula: During the ceremony, X is independently O or NR', and R', if present, is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.

[0028] Further provided herein is a compound in which the UAA residue UAA has the structure of formula (IV):

[0029] [ka] A conjugate is provided having the formula: During the ceremony, Each X is independently O or NR'; Y is a bond, -O-, -NR-, or -N=; A is a bond or -(CH2) n -, m is 1 or 2, and n is an integer from 1 to 4; each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; Ring A is a 5- to 6-membered aryl or heteroaryl; R A are each independently -OH, -OR X , halogen, NHR X , N(R X )2, or optionally substituted alkyl, and R X each is optionally substituted alkyl; L is -(CH2) P - or -C(O)NH-(CH2) P -, p is an integer from 1 to 6, and wherein m is 1 when Y is a bond, -O- or -NR-, and m is 2 when Y is -N=.

[0030] Further provided herein are conjugates wherein the payload comprises an imaging agent, a radioligand agent, or a cytotoxic agent. Further provided herein are conjugates wherein the cytotoxic moiety comprises a small molecule drug or a chemotherapeutic agent. Further provided herein are conjugates wherein the radioligand agent comprises: 153 Sm, 177 LU, 90 Y, 131 I, 149 Tb, 211 At, 212 Pb / 212 Bi, 213 Bi, 223 Ra, 225 Ac, and 227 Further provided herein is a conjugate wherein the radioligand agent further comprises a chelating agent. Further provided herein is a conjugate wherein the radioligand agent further comprises a chelating agent. 99m Tc, 131 I, 201 Tl, 111 In, and 67Ga. Further provided herein is a conjugate in which the payload is linked to the conjugate by a linker. Further provided herein is a conjugate in which the linker comprises a polymer. Further provided herein is a conjugate in which the linker is a cleavable or non-cleavable linker. Further provided herein is a conjugate in which the linker is 0.01 kDa to 2.5 kDa. Further provided herein is a conjugate in which the linker is 0.01 kDa to 2.5 kDa. Further provided herein is a conjugate in which the linker is linear, branched, polymeric, or dendritic. Further provided herein is a conjugate in which the linker is a bifunctional or multifunctional linker or a bifunctional or multifunctional polymer. Further provided herein is a conjugate in which the linker comprises a water-soluble polymer. Further provided herein is a conjugate wherein the water soluble polymer is polyethylene glycol (PEG). Further provided herein is a conjugate wherein the PEG has a molecular weight of 0.1 kDa to 2.5 kDa. Further provided herein is a conjugate wherein the PEG comprises 1 to 8 monomers. Further provided herein is a conjugate wherein the targeting domain comprises an antibody, an antibody fragment, or an antigen binding domain. Further provided herein is a conjugate wherein the targeting domain comprises an antigen binding domain, the conjugate comprises a CDR region, and at least one UAA residue is within or near the CDR region. Further provided herein is a conjugate wherein the UAA residue is comprised within the CDR region. Further provided herein is a conjugate wherein the targeting domain comprises a single domain antibody (sdAb).Further, as used herein, the cell surface molecule is PSMA, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endo180, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD166, CLL-1, ADAM9, cKIT, CD36, CD73, ITGa Vb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, ROR1, TROP- Conjugates selected from the group consisting of fibronectin, tissue factor, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, pCadherin, CEACAM6, CD47, and EpCAM are provided.

[0031] Further provided herein is a conjugate comprising (i) a payload and (ii) an engineered single domain antibody (sdAb) comprising an sdAb having CDR regions and at least one unnatural amino acid (UAA) residue within or near the CDR regions, wherein the sdAb comprises any one of SEQ ID NOs: 1-4 or 16-64. Further provided herein is a conjugate wherein the UAA residue comprises a fluorosulfate moiety. Further provided herein is a conjugate wherein the UAA residue comprises an aryl-fluorosulfate moiety. Further herein, the UAA residue is represented by formula I:

[0032] [ka] A conjugate comprising: Further herein, the UAA residue UAA has the structure:

[0033] [ka] A conjugate having the formula: Further provided herein is a UAA residue, UAA, having the structure:

[0034] [ka] A conjugate having the formula: Further herein, the UAA residue is represented by formula II:

[0035] [ka] A conjugate comprising: Further provided herein is a UAA residue, UAA, having the structure:

[0036] [ka] A conjugate having the formula: Further as defined herein, UAA has the structure:

[0037] [ka] A conjugate having the formula: Further herein, the UAA residue is represented by formula III:

[0038] [ka] A conjugate comprising: Further provided herein is a UAA residue, UAA, having the structure:

[0039] [ka] A conjugate having the formula: Further provided herein is a UAA residue, UAA, having the structure:

[0040] [ka] A conjugate having the formula:

[0041] Further provided herein is a compound in which the UAA residue UAA has the structure of formula (IA):

[0042] [ka] A conjugate is provided having the formula: During the ceremony, Each X is independently O or NR'; Y is a bond, -O-, -NR-, or -N=; A is a bond or -(CH2) n -, m is 1 or 2, and n is an integer from 1 to 4; each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, L is -(CH2) P - or -C(O)NH-(CH2) P -, p is an integer from 1 to 6, and wherein m is 1 when Y is -O- or -NR-, and m is 2 when Y is -N=.

[0043] Further provided herein is a compound represented by the formula (IA) having the structure (IA-a):

[0044] [ka] A conjugate having the formula: Further provided herein is a UAA of formula (IA) having the structure of formula (IA-b):

[0045] [ka] A conjugate having the formula: Further provided herein is a compound having the structure of formula (IB):

[0046] [ka] A conjugate having the formula: Further provided herein is a compound having the structure of formula (IC):

[0047] [ka] A conjugate having the formula:

[0048] Further provided herein is a UAA of formula (IA) having the structure of formula (ID):

[0049] [ka] A conjugate is provided having the formula: During the ceremony, Each X is independently O or NR'; Y is a bond, -O-, -NR-, or -N=; A is a bond or -(CH2) n -, m is 1 or 2, and n is an integer from 1 to 4; each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, and wherein m is 1 when Y is a bond, -O- or -NR-, and m is 2 when Y is -N=.

[0050] Further provided herein is a UAA of formula (IA) having the structure of formula (IE):

[0051] [ka] A conjugate is provided having the formula: During the ceremony, Each X is independently O or NR'; Y is a bond, -O-, -NR-, or -N=; A is a bond or -(CH2) n -, m is 1 or 2, and n is an integer from 1 to 4; each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, and wherein m is 1 when Y is a bond, -O- or -NR-, and m is 2 when Y is -N=.

[0052] Further provided herein is a UAA of formula (IA) having the structure of formula (IIA):

[0053] [ka] and providing engineered sdAbs having the formula: During the ceremony, X is independently O or NR', and R', if present, is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.

[0054] Further provided herein is a UAA of formula (IA) having the structure of formula (IIB):

[0055] [ka] and providing engineered sdAbs having the formula: During the ceremony, X is independently O or NR, and R', if present, is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.

[0056] Further provided herein is a compound in which the UAA residue UAA has the structure of formula (IV):

[0057] [ka] A conjugate is provided having the formula: During the ceremony, each X is independently O or NR'; and Y is a bond, -O-, -NR-, or -N=; A is a bond or -(CH2) n -, m is 1 or 2, and n is an integer from 1 to 4; each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; Ring A is a 5- to 6-membered aryl or heteroaryl; R A are each independently -OH, -OR X , halogen, NHR X , N(R X )2, or optionally substituted alkyl, and R X each is optionally substituted alkyl; L is -(CH2) P - or -C(O)NH-(CH2) P -, p is an integer from 1 to 6, and wherein m is 1 when Y is a bond, -O- or -NR-, and m is 2 when Y is -N=.

[0058] Further provided herein is a conjugate wherein the engineered sdAb comprises SEQ ID NO:1 and the UAA residue is comprised in any one of SEQ ID NOs:5, 6, and 7. Further provided herein is a conjugate wherein the UAA residue is present at an amino acid position selected from the group consisting of 26, 28, 29, 30, 99, 102, 103, 105, 108, 110, 111, 112, 113, 114, and 115 relative to SEQ ID NO:1. Further provided herein is a conjugate wherein the engineered sdAb comprises SEQ ID NO:2 and the UAA residue is present in any one of SEQ ID NOs:8, 9, and 10. Further provided herein is a conjugate wherein the UAA residue is present at an amino acid position selected from the group consisting of 50, 52, 53, 54, 56, 58, and 100 relative to SEQ ID NO:2. Further provided herein is a conjugate wherein the engineered sdAb comprises SEQ ID NO:3 and the UAA residue is comprised in any one of SEQ ID NOs:11, 12, and 13. Further provided herein is a conjugate wherein the UAA residue is present at an amino acid position selected from the group consisting of 58, 62, 101, 103, and 107 relative to SEQ ID NO:3. Further provided herein is a conjugate wherein the engineered sdAb comprises SEQ ID NO:16 and the UAA is present at amino acid position 109 relative to SEQ ID NO:16. Further provided herein is a conjugate wherein the engineered sdAb comprises SEQ ID NO:23 and the UAA is present at an amino acid position selected from the group consisting of 52, 53, 54, 55, 56, 58, 60, 62, and 64 relative to SEQ ID NO:23. Further provided herein is a conjugate wherein the engineered sdAb comprises SEQ ID NO:24 and the UAA is at an amino acid position selected from the group consisting of 53, 55, 56, 57, 58, 60, 64, and 67 relative to SEQ ID NO:24.

[0059] Further provided herein are conjugates in which the payload is not linked via a UAA residue side chain. Further provided herein are conjugates in which the payload comprises an imaging agent, a radioligand agent, or a cytotoxic agent. Further provided herein are conjugates in which the cytotoxic moiety comprises a small molecule drug or a chemotherapeutic agent. Further provided herein are conjugates in which the radioligand agent is 153 Sm, 177 LU, 90 Y, 131 I, 149 Tb, 211 At, 212 Pb / 212 Bi, 213 Bi, 223 Ra, 225 Ac, and 227 Further provided herein is a conjugate, wherein the radioligand agent further comprises a chelating agent. Further provided herein is a conjugate, wherein the imaging agent is a fluorophore, or 99m Tc, 131 I, 201 Tl, 111 In, and 67 Ga.

[0060] Provided herein are methods comprising administering a conjugate as described herein, wherein the conjugate covalently binds a target on the surface of a cell. Further provided herein are methods wherein the cell comprises a tumor cell. Further provided herein are methods wherein the conjugate kills or inhibits the growth of a tumor cell. Further provided herein are methods wherein the target is PSMA, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD166, CLL-1, ADAM9, cKIT, CD36, CD73, IT GaVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, ROR1, T The conjugates are selected from the group consisting of ROP-2, NECTIN4, cMET, CD19, CD22, CD30, CD33, CD123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM. Further provided herein are methods of treating a disease or disorder comprising administering a conjugate as described herein.Further, as used herein, the disease may be selected from the group consisting of PCa (prostate cancer), CRPCa (castration resistant prostate cancer), solid tumors (neovasculature), NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), ESCC (esophageal cancer), GC (gastric cancer), CRC (colorectal cancer), SCLC (small cell lung cancer), MPM (mesothelioma), PDAC (Pancreatic ductal adenocarcinoma), ALL (Acute Lymphoblastic Leukemia), AML (Acute Myeloid Leukemia), and the like. The methods include one or more of: leukemia (MDS), myelodysplastic syndromes (MDS), MSI-high tumor, melanoma, DLBCL (diffuse large B cell lymphoma), endometrial cancer, cervical cancer, bladder cancer, BrCa (breast cancer), TNBC (triple negative breast cancer), NE-PCa (neuroendocrine prostate cancer), GBM (glioblastoma), and RCC (renal cell carcinoma).

[0061] Provided herein is a method of making the conjugates described herein, comprising generating a targeting domain comprising at least one unnatural amino acid, and conjugating the targeting domain to a payload, optionally via a linker. Also provided herein is a method, wherein the targeting domain comprising at least one unnatural amino acid is synthesized in-vivo. Also provided herein is a method, wherein the generating step comprises the use of an orthogonal tRNA synthetase / suppressor tRNA pair. Also provided herein is a method, wherein the generating step comprises the use of an orthogonal tRNA synthetase / suppressor tRNA pair. Also provided herein is a method, wherein the generating step comprises the use of an orthogonal tRNA synthetase / suppressor tRNA pair. pyl (pyrrolysine tRNA synthetase / tRNA pyl ). Further provided herein are methods, wherein the orthogonal tRNA synthetase comprises SEQ ID NO: 84, 87, 92, or a variant thereof.

[0062] Provided herein is a method of delivering a cytotoxic payload to a cell, the method comprising administering a conjugate as described herein, where the conjugate covalently binds a target on the surface of the cell, thereby delivering the cytotoxic payload. Further provided herein is a method, where the cell is a tumor cell. Further provided herein is a method, where the cell is contained within a tumor microenvironment. Further provided herein is a method, where the cell is contained within a mammalian subject. Further provided herein is a method, where the cell is contained within a human subject. Further provided herein is a method, where the conjugate kills or inhibits the growth of a tumor cell. Further described herein, the targets include PSMA, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD166, CLL-1, ADAM9, cKIT, CD36, CD73, IT GaVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, ROR1, T The method provides for the detection of a marker selected from the group consisting of ROP-2, NECTIN4, cMET, CD19, CD22, CD30, CD33, CD123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM.Further provided herein is a method, wherein the human subject is suffering from or diagnosed with a disease or condition selected from the group consisting of PCa (prostate cancer), CRPCa (castration-resistant prostate cancer), solid tumor (angiogenesis), NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), ESCC (esophageal cancer), GC (gastric cancer), CRC (colorectal cancer), SCLC (small cell lung cancer), MPM (mesothelioma), PDAC (pancreatic ductal adenocarcinoma), ALL (acute lymphoblastic leukemia), AML (acute myeloid leukemia), MDS (myelodysplastic syndrome), MSI-high tumor, melanoma, DLBCL (diffuse large B-cell lymphoma), endometrial cancer, cervical cancer, bladder cancer, BrCa (breast cancer), TNBC (triple-negative breast cancer), NE-PCa (neuroendocrine prostate cancer), GBM (glioblastoma), and RCC (renal cell carcinoma).

[0063] Provided herein is a conjugate comprising (i) a first targeting domain, (ii) a second targeting domain, and (iii) a payload, wherein the first targeting domain comprises at least one first unnatural amino acid (UAA) such that the first targeting domain can be covalently bound to a first target at the site of the UAA, and the second targeting domain is configured to bind to a second target. In some embodiments, the first target and the second target are on the same cell. In some embodiments, the first targeting domain comprises an antibody, an antibody fragment, or an antigen-binding domain. In some embodiments, the first targeting domain comprises a single domain antibody (sdAb). In some embodiments, the first UAA is comprised within or in the vicinity of the region of the first targeting domain that interfaces with the first target. In some embodiments, the second targeting domain comprises an antibody, an antibody fragment, or an antigen-binding domain. In some embodiments, the second targeting domain comprises a single domain antibody (sdAb). In some embodiments, the first targeting domain and the second targeting domain are linked to form a fusion protein. In some embodiments, the first targeting domain and the second targeting domain are linked by chemical conjugation. In some embodiments, the first targeting domain and the second targeting domain are linked by a linker. In some embodiments, the first targeting domain and the second targeting domain bind the same target. In some embodiments, the first targeting domain and the second targeting domain bind different epitopes of the same target. In some embodiments, the first targeting domain and the second targeting domain bind the same epitope of the same target. In some embodiments, the same target is a monomer. In some embodiments, the same target is a multimeric molecule. In some embodiments, the first targeting domain and the second targeting domain bind different targets. In some embodiments, the first target is a first cell surface molecule. In some embodiments, the second target is a second cell surface molecule.In some embodiments, at least one first UAA comprises a fluorosulfate moiety. In some embodiments, at least one first UAA comprises an aryl fluorosulfate moiety. In some embodiments, at least one first UAA comprises Formula I:

[0064] [ka] Includes. In some embodiments, at least one first UAA has the structure:

[0065] [ka] has. In some embodiments, at least one first UAA has the structure:

[0066] [ka] has. In some embodiments, the at least one first UAA has formula II:

[0067] [ka] Includes. In some embodiments, at least one first UAA has the structure:

[0068] [ka] has. In some embodiments, at least one first UAA has the structure:

[0069] [ka] has. In some embodiments, the at least one first UAA has formula III:

[0070] [ka] Includes. In some embodiments, at least one first UAA has the structure:

[0071] [ka] has. In some embodiments, at least one first UAA has the structure:

[0072] [ka] has. In some embodiments, at least one first UAA has the structure of formula (IA):

[0073] [ka] wherein each X is independently O or NR', Y is a bond, -O-, -NR-, or -N=, and A is a bond or -(CH) n -, m is 1 or 2, n is an integer from 1 to 4, each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, and L is -(CH2) P - or -C(O)NH-(CH2) P -, p is an integer from 1 to 6, and where m is 1 when Y is -O- or -NR-, and m is 2 when Y is -N=. In some embodiments, at least one first UAA has the structure of formula (IA-a):

[0074] [ka] In some embodiments, at least one first UAA has the formula (IA-b):

[0075] [ka] In some embodiments, at least one first UAA has the structure of formula (IB):

[0076] [ka] In some embodiments, at least one first UAA has the structure of formula (IC):

[0077] [ka] In some embodiments, at least one first UAA has the structure of formula (ID):

[0078] [ka] wherein each X is independently O or NR', Y is a bond, -O-, -NR-, or -N=, and A is a bond or -(CH) n -, m is 1 or 2, n is an integer from 1 to 4, each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, and where m is 1 when Y is a bond, -O- or -NR-, and m is 2 when Y is -N=. In some embodiments, at least one first UAA has the structure of formula (IE):

[0079] [ka] wherein each X is independently O or NR', Y is a bond, -O-, -NR-, or -N=, and A is a bond or -(CH) n -, m is 1 or 2, n is an integer from 1 to 4, each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, and where m is 1 when Y is a bond, -O- or -NR-, and m is 2 when Y is -N=. In some embodiments, at least one first UAA has the structure of formula (IIA):

[0080] [ka] wherein X is independently O or NR', and R', if present, is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl. In some embodiments, at least one first UAA has the structure of formula (IIB):

[0081] [ka] wherein X is independently O or NR', and R', if present, is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl. In some embodiments, at least one first UAA has the structure of formula (IV):

[0082] [ka] wherein each X is independently O or NR', Y is a bond, -O-, -NR-, or -N=, and A is a bond or -(CH) n -, m is 1 or 2, n is an integer from 1 to 4, each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl, and ring A is a 5- to 6-membered aryl or heteroaryl; R A are each independently -OH, -OR X , halogen, NHR X , N(R X )2, or optionally substituted alkyl, p is 0, 1, 2, 3, or 4, and R X is optionally substituted alkyl, and L is -(CH2) P - or -C(O)NH-(CH2) P-, p is an integer from 1 to 6, and where m is 1 when Y is a bond, -O- or -NR-, and where m is 2 when Y is -N=. In some embodiments, the first cell surface molecule is PSMA, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD166, CLL-1, ADAM9, cKIT, CD36, CD73, ITGaVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, RO Selected from the group consisting of R1, TROP-2, NECTIN4, cMET, CD19, CD22, CD30, CD33, CD123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM.In some embodiments, the second cell surface molecule is PSMA, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD166, CLL-1, ADAM9, cKIT, CD36, CD73, ITGaVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, RO Selected from the group consisting of R1, TROP-2, NECTIN4, cMET, CD19, CD22, CD30, CD33, CD123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM. In some embodiments, the second domain comprises a second UAA, such that the second domain can covalently bind to a second target at the site of the second UAA. In some embodiments, the second UAA is different from at least one UAA included in the first targeting domain. In some embodiments, the second UAA is the same as at least one UAA included in the first targeting domain. In some embodiments, the second UAA comprises a fluorosulfate moiety. In some embodiments, the second UAA comprises an aryl-fluorosulfate moiety. In some embodiments, the second UAA has formula I:

[0083] [ka] In some embodiments, the second UAA comprises the structure:

[0084] [ka] In some embodiments, the second UAA has the structure:

[0085] [ka] In some embodiments, the second UAA has formula II:

[0086] [ka] In some embodiments, the second UAA comprises the structure:

[0087] [ka] In some embodiments, the second UAA has the structure:

[0088] [ka] In some embodiments, the second UAA has formula III:

[0089] [ka] In some embodiments, the second UAA comprises the structure:

[0090] [ka] In some embodiments, the second UAA has the structure:

[0091] [ka] In some embodiments, the second UAA has the structure of formula (IA):

[0092] [ka] wherein each X is independently O or NR', Y is a bond, -O-, -NR-, or -N=, and A is a bond or -(CH) n -, m is 1 or 2, n is an integer from 1 to 4, each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, and L is -(CH2) P - or -C(O)NH-(CH2) P -, where p is an integer from 1 to 6, and where, when Y is -O- or -NR-, m is 1, and when Y is -N=, m is 2. In some embodiments, the second UAA has the structure of formula (IA-a):

[0093] [ka] In some embodiments, the second UAA has the structure of formula (IA-b):

[0094] [ka] In some embodiments, the second UAA has the structure of formula (IB):

[0095] [ka] In some embodiments, the second UAA has the structure of formula (IC):

[0096] [ka] In some embodiments, the second UAA has the structure of formula (ID):

[0097] [ka] wherein Each X is independently O or NR'; Y is a bond, -O-, -NR-, or -N=; A is a bond or -(CH2) n - and m is 1 or 2; n is an integer from 1 to 4; each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, and where, when Y is a bond, -O- or -NR-, then m is 1; When Y is -N=, m is 2. In some embodiments, the second UAA has the structure of formula (IE):

[0098] [ka] wherein each X is independently O or NR', Y is a bond, -O-, -NR-, or -N=, and A is a bond or -(CH) n-, m is 1 or 2, n is an integer from 1 to 4, each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, and where m is 1 when Y is a bond, -O- or -NR-, and m is 2 when Y is -N=. In some embodiments, the second UAA has the structure of formula (IIA):

[0099] [ka] wherein X is independently O or NR', and R', if present, is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl. In some embodiments, the second UAA has the structure of formula (IIB):

[0100] [ka] wherein X is independently O or NR', and R', if present, is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl. In some embodiments, the second UAA has the structure of formula (IV):

[0101] [ka] wherein each X is independently O or NR', Y is a bond, -O-, -NR-, or -N=, and A is a bond or -(CH) n-, m is 1 or 2, n is an integer from 1 to 4, each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl, ring A is a 5- to 6-membered aryl or heteroaryl, and R A are each independently -OH, -OR X , halogen, NHR X , N(R X )2, or optionally substituted alkyl, p is 0, 1, 2, 3, or 4, and R X is optionally substituted alkyl, and L is -(CH2) P - or -C(O)NH-(CH2) P-, p is an integer from 1 to 6, and wherein m is 1 when Y is a bond, -O- or -NR-, and m is 2 when Y is -N=. In some embodiments, the first targeting domain comprises any one of SEQ ID NOs: 1-4 or 16-64. In some embodiments, the first targeting domain comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1-4 or 16-64. In some embodiments, the second targeting domain comprises any one of SEQ ID NOs: 1-4 or 16-64. In some embodiments, the second targeting domain comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1-4 or 16-64. In some embodiments, the conjugate comprises a SEQ ID NO: 66-72. In some embodiments, the conjugate comprises a sequence having at least 70% sequence identity to SEQ ID NOs: 66-72. In some embodiments, the conjugate comprises any one of SEQ ID NOs: 1-4 or 16-64. In some embodiments, the conjugate comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1-4 or 16-64. In some embodiments, the conjugate comprises a sequence having at least 70% sequence identity to SEQ ID NOs: 66-72. In some embodiments, at least one of the first targeting domain and the second targeting domain comprises SEQ ID NO: 1. In some embodiments, the UAA is present at an amino acid position selected from the group consisting of 26, 28, 29, 30, 99, 102, 103, 105, 108, 110, 111, 112, 113, 114, and 115 relative to SEQ ID NO: 1. In some embodiments, at least one of the first targeting domain and the second targeting domain comprises SEQ ID NO: 2. In some embodiments, the UAA is present at an amino acid position selected from the group consisting of 50, 52, 53, 54, 56, 58, and 100 relative to SEQ ID NO: 2. In some embodiments, at least one of the first targeting domain and the second targeting domain comprises SEQ ID NO:3.In some embodiments, the UAA is at an amino acid position selected from the group consisting of 58, 62, 101, 103, and 107 relative to SEQ ID NO:3. In some embodiments, at least one of the first targeting domain and the second targeting domain comprises SEQ ID NO:16. In some embodiments, at least one of the first targeting domain and the second targeting domain that comprises SEQ ID NO:16 further comprises a non-natural amino acid at position 109 relative to SEQ ID NO:16. In some embodiments, at least one of the first targeting domain and the second targeting domain comprises SEQ ID NO:18. In some embodiments, the payload comprises an imaging agent, a radioligand agent, or a cytotoxic agent. In some embodiments, the cytotoxic moiety comprises a small molecule drug or a chemotherapeutic drug. In some embodiments, the radioligand agent is. 153 Sm, 177 LU, 90 Y, 131 I, 149 Tb, 211 At, 212 Pb / 212 Bi, 213 Bi, 223 Ra, 225 Ac, and 227 In some embodiments, the radioligand agent further comprises a chelating agent. In some embodiments, the radioligand agent is selected from the group consisting of 99 mTc, 131 I, 201 Tl, 111 In, and 67Ga. In some embodiments, the payload is attached to the conjugate by a linker. In another embodiment, a method is provided herein that includes administering a conjugate, wherein the conjugate covalently binds a first target on the surface of a first cell. In some embodiments, the conjugate binds a second target on the surface of a first cell. In some embodiments, the first targeting domain and the second targeting domain bind to the same target. In some embodiments, the first targeting domain and the second targeting domain bind to the same epitope of the same target. In some embodiments, the first targeting domain and the second targeting domain bind to different epitopes of the same target. In some embodiments, the first targeting domain and the second targeting domain bind to different targets on the surface of a first cell. In some embodiments, the second domain comprises a second UAA, and the second UAA is covalently bound to a second target. In some embodiments, the first cell is a tumor cell. In some embodiments, the conjugate kills or inhibits the growth of a tumor cell when bound to a first target. In some embodiments, the conjugate kills or inhibits the growth of a tumor cell when bound to a second target. In some embodiments, the conjugate kills or inhibits the growth of a tumor cell when bound to a first target and a second target. In some embodiments, the first target is PSMA, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD166, CLL-1, ADAM9, cKIT, CD36, CD73, ITGaVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, RO Selected from the group consisting of R1, TROP-2, NECTIN4, cMET, CD19, CD22, CD30, CD33, CD123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM.In some embodiments, the second target is PSMA, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD166, CLL-1, ADAM9, cKIT, CD36, CD73, ITGaVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, RO The payload is selected from the group consisting of R1, TROP-2, NECTIN4, cMET, CD19, CD22, CD30, CD33, CD123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM. In some embodiments, the payload is a radiolabeled agent or a cytotoxic agent. In some embodiments, the payload is an imaging agent. In some embodiments, the method images or identifies the first cell when the conjugate is bound to the first target and the second target on the first cell. In yet another aspect, provided herein are methods of producing a conjugate provided herein, the methods comprising: (a) synthesizing in vivo a first targeting domain comprising at least one unnatural amino acid; and (b) conjugating a payload to the first targeting domain or the second targeting domain, optionally via a linker. In some embodiments, the methods further comprise synthesizing in vivo the second targeting domain as a fusion protein to the first targeting domain.In some embodiments, the synthesizing step comprises the use of an orthogonal tRNA synthetase / suppressor-tRNA pair. In some embodiments, the synthesizing step comprises the use of an orthogonal tRNA synthetase / suppressor-tRNA pair. pyl The orthogonal tRNA synthetase / suppressor tRNA pair is derived from

[0102] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0103] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the invention are utilized and the accompanying drawings. [Figure 1A] Various SDS-PAGE analyses of crosslinks between FSY-modified sdAb with FSY at different positions and PSMA at molar ratios of about 7:1 sdAb to PSMA (final PSMA concentration was 0.125 mg / mL, 1.25 uM) at 37° C. are shown. FIG 1A represents a gel showing the C1 construct containing FSY in the presence or absence of target PSMA. PSMA-C1-FSY crosslinks, PSMA, and C1-FSY are labeled. [Figure 1B] Various SDS-PAGE analyses of crosslinks between FSY-modified sdAb with FSY at different positions and PSMA at molar ratios of about 7:1 sdAb to PSMA (final PSMA concentration was 0.125 mg / mL, 1.25 uM) at 37° C. are shown. FIG. 1B represents a gel showing the C1 construct containing FSY in the presence or absence of target PSMA. PSMA-C1-FSY crosslinks, PSMA, and C1-FSY are labeled. [Figure 1C]Various SDS-PAGE analyses of crosslinks between FSY-modified sdAb with FSY at different positions and PSMA at molar ratios of about 7:1 sdAb to PSMA (final PSMA concentration was 0.125 mg / mL, 1.25 uM) at 37° C. are shown. FIG. 1C represents a gel showing the C1 construct containing FSY in the presence or absence of target PSMA. PSMA-C1-FSY crosslinks, PSMA, and C1-FSY are labeled. [Figure 1D] Various SDS-PAGE analyses of crosslinks between FSY-modified sdAb with FSY at different positions and PSMA at molar ratios of about 7:1 sdAb to PSMA (final PSMA concentration was 0.125 mg / mL, 1.25 uM) at 37° C. are shown. FIG. ID represents a gel showing the C1 construct containing FSY in the presence or absence of target PSMA. PSMA-C1-FSY crosslinks, PSMA, and C1-FSY are labeled. [Figure 1E] Various SDS-PAGE analyses of crosslinks between FSY-modified sdAb with FSY at different positions and PSMA at molar ratios of about 7:1 sdAb to PSMA (final PSMA concentration was 0.125 mg / mL, 1.25 uM) at 37° C. are shown. FIG. 1E represents a gel showing the C1 construct containing FSY in the presence or absence of target PSMA. PSMA-C1-FSY crosslinks, PSMA, and C1-FSY are labeled. [Figure 2A] Various SDS-PAGE analyses of cross-linking between C2 (a PSMA-specific sdAb) and PSMA at 37°C with a molar ratio of 8:1 sdAb:PSMA are shown. Figure 2A represents a gel showing the C2 construct containing FSY at positions 50-61 in the presence of target PSMA, as well as PSMA and wild-type C2-CDR2 pool control. The thick arrow indicates the presence of the cross-linked product. PSMA and C2 are labeled with thin arrows. [Figure 2B] Figure 2B shows various SDS-PAGE analyses of cross-linking between C2 (a PSMA-specific sdAb) and PSMA at 8:1 molar ratio of sdAb:PSMA at 37° C. Figure 2B represents a gel showing the C2 construct containing FSY in the presence of target PSMA. [Figure 2C]Various SDS-PAGE analyses of cross-linking between C2 (a PSMA-specific sdAb) and PSMA at 8:1 molar ratio of sdAb:PSMA at 37°C are shown. Figure 2C represents a gel showing the C2 construct containing FSY in the presence of target PSMA, as well as a wild-type C2-CDR3 pool control. The thick arrow indicates the presence of the cross-linked product. PSMA and C2-FSY are labeled with thin arrows. [Figure 3A] Figure 2 shows SDS-PAGE analysis of the kinetics of cross-linking between FSY modified sdAb (FSY at Cl position 28 or 102) and PSMA over 180 minutes at 37°C at a molar ratio of 5:1 sdAb to PSMA (final PSMA concentration was 0.125mg / mL, 1.25uM). [Figure 3B] Kinetic plots of PSMA crosslinking percentage versus time are shown for crosslinking experiments between FSY-modified sdAb and PSMA. The y-axis represents % crosslinked PSMA (0-60 in 10 unit intervals) and the x-axis represents time (min, 0-180 in 30 min intervals). Constructs tested were C1-28FSY (circles), C1-102FSY (squares), C1-112FSY (triangles), and C1-113FSY (inverted triangles). [Figure 4] Diagram of monomer-C1 related sdAb constructs: C1 (wild type sequence containing two cysteine ​​disulfide bonds), C39 (one cysteine ​​disulfide bond, C1-C101A / C104A, is replaced with two alanines, eliminating the disulfide bond), C1-102FSY (construct C1 with histidine 102 replaced with the unnatural amino acid FSY), and C39-102FSY (Cl-C101A / C104A / H102(FSY), replacement of both C101 and C104 cysteines with alanine and replacement of histidine 102 with FSY). [Figure 5A]Figure 1 shows SDS-PAGE analysis of the kinetics of crosslinking of either the biparatopic construct C40-102FSY (made by linking the C39-102FSY sequence with a copy of C39 that does not contain FSY) or the monoparatopic construct of C39-102FSY with PSMA over 180 min at 37°C. Bands corresponding to C39-102FSY, C40-102FSY, receptor, and crosslink species are labeled. Coupling rates are higher for the biparatopic construct compared to the monoparatopic construct. [Figure 5B] 4 shows kinetics plots of PSMA crosslinking versus time for crosslinking of PSMA with either the biparatopic construct C40-102FSY, or the monoparatopic construct C39-102FSY. [Figure 6A] SDS-PAGE analysis of cross-linking between various FSY-modified C3 constructs. Results for C3 constructs with FSY at positions 26-35 and 50-60 are shown, with the cross-linked product of construct C3-58FSY indicated with an asterisk. [Figure 6B] SDS-PAGE analysis of cross-linking between various FSY-modified C3 constructs. Results are shown for C3 constructs with FSY at positions 61-66 and 99-113, with cross-linking products observed for at least constructs C3-62FSY, C3-101FSY, C3-103FSY, C3-107FSY. [Figure 7A] 1 shows SDS-PAGE analysis of cross-linking between various pooled FSK-modified C8 constructs and the FAP receptor. [Figure 7B] 1 shows SDS-PAGE analysis of cross-linking between various individual FSK-modified C8 constructs and the FAP receptor. [Figure 7C] 1 shows an SDS-PAGE analysis of the kinetics of cross-linking between various FSK-modified C8 constructs and the FAP receptor. [Figure 8A]1 shows SDS-PAGE analysis of cross-linking between various FSY-modified C9 constructs and the Her3 receptor, and the results of pooling C9 constructs with FSY at various positions with the Her3 receptor. [Figure 8B] 1 shows SDS-PAGE analysis of cross-linking between various FSY-modified C9 constructs and the Her3 receptor. 2 shows the results of a C9 construct with FSY at positions 52-68 and the Her3 receptor. [Figure 8C] 1 shows SDS-PAGE analysis of cross-linking between various FSY-modified C9 constructs and Her3 receptor. Results are shown for C9 constructs with FSY at positions 53, 55, 56, 57, 58, 60, 64 or 67 and Her3 receptor. [Figure 8D] Figure 1 shows an SDS-PAGE analysis of cross-linking between various FSY-modified C9 constructs and the Her3 receptor.Figure 2 shows an SDS-PAGE analysis of the kinetics of cross-linking between an FSY-modified C9 construct (C9-55FSY) and the Her3 receptor. [Figure 9] 1 shows the results of a binding assay of C2-54FSY and C2-54TYR sdAbs to human prostate tumor cell lines LNCaP (PSMA+) and PC3 (PSMA-) using flow cytometry. [Figure 10A] SDS-PAGE analysis of FSY modified sdAb crosslinked to cells. Western blots with LNCaP cells and C2-54 FSY incubated at different concentrations and time points. Blots are analyzed using anti-PSMA antibody (top panel), anti-sdAb antibody (middle panel), and anti-GAPDH (bottom panel) as loading controls. [Figure 10B] SDS-PAGE analysis of FSY modified sdAb crosslinked to cells is shown. Western blots with LNCaP cells and C2-54TYR are shown to show no crosslinking to PSMA. The rightmost lane is a control showing a sample of C2-54FSY incubated at 1 uM for 9 hours with LNCaP cells. [Figure 10C] 1 shows a graph of the kinetics of crosslinking with various concentrations of C2-54FSY at various time points. [Figure 11A]1 shows a Western blot analysis of the kinetics of cross-linking of C2-54FSY and C3-101FSY with PSMA at 1 uM and 24 nM in LNCaP cells at different times, the top panel shows a blot with anti-PSMA antibody and the bottom panel shows an anti-GAPDH control. [Figure 11B] 1 shows a graph of the kinetics of cross-linking of C2-54FSY and C3-101FSY to PSMA in LNCaP cells. [Figure 12A] The test design and the results of in vivo cross-linking assay are shown. Figure 12A shows the test design and Western blots of LNCaP and PC3 tumor tissue samples from mice administered C2-54 TYR and C2-54 FSY. The top panel shows the PSMA area of ​​the gel blotted with anti-VHH antibody, the middle panel shows the free VHH area of ​​the gel blotted with anti-VHH antibody, and the bottom panel shows the anti-GAPDH blot. The left panel shows the sample from an animal with LNCaP tumor, and the right panel shows the PC3 sample. Cross-linking is observed only in LNCaP (PSMA+) tumor and in the presence of C2-54FSY. [Figure 12B] 1 shows the study design and results of an in vivo cross-linking assay. 2 shows graphs of plasma concentrations of C2-54 TYR and C2-54 FSY after administration. [Figure 13] SDS-PAGE analysis of cross-linking kinetics for C8-54FSY, C8-55FSY, and C8-56FSY (left to right) is shown. Band positions representing cross-linking and C8-FSY are labeled. For each construct, lanes represent (left to right) 0, 15, 30, 60, 120, 180 minutes, and ladder. [Figure 14]SDS-PAGE analysis of crosslinks for various C15-FSY containing constructs is shown. Left gel: lanes (left to right) represent constructs with FSY substitutions at positions 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 33, 34, 35, ladder, and Her2. Right gel: lanes (left to right) represent constructs with FSY substitutions at positions 36, 37, 66, 67, 68, 69, wt ladder, and Her2. Band positions representing crosslinks and constructs containing FSY are labeled. [Figure 15] SDS-PAGE analysis of cross-linking of biparatopic construct C38-FSY with Her2-Fc. Lanes (left to right) are time at 0, 0.5, 1.5, 2.5, 3.5, 4.5, 5.5, Her2, and ladder. Band positions representing cross-linking, Her2-Fc, and C38-FSY are labeled. [Figure 16] Fluorescence versus concentration plots from flow cytometric binding studies of constructs in A431 and Colo320DM cells, A431-C23-TYR (filled squares), A431-C23-FSY (filled triangles), Colo320DM-C23-TYR ("x" symbol), and Colo320DM-C23-FSY (open circles). The y-axis represents AF680-GeoMean from 0 to 200,000 in 40,000 unit intervals. The x-axis represents concentration (M) from 10-11 to 10-5 on a base 10 logarithmic scale. [Figure 17] Figure 1 shows intratumoral free and EGFR crosslinking AF680-labeled compounds C23-TYR and C23-FSY in a time-dependent manner in A431 (EGFR+) and COLO320DM (EGFR-) tumors. Free AF-680-labeled C23-TYR and C23-FSY were detected in the region of -15 kD (lower panel), and EGFR crosslinking sdAb bands were detected in the region of -175 kD (upper panel). In EGFR+ A431 tumors, time-dependent crosslinking of EGFR was observed with C23-FSY, but not with C23-TYR. In EGFR- COLO320DM tumors, no retention or crosslinking of free sdAb was observed. [Figure 18A] Plots of photons / sec / g tissue in triplicate biological replicates from A431 tumors derived from animals dosed with C23-TYR or C23-FSY test articles are shown. Values ​​from individual animals are shown, with the center bar indicating mean intensity and errors indicating SEM (* indicates p=0.024, ** indicates p=0.002 by paired t-test). C23-FSY was present at significantly higher levels in A431 tumors at both 8 and 24 hours compared to non-FSY-containing C23-TYR protein. The y-axis represents 0-1.5x1010 photons / s / g tissue in 0.5x1010 intervals. The x-axis represents time (hr) at 8 and 24 hours post-dose. Stars indicate statistical significance by paired t-test (2-tailed) (*=p≦0.05, **=p≦0.005). [Figure 18B] Plots of quantitative ex vivo fluorescence intensity of A431 and COLO320DM tumors from animals administered C23-FSY 8 and 24 hours after dosing are shown. Plots show photons / sec / g tissue in triplicate biological replicates. Values ​​from individual animals are shown, with the middle bar showing the mean intensity and error bars showing SEM. C23-FSY was present at significantly higher levels in A431 tumors at both the 8 and 24 hour time points compared to the amount present in EGFR-COLO320DM tumors, demonstrating the specificity of tumor locking of C23-FSY protein. The y-axis represents photons / sec / g tissue from 0 to 1x1010 in 0.5x1010 intervals. The x-axis represents time (hr) after dosing at 8 and 24 hours for each set of bars. The left set is A431 model tumors and the right set is Colo320DM model tumors. [Figure 19]Fluorescent images of SDS PAGE gels are shown, which show tumor-associated free and PSMA cross-linked sdAb in a time-dependent manner. Mice bearing LNCaP tumors were administered C30-TYR or C30-FSY in triplicate. At the indicated time points after treatment, tumors were harvested and processed for gel electrophoresis to detect fluorophore-conjugated sdAb test articles. Free (uncross-linked) sdAb-AF680 was detected in the approx. 20 kD region (lower panel), whereas the PSMA cross-linked sdAb-AF680 species of C30-FSY migrated in the 100 kD region (upper panel). Lanes marked with * indicate vehicle samples. [Figure 20] Quantitative analysis of tumor-associated free and PSMA cross-linked test articles C30-TYR and C30-FSY is shown. Fluorescent band intensity of the free and PSMA cross-linked species bands from the gels above was quantified by densitometry and comparison to a standard curve. Total intratumor test article concentration (free and PSMA cross-linked, pg / mg tumor tissue) is plotted versus time (h). Data points marked with * indicate samples that were below the limits of detection and quantitation. Tumor exposure of C30-FSY was increased approximately 3-fold relative to non-covalently bound C30-TYR. [Figure 21A] 1 shows a plot of the comparative cytotoxicity of C26-54TYR and C26-54FSY test articles in PC3PIP (PSMA positive) and PC3flu (PSMA negative) cell lines, showing concentration vs. cell viability curves of sdAb conjugated to MMAE. The y-axis represents % viability from 0 to 120 in 20 unit intervals. The x-axis represents test article concentration (M) from 10-11 to 10-6 in base 10 log intervals. [Figure 21B] 1 shows a plot of the comparative cytotoxicity of C28-101TYR and C28-101FSY test articles in PC3PIP (PSMA positive) and PC3flu (PSMA negative) cell lines, showing concentration vs. cell viability curves of sdAb conjugated to MMAE. The y-axis represents % viability from 0 to 120 in 20 unit intervals. The x-axis represents test article concentration (M) from 10-11 to 10-6 in base 10 log intervals. [Figure 22]SDS-PAGE analysis of FcFc cross-linking kinetics is shown, in which 52FSY and 54FSY variants of C17 were incubated with Her2 receptor and examined for cross-linking efficiency. Lanes 1-5 represent C17-52FSY at 0, 30, 60, 120, and 180 min, lanes 6-10 represent C17-54FSY at 0, 30, 60, 120, and 180 min, and lanes 11 and 12 are FcHer2 and ladder, respectively. Band positions corresponding to C17-FSY, receptor, and cross-linked product are labeled. [Figure 23A] 1 shows a plot of comparative cytotoxicity using a 5 hour washout for C33-52TYR, C33-52FSY, C33-54TYR and C33-54FSY test articles, showing concentration vs. cell viability curves for sdAb conjugated to MMAE in BT474 cells. The y-axis represents % viability from -20 to 120 in 20 unit intervals. The x-axis represents test article concentration (nM) from 10-3 to 103 in base 10 log intervals. [Figure 23B] Figure 1 shows a plot of comparative cytotoxicity using 6 consecutive day exposure of C33 test article, which shows the concentration vs. cell viability curve of sdAb conjugated to MMAE in BT474 cells. The y-axis represents % viability from -20 to 120 in 20 unit intervals. The x-axis represents test article concentration (nM) from 10-2 to 102 in base 10 log intervals. [Figure 24A] Plots of PSMA crosslinking for monoparatopic and biparatopic constructs (C3-101FSY, squares; C34-FSY, triangles; C36-FSY, circles) are shown. The y-axis represents crosslinked PSMA (total %) from 0 to 100 in 20 unit intervals. The x-axis represents test article concentration (nM) from 0.01 to 1000 in base 10 intervals. The plot corresponds to crosslinking at 1 hour. [Figure 24B]Plots of PSMA crosslinking for monoparatopic and biparatopic constructs (C3-101FSY, squares; C34-FSY, triangles; C36-FSY, circles) are shown. The y-axis represents crosslinked PSMA (total %) from 0 to 100 in 20 unit intervals. The x-axis represents test article concentration (nM) from 0.01 to 1000 in base 10 intervals. The plot corresponds to crosslinking at 6 hours. [Figure 25A] Western blot analysis of FSY cross-linking kinetics at various concentrations of C3-101FSY and C34-FSY constructs at 1 and 6 h is shown. Lanes 1-6 represent C3-101FSY at 1 h, lanes 7-12 represent C34-FSY at 1 h, lanes 13-18 represent C3-101FSY at 6 h, and lanes 19-24 represent C34-FSY at 6 h. Each grouping of six lanes represents increasing construct concentrations (from left to right): UTC, 0.1, 1, 10, 100, 1000 nM. The top set of blots shows a-PSMA, and the bottom set of blots shows a-GAPDH. Band positions of GAPDH, PSMA, cross-linked monomer, and cross-linked dimer are labeled. [Figure 25B] Western blot analysis of FSY cross-linking kinetics at various concentrations of C3-101FSY and C36-FSY constructs at 1 and 6 h is shown. Lanes 1-6 represent C3-101FSY at 1 h, lanes 7-12 represent C36-FSY at 1 h, lanes 13-18 represent C3-101FSY at 6 h, and lanes 19-24 represent C36-FSY at 6 h. Each grouping of six lanes represents increasing construct concentrations (from left to right): UTC, 0.1, 1, 10, 100, 1000 nM. The top set of blots shows a-PSMA, and the bottom set of blots shows a-GAPDH. Band positions of GAPDH, PSMA, cross-linked monomer, and cross-linked dimer are labeled. [Figure 26A]SDS-PAGE analysis of cross-linking kinetics between EGFR and the monoparatopic construct C4-109FSY is shown. The t half-max was approximately 120 min. Bands corresponding to the cross-linked product, EGFR, and sdAb monomer are labeled. A cartoon of receptor engaged with monomeric FSY-containing sdAb is shown on the right. [Figure 26B] SDS-PAGE analysis of the kinetics of cross-linking between EGFR and the biparatopic construct C37-FSY. Bands corresponding to the cross-linked product, EGFR, and sdAb dimer are labeled. A cartoon of receptor coupled with the biparatopic FSY-containing sdAb construct is shown on the right. [Figure 27] Plots of EGFR crosslinking kinetics are shown for the monoparatopic construct C4-109FSY (squares) or the biparatopic construct C37-FSY (circles). The y-axis represents % EGFR crosslinking from 0 to 100%. The x-axis represents time (min) from 0 to 400 in 100 min intervals. [Figure 28A] Illustrated are exemplary conjugates described herein that include a first targeting domain (e.g., constructs C1-C4) and a second targeting domain (e.g., constructs C1-C4), where the conjugate includes an unnatural amino acid (e.g., FSY). One exemplary conjugate also includes a payload. Exemplary conjugates can include an unnatural amino acid on the first targeting domain, the second targeting domain, or both targeting domains. [Figure 28B] Illustrated are exemplary conjugates described herein that include a first targeting domain (e.g., constructs C1-C4) and a second targeting domain (e.g., constructs C1-C4), where the conjugate includes an unnatural amino acid (e.g., FSY). One exemplary conjugate also includes a payload. Exemplary conjugates can include an unnatural amino acid on the first targeting domain, the second targeting domain, or both targeting domains. [Figure 28C]Illustrated are exemplary conjugates described herein that include a first targeting domain (e.g., constructs C1-C4) and a second targeting domain (e.g., constructs C1-C4), where the conjugate includes an unnatural amino acid (e.g., FSY). One exemplary conjugate also includes a payload. Exemplary conjugates can include an unnatural amino acid on the first targeting domain, the second targeting domain, or both targeting domains. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0104] Antibodies, antibody fragments, and antibody-related constructs such as antibody-drug conjugates (ADCs) can be useful tools for research and clinical applications. However, in some instances, the use of these molecules is limited by their on / off rates and stability on targets. Provided herein are compositions and methods for specific and covalent attachment of conjugates to targets. In some instances, the conjugates include at least one targeting domain with at least one unnatural amino acid (UAA) residue near the interface between the target and the targeting domain, such that upon binding of the targeting domain to the target, a covalent bond is formed between the target and the UAA residue. In some instances, the conjugates include at least one targeting domain and at least one payload. In some instances, the targeting domain includes at least one unnatural amino acid (UAA) residue near the interface between the target and the targeting domain, such that upon binding of the targeting domain to the target, a covalent bond is formed between the target and the UAA residue. In some instances, once the conjugate reaches its target cell / tumor, the payload is now covalently attached to the target via the targeting domain. In some instances, the covalent interaction eliminates or reduces the off-rate of the conjugate that binds to the target and increases contact with the attached payload.

[0105] In some examples, the conjugate comprises (i) a first targeting domain and (ii) a second targeting domain, where the first or second targeting domain comprises at least one unnatural amino acid (UAA). In some examples, the first or second targeting domain comprises at least one UAA present near the interface between the target and the first or second targeting domain, and a covalent bond is formed between the target and the at least one UAA upon binding of the first or second targeting domain to the target. In some examples, the conjugate further comprises a payload. In some examples, when the conjugate reaches a target cell, such as a cell in a tumor or tumor microenvironment, the payload is covalently bound to the target via one of the targeting domains (e.g., the first or second targeting domain).

[0106] In some examples, the covalent interaction eliminates or reduces the off-rate of the conjugate that binds to the target, or stabilizes contact with the target. In further examples, the first and second targeting domains in the conjugates provided herein target the same target or different targets. In some cases, only the second targeting domain, but not the first targeting domain, contains a UAA. In some cases, only the first targeting domain, but not the second targeting domain, contains a UAA. In some cases, both the first targeting domain and the second targeting domain contain a UAA. In some examples, the payload can be attached to the first targeting domain or the second targeting domain. In some cases, the payload is attached to the first targeting domain. In some cases, the payload is attached to the second targeting domain. Conjugate

[0107] The conjugate may include a targeting domain and a payload. In some embodiments, the payload is connected to the conjugate with a linker. In some examples, the conjugate includes at least one unnatural amino acid. In some examples, the targeting domain is configured to bind to a target, and one of the unnatural amino acids in the targeting domain forms a covalent bond with the target.

[0108] The conjugates provided herein may include (i) a first targeting domain and (ii) a second targeting domain. The conjugates provided herein may include (i) a first targeting domain, (ii) a second targeting domain, and (iii) a payload. The conjugates described herein may include a first targeting domain and a second targeting domain, where the first targeting domain and the second targeting domain are configured to bind to the same cell. In some examples, the conjugates include at least one unnatural amino acid (UAA) included in the first targeting domain, the second targeting domain, or each of the first targeting domain and the second targeting domain includes at least one UAA. In some examples, the targeting domain is configured to bind to a target, and one of the UAAs in the targeting domain (e.g., the first and / or second targeting domain) forms a covalent bond with the target. In some examples, the first targeting domain comprises a UAA and is configured to form a covalent bond with the first target, such that at least one UAA is present in the first targeting domain near the interface between the first target and the first targeting domain. In some examples, the first target and the first targeting domain are bound, and a covalent bond is formed between the first target and a UAA in the first targeting domain of the conjugate. In some examples, the second targeting domain comprises a UAA and is configured to form a covalent bond with the second target, such that at least one UAA is present in the second targeting domain near the interface between the second target and the second targeting domain. In some examples, the second target and the second targeting domain are bound, and a covalent bond is formed between the second target and a UAA in the second targeting domain of the conjugate. In some examples, the first target is the same as the second target. In some examples, the first target and the second target are on the surface of the same cell.

[0109] In some embodiments, the first targeting domain and the second targeting domain bind the same target, such as on the surface of a cell, and engagement of one of the targeting domains brings the other targeting domain into proximity with the corresponding target. In some cases, the first targeting domain binds to the first target, and the second targeting domain binds to the second target, and at least one of the first and second targeting domains comprises a UAA, which forms a covalent bond with the corresponding target. In some examples, one targeting domain forms a covalent bond with its corresponding target, and the other targeting domain binds non-covalently to its corresponding target. In some examples, both the first targeting domain and the second targeting domain each bind covalently to their corresponding target.

[0110] The conjugates provided herein may be configured to bind to more than one target. In some examples, the second targeting domain may bind a target other than the first targeting domain. In some examples, the conjugates include at least 2, 3, 4, 5, 6, or more than 7 targeting domains. In some examples, the targeting domains (e.g., the first targeting domain and the second targeting domain) are linked to each other by a linker (e.g., a chemical linker, a fusion protein, or other linkers provided herein). In some examples, the multiple targeting domains (e.g., the first targeting domain and the second targeting domain) in the conjugates provided herein may provide a monoparatopic or biparatopic construct. In some embodiments, the first targeting domain and the second targeting domain bind to the same target. In some examples, the first targeting domain and the second targeting domain bind to different epitopes of the same target. In some embodiments, the first targeting domain and the second targeting domain bind to different targets. In some embodiments, the first targeting domain and the second domain combine to form a fusion protein.

[0111] The targeting domain (e.g., the first targeting domain or the second targeting domain) may guide the payload attached to the conjugate to a target. In some examples, the targeting domain comprises an antibody or a fragment thereof. In some examples, the targeting domain comprises a monospecific Fab2, a bispecific Fab2, a trispecific Fab3, a monovalent IgG, an scFv, a bispecific diabody, a trispecific triabody, an scFv-Fc, a nanobody (i.e., a single domain antibody, sdAb), a minibody, an IgNAR, a V-NAR, a hcIgG, a VhH, or a peptibody, a Darpin, a monobody / FN3, a VNAR, a Repebody, a Darpin. In some examples, the targeting domain comprises a nanobody. In some embodiments, the targeting domain comprises a single domain antibody (sdAb). In some examples, the targeting domain comprises one or more CDR regions. In some embodiments, the targeting domain binds to a cell surface molecule. In some examples, the conjugate is biparatopic. In some embodiments, the first targeting domain and the second targeting domain each comprise an antibody or an antigen-binding fragment, and the structure of such an antibody or an antigen-binding fragment can be the same or different.In some examples, the first targeting domain can be a single chain (e.g., Fv) antibody fragment, and the second targeting domain can be an sdAb, or in other examples, the first targeting domain can be an sdAb, and the second targeting domain can be an sdAb.

[0112] In some examples, the targeting domain (eg, the first targeting domain and / or the second targeting domain) is an antibody mimic, such as an affibody, a DARPin, or a minibinder.

[0113] In some examples, the target comprises a cell surface protein. In some examples, the target comprises PSMA. In some examples, the targeting domain (e.g., the first targeting domain and / or the second targeting domain) comprises any one of SEQ ID NOs: 1-4 or 16-64. In some examples, the targeting domain comprises a sequence having at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% identity to any one of SEQ ID NOs: 1-4 or 16-64. In some examples, the targeting domain comprises a sequence having at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% identity to any one of SEQ ID NOs: 1-4 or 16-64, and at least one unnatural amino acid.

[0114] The unnatural amino acid may be located at any position in the conjugate. In some examples, one or both of the first or second targeting domains (e.g., the first targeting domain or the second targeting domain) comprises an unnatural amino acid. In some examples, the targeting domain is an antibody or antigen-binding fragment comprising one or more complementarity determining regions (CDRs). In some examples, the one or more unnatural amino acids are included within or near the CDRs of only one or both of the targeting domains. In some examples, the targeting domain comprises an unnatural amino acid. In some examples, the targeting domain comprises C1, C2, or C3. In some examples, the conjugate comprises C1 with an unnatural amino acid in CDR1, CDR2, or CDR3. In some examples, the conjugate comprises a C1 with an unnatural amino acid at any one of positions 26, 28, 29, 30, 99, 102, 103, 105, 108, 110, 111, 112, 113, 114, and 115. In some examples, the conjugate comprises a C1 with an unnatural amino acid at any one of positions 28, 102, 112, and 113. In some examples, the conjugate comprises a C2 with an unnatural amino acid at CDR1, CDR2, or CDR3. In some examples, the conjugate comprises a C2 with an unnatural amino acid at any one of positions 50, 52, 53, 54, 56, 58, or 100. In some examples, the conjugate comprises a C4 with an unnatural amino acid at CDR1, CDR2, or CDR3. In some examples, the conjugate comprises a C4 with an unnatural amino acid at position 109. In some examples, the conjugate comprises a C3 with an unnatural amino acid in CDR1, CDR2, or CDR3. In some examples, the conjugate comprises a C3 with an unnatural amino acid at any one of positions 58, 62, 101, 103, or 107. In some examples, the conjugate comprises a C8 with an unnatural amino acid at any one of positions 52, 53, 54, 55, 56, 58, 60, 62, 64.In some examples, the conjugate comprises a C9 having an unnatural amino acid at any one of positions 53, 55, 56, 57, 58, 60, 64, 67. In some examples, the conjugate comprises a single domain antibody (sdAb) comprising an sdAb having a CDR region and at least one unnatural amino acid (UAA) residue within or near the CDR region, where the sdAb comprises any one of SEQ ID NOs: 1-4 or 14-64. In some examples, the conjugate comprises a single domain antibody (sdAb) comprising an sdAb having a CDR region and at least one unnatural amino acid (UAA) residue within or near the CDR region, where the sdAb comprises a sequence having 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, at least 65% identity to any one of SEQ ID NOs: 1-4 or 16-64. In some embodiments, the CDR regions comprise one or more of SEQ ID NOs: 5-13. In some embodiments, the CDR regions comprise a sequence having at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% identity to one or more of SEQ ID NOs: 5-13.

[0115] In some examples, the conjugate includes a tag (e.g., a His6 tag) for purification or the like. In some examples, the conjugate does not include a tag, or the tag is removed prior to administration. In some examples, the conjugate includes a leader sequence, for expression or secretion, or the like. In some examples, the conjugate does not include a leader sequence, or the leader sequence is removed from the first targeting domain or the second targeting domain prior to formation of the conjugate, attachment of the payload, or administration of the conjugate.

[0116] In some examples, the conjugate includes a signal sequence. The signal sequence may allow for expression, folding, or oxidation of the conjugate in the bacterial cell. In some examples, the signal sequence may allow for transport of the conjugate expressed in the bacterial cell to another location or environment to facilitate folding or function of the conjugate. In some examples, the signal sequence may be a PelB sequence. In some examples, the signal sequence may allow for transport of the conjugate to the periplasm of the bacterial cell. In some examples, the environment may be oxidizing or reducing to allow for disulfide formation or disulfide reduction.

[0117] target The conjugates provided herein can be configured to bind to one or more targets. In some examples, the conjugate comprises a single targeting domain comprising a UAA, and the conjugate binds to the target. In some examples, the conjugate comprises two (or at least two) targeting domains, and the first and second targeting domains bind to one or more targets. In some examples, the first targeting domain binds to a first target, and the second targeting domain binds to a second target. In some examples, the UAA included in the targeting domain (e.g., the single targeting domain, or the first targeting domain, or the second targeting domain) forms a covalent bond between the targeting domain (e.g., the single targeting domain, or the first targeting domain, or the second targeting domain) and the target (corresponding to the targeting domain, e.g., the first or second target). In some examples, the conjugate comprises a first targeting domain comprising a first UAA and a second targeting domain comprising a second UAA. In some cases, such UAAs are the same, or in other cases, different. In some cases, the target is a cell surface molecule (i.e., present entirely or partially on the outer surface of a cell). In some cases, the first targeting domain and the second targeting domain bind to the same target, such as the same cell surface molecule. In some cases, the first targeting domain and the second targeting domain each bind to a different target, such as binding to a different cell surface molecule. The different cell surface molecules can be on the same cell.

[0118] In some examples, the target is a cell surface molecule present on a tumor cell. In some examples, the target is a monomer. In some examples, the target is included in a multimeric structure of homogeneous or heterogeneous units. In some examples, the target is PSMA, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD166, CLL-1, ADAM9, cKIT, CD36, CD73, I TGaVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, ROR1, TROP-2, NECTIN4, cMET, CD19, CD22, CD30, CD33, CD123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM are provided.

[0119] In some examples, the conjugate comprises a first targeting domain and a second targeting domain, both of which bind to the same target, where the target is a cell surface molecule on a tumor cell. For example, both targeting domains bind to PSMA, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD166, CLL-1, ADAM9, cKIT, CD36, CD73, IT GaVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, ROR1, T The conjugate binds to a target selected from the group consisting of ROP-2, NECTIN4, cMET, CD19, CD22, CD30, CD33, CD123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM. In some examples, the conjugate comprises a first targeting domain and a second targeting domain that bind to different targets, where one or more of the different targets are cell surface molecules on a tumor cell.For example, the first and second targeting domains can be PSMA, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD166, CLL-1, ADAM9, cKIT, CD36, CD73, ITG aVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, ROR1, TROP Each of the conjugates binds to a different target selected from the group consisting of IL-2, NECTIN4, cMET, CD19, CD22, CD30, CD33, CD123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM. In some examples, the conjugate carries a payload on the cell surface when the first or second target (or both) is bound. In some examples, the conjugate does not have a payload and the conjugate acts as a blocker or antagonist when covalently bound to the first target, the second target, or both the first target and the second target.

[0120] In some examples, the conjugate comprises a single targeting domain, and the targeting domain binds to a target, where the target is a cell surface molecule on a tumor cell. For example, the targeting domain may be PSMA, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD166, CLL-1, ADAM9, cKIT, CD36, CD73, IT GaVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, ROR1, T The conjugate binds to a target selected from the group consisting of ROP-2, NECTIN4, cMET, CD19, CD22, CD30, CD33, CD123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM. In some examples, the single targeting domain carries a payload to the target. In some examples, the single targeting domain does not have a payload, and the conjugate acts as a blocker or antagonist when covalently bound to the target.

[0121] The target to which the targeting domain binds may include various structures. In some examples, the target includes one or more epitopes to which the targeting domain can bind. In some examples, the target includes multiple epitopes and may be bound by multiple targeting domains, such as by a first targeting domain that binds to a first epitope and a second targeting domain that binds to a second epitope. In some examples, the target includes a monomer. In some examples, the target includes a single-chain peptide. In some examples, the target includes a multimeric molecule. In some examples, the multimeric molecule includes two or more subunits. In some examples, the subunits have the same structure. In some examples, the subunits are of different structures or a combination of the same structure and different structures. In some examples, the multimeric molecule includes two or more molecules in a complex. In some examples, the multimeric molecule includes two proteins that complex or interact with each other.

[0122] Exemplary Targeting Domains and Exemplary Conjugates The conjugates provided herein include a targeting domain that can be assembled from molecules that bind to a target. In some examples, the targeting domain (a single targeting domain or a first targeting domain and / or a second targeting domain) includes an antigen-binding region, which binds to a specific target. Such an antigen-binding region can include CDRs, such as the three CDRs commonly found in the heavy or light chain of an antibody. In some examples, the targeting domain includes an antigen-binding fragment that includes CDRs, such as a VHH (also called a nanobody or single domain antibody). In some examples, a single domain antibody includes one or more UAAs. In some examples, for a conjugate having a single targeting domain or multiple targeting domains, one of the targeting domains (e.g., the first targeting domain or the second targeting domain) is selected from a single domain antibody in Table 1, such as C1, C2, C3, C4, or C5. In some examples, for a conjugate having multiple targeting domains, each of the targeting domains (e.g., the first targeting domain and the second targeting domain) is independently selected from a single domain antibody in Table 1, such as, for example, any of C1, C2, C3, C4, or C5, and one or more UAAs are present in or near a CDR of the single domain antibody. In some examples, the first targeting domain is selected from a single domain antibody in Table 1, such as, any of C1, C2, C3, C4, or C5. In some examples, the second targeting domain is selected from a single domain antibody in Table 1, such as, any of C1, C2, C3, C4, or C5.

[0123] In some examples, the conjugate comprises a single targeting domain, a first targeting domain, a second targeting domain, or both targeting domains, including construct C1. In some examples, the conjugate comprises construct C1 with an unnatural amino acid in CDR1, CDR2, or CDR3. In some examples, the conjugate comprises construct C1 with an unnatural amino acid at any one of positions 26, 28, 29, 30, 99, 102, 103, 105, 108, 110, 111, 112, 113, 114, and 115 of SEQ ID NO: 1, 4, 19, or 73. In some examples, the conjugate comprises construct C1 with an unnatural amino acid at any one of positions 28, 102, 112, and 113 of SEQ ID NO: 1, 4, 19, or 73. In some examples, the conjugate comprises a single domain antibody (sdAb) and at least one unnatural amino acid (UAA) within or near a CDR region in the sdAb. In some examples, such an sdAb comprises SEQ ID NO:1. In some examples, the conjugate comprises a single domain antibody (sdAb) and at least one unnatural amino acid (UAA) within or near a CDR region in the sdAb, the sdAb comprising a sequence having at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, at least 65% sequence identity to SEQ ID NO:1. In some examples, the conjugate comprises a single targeting domain, a first targeting domain, or a second targeting domain, or both, having at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% identity to SEQ ID NO:1.

[0124] In some examples, the conjugate comprises a single targeting domain, a first targeting domain, a second targeting domain, or both targeting domains, comprising construct C2. In some examples, the conjugate comprises construct C2 with an unnatural amino acid in CDR1, CDR2, or CDR3. In some examples, the conjugate comprises construct C2 with an unnatural amino acid at any one of positions 50, 52, 53, 54, 56, 58, or 100 of SEQ ID NO: 2 or 22. In some examples, the conjugate comprises a single domain antibody (sdAb) comprising an sdAb and at least one unnatural amino acid (UAA) within or near a CDR region within the sdAb, wherein the sdAb comprises SEQ ID NO: 2. In some examples, the conjugate comprises a single domain antibody (sdAb) and at least two unnatural amino acids (UAAs) in or near the CDR regions in the sdAb, where the sdAb comprises a sequence having at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, at least 65% sequence identity to SEQ ID NO: 2. In some examples, the conjugate comprises a single targeting domain, a second targeting domain, or both, that have at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% sequence identity to SEQ ID NO: 2.

[0125] In some examples, the conjugate comprises a single targeting domain, a first targeting domain, a second targeting domain, or both targeting domains, including construct C3. In some examples, the conjugate comprises construct C3 with an unnatural amino acid in CDR1, CDR2, or CDR3. In some examples, the conjugate comprises construct C3 with an unnatural amino acid at any one of positions 58, 62, 101, 103, or 107 of SEQ ID NO:3. In some examples, the conjugate comprises an unnatural amino acid in CDR1, CDR2, or CDR3 of C3. In some examples, the conjugate comprises an unnatural amino acid at position 109. In some examples, the conjugate comprises a single domain antibody (sdAb) with at least one unnatural amino acid (UAA) within or near a CDR region within the sdAb, wherein the sdAb comprises SEQ ID NO:3. In some examples, the conjugate comprises a single domain antibody (sdAb) and at least three unnatural amino acids (UAAs) in or near the CDR regions in the sdAb, where the sdAb comprises a sequence having at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, at least 65% sequence identity to SEQ ID NO: 1. In some examples, the conjugate comprises a single targeting domain, a second targeting domain, or a third targeting domain, or both, having at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% sequence identity to SEQ ID NO:2.

[0126] In some examples, the conjugate comprises a single targeting domain, a first targeting domain, a second targeting domain, or both targeting domains, including construct C4. In some examples, the conjugate comprises construct C4 with an unnatural amino acid in CDR1, CDR2, or CDR3. In some examples, the conjugate comprises a single domain antibody (sdAb), and at least one unnatural amino acid (UAA) within or near a CDR region within, where the sdAb comprises SEQ ID NO: 16. In some examples, the conjugate comprises a single domain antibody (sdAb), and at least one unnatural amino acid (UAA) within or near a CDR region within, where the sdAb comprises a sequence having at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, at least 65% sequence identity to SEQ ID NO: 16. In some examples, the conjugate includes a single targeting domain, a second targeting domain, or a sixteenth targeting domain, or both, that have at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% sequence identity to SEQ ID NO:16.

[0127] In some examples, the conjugate comprises a single targeting domain, a first targeting domain, a second targeting domain, or both targeting domains, including construct C5. In some examples, the conjugate comprises construct C5 with an unnatural amino acid in CDR1, CDR2, or CDR3. In some examples, the conjugate comprises a single domain antibody (sdAb), and at least one unnatural amino acid (UAA) within or near a CDR region within, where the sdAb comprises SEQ ID NO: 18. In some examples, the conjugate comprises a single domain antibody (sdAb), and at least one unnatural amino acid (UAA) within or near a CDR region within, where the sdAb comprises a sequence having at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, at least 65% sequence identity to SEQ ID NO: 18. In some examples, the conjugate includes a single targeting domain, a second targeting domain, or an 18th targeting domain, or both, that have at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% sequence identity to SEQ ID NO:18.

[0128] The conjugates described herein may include two or more targeting domains. In some examples, the two or more targeting domains (e.g., a first targeting domain or a second targeting domain) are connected to each other via a linker. In some examples, the conjugate is a fusion protein. In some examples, the linker includes linker-L1 (SEQ ID NO: 14). In some examples, the conjugate includes any one of SEQ ID NOs: 65-72. In some embodiments, the conjugate has at least 99%, 98%, 97%, 95%, 90%, 85%, 80%, 70%, or at least 65% sequence identity to one of SEQ ID NOs: 65-72.

[0129] In some examples, the first targeting domain comprises any one of SEQ ID NOs: 1-4 or 16-64. In some examples, the first targeting domain comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1-4 or 16-64. In some examples, the second targeting domain comprises any one of SEQ ID NOs: 1-4 or 16-64. In some examples, the second targeting domain comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1-4 or 16-64. In some examples, the conjugate is comprised of a single targeting domain, the single targeting domain comprises any one of SEQ ID NOs: 1-4 or 16-64. In some examples, the single targeting domain comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1-4 or 16-64.

[0130] In some examples, the conjugate comprises SEQ ID NO: 65-72. In some examples, the conjugate comprises a sequence having at least 70% sequence identity to any one of SEQ ID NO: 65-72. In some examples, the conjugate comprises any one of SEQ ID NO: 1-4 or 16-64. In some examples, the conjugate comprises a sequence having at least 70% sequence identity to any one of SEQ ID NO: 1-4 or 16-64. In some examples, the conjugate comprises a sequence having at least 70% sequence identity to SEQ ID NO: 65-72.

[0131] In some examples, the amino acid sequences associated with the conjugates described herein are in Table 1A.

[0132] [Table 1-1]

[0133] [Table 1-2]

[0134] [Table 1-3]

[0135] [Table 1-4]

[0136] [Table 1-5]

[0137] [Table 1-6]

[0138] [Table 1-7]

[0139] [Table 1-8] X indicates the position of the FSY incorporation site. Sequences in the table that have a His6 purification tag and / or a PelB leader sequence are also embodied herein.

[0140] In some examples, DNA sequences associated with the conjugates described herein are in Table 2.

[0141] [Table 2-1]

[0142] [Table 2-2]

[0143] [Table 2-3]

[0144] [Table 2-4]

[0145] [Table 2-5]

[0146] [Table 2-6]

[0147] [Table 2-7]

[0148] [Table 2-8]

[0149] [Table 2-9]

[0150] Payload The conjugate may include a payload. In some examples, a targeting domain included in the conjugate (e.g., a single targeting domain, a first targeting domain, a second targeting domain) may target the payload. In some examples, the targeting domain (e.g., a single targeting domain, a first targeting domain, a second targeting domain) includes an antibody or fragment thereof that targets the payload to a cell, such as a tumor cell. In some embodiments, the payload includes an imaging agent, a radioligand agent, or a cytotoxic agent. In some embodiments, the cytotoxic moiety includes a small molecule drug, a peptide, a protein, or a chemotherapeutic drug. In some embodiments, the payload includes a radioligand agent. In some embodiments, the radioligand agent is 35 S, 3 H, 111In, 112 In, 14 C. 186 Re, 188 Re, 32 P, 153 Sm, 177 Lu, 86 Y, 88 Y, 90 Y, 131 I, 123 I, 124 I, 125 I, 149 Tb, 211 At, 212 Pb / 212 Bi, 213 Bi, 223 Ra, 225 Ac, 64 Cu, 67 Cu, and 227 In some embodiments, the radioligand agent further comprises a chelating agent. Examples of chelating agents include DOTA, DOTAGA, NOTA, MACROPA, THP, and TRAP. In some embodiments, the radioligand agent is 99 mTc, 131 I, 201 Tl, 111 In, and 67 In some examples, the imaging agent comprises a dye. In some examples, the conjugate comprises two or more payloads. For example, the conjugate comprises a payload dye for visualizing tissue penetration and a payload chemotherapeutic agent for killing a tumor.

[0151] The payload may be attached to the conjugate. The payload may be covalently attached to the conjugate. In some examples, the payload is not attached to the conjugate via an unnatural amino acid (UAA) residue. In some embodiments, the payload is attached to amino acids n+x (towards the C-terminus) from the UAA residue. In some embodiments, the attachment site is defined as x amino acids away from the unnatural amino acid position n. In some embodiments, the payload is attached to amino acids nx (towards the N-terminus) from the UAA residue. In some embodiments, the UAA residue is included within or near the region of the targeting domain (e.g., the single targeting domain or the first or second targeting domain) that contacts the target. In some examples, the payload is attached at least 2, 5, 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 125 amino acids away from the unnatural amino acid in the conjugate.

[0152] Unnatural Amino Acids (UAA) Unnatural amino acids (UAAs) can be incorporated into the conjugates described herein. In some examples, the UAA residue is within the targeting domain of the conjugate. In some examples, the UAA is configured to covalently bind to the target. In some examples, the UAA is configured to covalently bind to an amino acid present in the target. In some examples, the conjugate comprises one, two, three, or more UAAs. In some examples, the conjugate comprises a first targeting domain comprising a first UAA and a second targeting domain comprising a second UAA. In some examples, the UAA is configured within the targeting domain (e.g., the first targeting domain or the second targeting domain) to covalently bind to an amino acid present in the target when the targeting domain binds to the target. In some examples, such amino acids include nucleophilic amino acids. In some examples, the UAA residue forms a covalent bond with lysine, histidine, or tyrosine. In some examples, the UAA residue is located in a target binding domain. In some examples, the UAA, once incorporated into the conjugate, is located in a CDR of a targeting domain, such as an antibody or antigen-binding fragment, e.g., a targeting domain comprising a single domain antibody. In some embodiments, the UAA residue comprises an aryl-fluorosulfate moiety. In some examples, the UAA residue is genetically encoded into the conjugates described herein. In some examples, the UAA residue comprises a variant of tyrosine or lysine. In some embodiments, the unnatural amino acid residue has the structure of Formula I:

[0153] [ka] In some embodiments, the unnatural amino acid residue comprises the structure of Formula II:

[0154] [ka] In some embodiments, the UAA residue is from a UAA incorporation described herein. In some embodiments, the unnatural amino acid is 2-amino-3-(4-((fluorosulfonyl)oxy)phenyl)propanoic acid:

[0155] [ka] In some embodiments, the unnatural amino acid is fluorosulfonyl tyrosine (FSY):

[0156] [ka] In some embodiments, the unnatural amino acid is N6-(4-((fluorosulfonyl)oxy)benzoyl)lysine:

[0157] [ka] In some embodiments, the unnatural amino acid is fluorosulfonyloxybenzoyl-L-lysine (FSK):

[0158] [ka] In some embodiments, the unnatural amino acid residue has the structure of Formula III:

[0159] [ka] In some embodiments, the unnatural amino acid residue comprises:

[0160] [ka] In some embodiments, the unnatural amino acid residue is:

[0161] [ka] It is.

[0162] In some embodiments, the unnatural amino acid (UAA) of the UAA residue has the structure of formula (IA): (IA) having During the ceremony, Each X is independently O or NR'; Y is a bond, -O-, -NR-, or -N=; A is a bond or -(CH2) n -, m is 1 or 2, and n is an integer from 1 to 4; each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, L is -(CH2) P - or -C(O)NH-(CH2) P -, p is an integer from 1 to 6, and wherein m is 1 when Y is -O- or -NR-, and m is 2 when Y is -N=.

[0163] In some embodiments, the UAA of formula (IA) has the structure of formula (IA-a):

[0164] [ka] In some embodiments, the UAA of formula (IA) has the structure of formula (IA-b):

[0165] [ka] has.

[0166] In some embodiments, the UAA of formula (IA) has the structure of formula (IB):

[0167] [ka] has.

[0168] In some embodiments, the UAA of formula (IB) has the structure of formula (IB-a):

[0169] [ka] In some embodiments, the UAA of formula (IB) has the structure of formula (IB-b):

[0170] [ka] has.

[0171] In some embodiments, the UAA of formula (IA) has the structure of formula (IC):

[0172] [ka] has.

[0173] In some embodiments, the UAA of formula (IC) has the structure of formula (IC-a):

[0174] [ka] In some embodiments, the UAA of formula (IB) has the structure of formula (IB-b):

[0175] [ka] In some preferred embodiments, R is hydrogen.

[0176] In some embodiments, the UAA of formula (IA) has the structure of formula (ID):

[0177] [ka] wherein Each X is independently O or NR'; Y is a bond, -O-, -NR-, or -N=; A is a bond or -(CH2) n -, m is 1 or 2, and n is an integer from 1 to 4; each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, and wherein m is 1 when Y is a bond, -O- or -NR-, and m is 2 when Y is -N=.

[0178] In some embodiments, the UAA of formula (ID) has the structure of formula (ID-a):

[0179] [ka] In some embodiments, the UAA of formula (ID) has the structure of formula (ID-b):

[0180] [ka] has.

[0181] In some embodiments, the UAA of formula (IA) has the structure of formula (IE):

[0182] [ka] wherein Each X is independently O or NR'; Y is a bond, -O-, -NR-, or -N=; A is a bond or -(CH2) n -, m is 1 or 2, and n is an integer from 1 to 4; each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; R 1 is hydrogen, fluoro or iodo, R 2 is hydrogen or methyl, and wherein m is 1 when Y is a bond, -O- or -NR-, and m is 2 when Y is -N=.

[0183] In some embodiments, the UAA of formula (IE) has the structure of formula (IE-a):

[0184] [ka] In some embodiments, the UAA of formula (IE) has the structure of formula (IE-b):

[0185] [ka] has.

[0186] In certain embodiments, Y is a bond, -O- or -NR- and m is 1. In other embodiments, Y is -N= and m is 2. In certain preferred embodiments, Y is -O- and m is 1. In other embodiments, Y is -NR= and m is 1. In other embodiments, Y is a bond and m is 1. In other embodiments, Y is O- or -NR- and m is 1.

[0187] In some embodiments, the UAA of formula (IA) has the structure of formula (IIA):

[0188] [ka] wherein X is independently O or NR', and R', if present, is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.

[0189] In some embodiments, the UAA of formula (IIA) has the structure of formula (IIA-a):

[0190] [ka] In some embodiments, the UAA of formula (IIA) has the structure of formula (IIA-b):

[0191] [ka] has.

[0192] In some embodiments, the UAA of formula (IA) has the structure of formula (IIB):

[0193] [ka] wherein X is independently O or NR', and R', if present, is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl.

[0194] In some embodiments, the UAA of formula (IIB) has the structure of formula (IIB-a):

[0195] [ka] In some embodiments, the UAA of formula (IIB) has the structure of formula (IIB-b):

[0196] [ka] has.

[0197] In some embodiments, the unnatural amino acid (UAA) has the structure of Formula (IV):

[0198] [ka] having During the ceremony, Each X is independently O or NR'; Y is a bond, -O-, -NR-, or -N=; A is a bond or -(CH2) n -, m is 1 or 2, and n is an integer from 1 to 4; each of R and R', if present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; Ring A is a 5- to 6-membered aryl or heteroaryl; R A are each independently -OH, -OR X , halogen, NHR X , N(R X )2, or optionally substituted alkyl; R X each is optionally substituted alkyl; L is -(CH2) P - or -C(O)NH-(CH2) P -, p is an integer from 1 to 6, and wherein m is 1 when Y is a bond, -O- or -NR-, and m is 2 when Y is -N=.

[0199] In some embodiments, A is a bond. In other embodiments, A is -(CH2). nIn some embodiments, n is 1, 2, 3, or 4. In one embodiment, n is 1. In another embodiment, n is 2. In yet another embodiment, n is 3. In yet another embodiment, n is 4.

[0200] In some embodiments, ring A is a 6-membered ring. In some embodiments, ring A is a 6-membered ring. In some embodiments, ring A is aryl. In some embodiments, ring A is heteroaryl. In some embodiments, ring A is a 6-membered aryl. In some embodiments, ring A is a 6-membered heteroaryl. In one preferred embodiment, ring A is phenyl.

[0201] In some embodiments, p is 0. In some embodiments, p is an integer from 1 to 4. In some embodiments, p is an integer from 1 to 3. In one embodiment, p is 1. In another embodiment, p is 2. In yet another embodiment, p is 3. In yet another embodiment, p is 4.

[0202] In some embodiments, R A are each independently -OH, -OR X , halogen, NHR X , N(R X )2, or optionally substituted alkyl, and R X Each is optionally substituted alkyl. In some embodiments, R A Each is independently -OH, halo-, or optionally substituted alkyl. In some embodiments, R A Each is independently halo- or optionally substituted alkyl. In some embodiments, R A are each independently -OH, or optionally substituted alkyl. In some embodiments, R A Each independently is optionally substituted alkyl. In some embodiments, R A Each independently is optionally substituted alkyl. In some embodiments, RA Each is independently unsubstituted alkyl. In one embodiment, R A Each is iodo. In another embodiment, R A Each is methyl. In one specific embodiment where p is 1, R A In another specific embodiment where p is 2, R A are each methyl.

[0203] In some embodiments, Y is a bond, -O-, -NR-, or -N=. In some embodiments, Y is -O-, -NR-, or -N=. In some embodiments, Y is a bond, -O-, or -NR-. In some embodiments, Y is a bond, -O-, or -N=. In some embodiments, Y is a bond, NR-, or -N=. In some embodiments, Y is -O- or -NR-. In some embodiments, Y is -O- or -N=. In some embodiments, Y is -NR- or -N=. In some embodiments, Y is -O- or -NR-. In some embodiments, Y is -O- or -N=. In some embodiments, Y is -NR- or -N=. In one embodiment, Y is a bond. In another embodiment, Y is -O-. In yet another embodiment, Y is -NR-. In yet another embodiment, Y is -N=.

[0204] In some embodiments, L is -(CH2). P In other embodiments, L is -C(O)NH-(CH2). P In some embodiments, p is an integer from 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In one embodiment, p is 1 to 4. In another embodiment, p is 1 or 2. In yet another embodiment, p is 1 or 4. In one preferred embodiment, L is -CH2-. In another preferred embodiment, L is -C(O)NH-(CH2)4-.

[0205] In some embodiments, R is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl. In some embodiments, R is hydrogen or substituted or unsubstituted alkyl. In one preferred embodiment, R is hydrogen. In another embodiment, R is substituted or unsubstituted C 1-6 In yet another embodiment, R is unsubstituted C 1-6 In one embodiment, R is alkyl. In one embodiment, R is methyl. In another preferred embodiment, R is hydrogen or methyl.

[0206] In some embodiments, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl. In some embodiments, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl. In some embodiments, R' is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl. In some embodiments, R' is hydrogen. In another embodiment, R' is substituted or unsubstituted. In yet another embodiment, R' is substituted or unsubstituted aryl.

[0207] In some embodiments, R 1 is hydrogen. In some embodiments, R 1 is fluoro. In some embodiments, R 1 is iodo. In some embodiments, R 2 is hydrogen. In other embodiments, R 2 is methyl. In some embodiments, R 1 is hydrogen, and R2 is hydrogen. In some embodiments, R 1 is fluoro and R 2 is hydrogen. In some embodiments, R 1 is hydrogen, and R 2 is methyl. In some embodiments, R 1 is iodine, R 2 is hydrogen.

[0208] linker- In some embodiments, the conjugates provided herein include more than one linker. In some embodiments, the targeting domains (e.g., the first and second targeting domains) are connected by a first linker. The first linker and the second linker can be any linker described herein. In some embodiments, the payload is connected to one of the targeting domains (the first and second targeting domains) by a second linker. In one preferred embodiment, the first linker is a peptide linker. In another preferred embodiment, the second linker is formed by chemical conjugation. In some embodiments, the second linker is a bifunctional linker for chemical conjugation. In another preferred embodiment, the second linker is a non-peptide linker.

[0209] In some embodiments, useful functional reactive groups for conjugating or attaching a targeting domain to a payload described herein include, for example, zero or higher order linkers. In some examples, the conjugate moiety includes a functional reactive group that reacts with a linker described herein (optionally previously attached to the payload, targeting domain, or other portion of the conjugate). In some embodiments, the linker includes a reactive group that reacts with a naturally occurring amino acid in a payload or targeting domain described herein. The targeting domain (e.g., the first targeting domain and the second targeting domain), or one of the targeting domains (e.g., the first targeting domain or the second targeting domain) and the payload can be conjugated together by reacting a nucleophilic reactive moiety on the first targeting domain with an electrophilic reactive moiety on the second targeting domain, or by reacting a nucleophilic reactive moiety on the targeting domain with an electrophilic reactive moiety on the payload. In an alternative embodiment, the first targeting domain and the second targeting domain, or one of the targeting domains (e.g., the first targeting domain or the second targeting domain) and the payload are conjugated together by reacting an electrophilic reactive moiety on the first targeting domain with a nucleophilic moiety on the second targeting domain, or by reacting an electrophilic reactive moiety on the targeting domain with a nucleophilic moiety on the payload. In some embodiments, an amide bond may form upon reaction of an amine on a targeting domain (e.g., the ε-amine of a lysine residue) with a carboxyl group on another targeting domain, or with an amine on a targeting domain with a carboxyl group on a payload. In alternative embodiments, the targeting domain and / or payload are derivatized with a derivatizing agent prior to conjugation.

[0210] In some cases, the higher order linker comprises a bifunctional linker, such as a homobifunctional linker or a heterobifunctional linker. Exemplary homobifunctional linkers include Lomant's reagent dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfoDST), ethylene glycol tartrate (EGCG), and ethylene glycol tartrate (EGCG). Lycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), l,4-di-3'-(2'-pyridyldithio)propionamido)buffer benzene (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB), such as l,5-difluoro-2,4-dinitrobenzene or l,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenylsulfone (DFDNPS), bis-[β-3-(4-azidosalicylamido)ethyl] disulfide (BASED), formaldehyde, glucuronide, etc. Examples of suitable iodoacetamides include, but are not limited to, butylaldehyde, 1,4-butanediol diglycidyl ether, adipic dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide).

[0211] In some embodiments, the bifunctional linker comprises a heterobifunctional linker. Exemplary heterobifunctional linkers include amine-reactive and sulfhydryl crosslinkers, such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexyl ester (sulfo-LC-sMPT), and succinimidyl-4-(N-maleimidomethyl)cyclohexyl ester (sulfo-LC-sMPT). San-l-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MB), N-succinimidyl (4-iodoactyl) aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoactyl) aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl) N-(γ-maleimidobutyryloxy)succinimide ester (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino] ... succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH),4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive and photoreactive crosslinkers such as N-hydroxysuccinimidyl-4-azidosalicylate (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(ρ-azidosalicylamido)ethyl-1,3′-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl -4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-SANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-nitrobenzamido)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), ρ-nitrophenyl diazopyruvate (ρNPDP), sulfhydryl-reactive and photoreactive crosslinkers such as ρ-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP),Benzophenone-4-iodoacetamide, Benzophenone-4-maleimide Carbonyl-reactive and photoreactive crosslinkers, such as ρ-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive crosslinkers, such as 4-(ρ-azidosalicylamido)butylamine (AsBA), arginine-reactive and photoreactive crosslinkers, such as ρ-azidophenylglyoxal (APG),

[0212] In some examples, the reactive functional group comprises a nucleophilic group that is reactive to an electrophilic group present on a binding moiety (e.g., a payload moiety or a targeting domain). Exemplary electrophilic groups include carbonyl groups, such as aldehydes, ketones, carboxylic acids, esters, amides, enones, acyl halides, or acid anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophilic groups include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and arylhydrazides. In some embodiments, the unnatural amino acids incorporated into the conjugates described herein comprise an electrophilic group.

[0213] In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker is a dipeptide linker. In some embodiments, the dipeptide linker is valine-citrulline (Val-Cit), phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala) and valine-lysine (Val-Lys). In some embodiments, the dipeptide linker is valine-citrulline. The linker may include a cleavable sequence or a sequence recognized by a protease.

[0214] In various embodiments, the targeting domains (e.g., the first targeting domain and the second targeting domain) include a polypeptide linker sequence. The linker can be C-terminal to the targeting domain. The linker can be expressed via recombinant techniques and can be coded using a nucleic acid sequence in conjunction with the expression of the targeting domain. The linker can link the targeting domains to form a fusion protein. The presence of the linker in the conjugate can allow the targeting domains to function properly without steric interference from other targeting domains. The linker can link the targeting domain to a tag, such as an expression tag or a purification tag. The linker can be a flexible linker. The flexibility of the linker can allow the targeting domains to adopt independent conformations with minimal interference from other targeting domains. In some embodiments, the linker is a peptide linker that contains, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids. In some examples, the peptide linker contains up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, or fewer amino acids. In additional cases, the peptide linker contains about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In additional cases, the polypeptide linker comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some examples, the polypeptide linker comprises (GGGGSGGGS)x (SEQ ID NO: 14), where x is 1-10. In some embodiments, the linker 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 polypeptide linkers. The linker may comprise SPSTPPTPSPSTPP, and the polypeptide linker may repeat (GGGGS)x, (GGGS)x. The linker may comprise glycine, serine, threonine, or proline.In some embodiments, the N-terminus of one targeting domain is fused to the C-terminus of a linker polypeptide, and the N-terminus of the linker polypeptide is fused to the N-terminus of another targeting domain.

[0215] In various embodiments, the targeting domains (e.g., the first targeting domain and the second targeting domain) are connected or separated by a linker. The linker can be a polypeptide linker. The linker can be expressed via recombinant technology and can be coded using a nucleic acid sequence in conjunction with the expression of the targeting domain. The linker can link the targeting domains to form a fusion protein. The presence of the linker in the conjugate can allow the targeting domains to function properly without steric interference from other targeting domains. The linker can be a flexible linker. The flexibility of the linker can allow the targeting domains to adopt independent conformations with minimal interference from other targeting domains. In some embodiments, the linker is a peptide linker, for example, comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids. In some examples, the peptide linker comprises up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, or fewer amino acids. In additional cases, the peptide linker comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In additional cases, the polypeptide linker comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some examples, the polypeptide linker comprises (GGGGSGGGS)x (SEQ ID NO: 14), where x is 1-10. In some embodiments, the linker is a polypeptide linker that 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. The linker may include SPSTPPTPSPSTPP, and the polypeptide linker may include repeats of (GGGGS)x, (GGGS)x. The linker may include glycine, serine, threonine, or proline.In some embodiments, the N-terminus of one targeting domain is fused to the C-terminus of a linker polypeptide, and the N-terminus of the linker polypeptide is fused to the N-terminus of another targeting domain.

[0216] In some embodiments, the linker comprises a self-immolative linker moiety. In some embodiments, the self-immolative linker moiety comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), or a derivative or analog thereof. In some embodiments, the linker comprises a dipeptide linker moiety and a self-immolative linker moiety. In some embodiments, the self-immolative linker moiety is as described in U.S. Pat. No. 9,089,614 and IPO Application No. WO2015038426.

[0217] In some embodiments, the cleavable linker is a glucuronide. In some embodiments, the cleavable linker is an acid-cleavable linker. In some embodiments, the acid-cleavable linker is a hydrazine. In some embodiments, the cleavable linker is a reducible linker.

[0218] In some embodiments, the linker comprises a maleimide group. In some examples, the maleimide group is also referred to as a maleimide spacer. In some examples, the maleimide group further comprises caproic acid to form maleimidocaproyl (me). In some cases, the linker comprises maleimidocaproyl (me). In some cases, the linker is maleimidocaproyl (me). In other examples, the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate (sulfo-sMCC) described above.

[0219] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some examples, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide and provide intramolecular catalysis of thiosuccinimide ring hydrolysis, thereby precluding the maleimide from undergoing elimination via a retro-Michael reaction. In some examples, the self-stabilizing maleimide is a maleimide group described in Lyon et al., "Self-hydrolyzing maleimides improves the stability and pharmacological properties of antibody-drug conjugates," Nat. Biotechnol. 32(10):1059-1062 (2014). In some examples, the linker comprises a self-stabilizing maleimide. In some examples, the linker is a self-stabilizing maleimide.

[0220] In some examples, the linker comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more of a carbonyl or dicarbonyl group, an oxime group, a hydroxylamine group, or a protected form thereof. The TLR agonist linker derivatives or targeting domains 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.

[0221] As described herein, the disclosure provides a targeting domain linked to another molecule having the formula "targeting domain-L-payload", where L is a linking group or a chemical bond. In some embodiments, L is stable in vivo. In some embodiments, L is hydrolyzable in vivo. In some embodiments, L is metastable in vivo.

[0222] The targeting domain and payload can be linked together via L using standard linking agents and procedures known to those of skill in the art. In some aspects, the targeting domain and payload are directly fused and L is a bond. In other aspects, the targeting domain and payload are fused by a linking group L. For example, in some embodiments, the targeting domain and payload are linked together via a peptide bond, optionally with a peptide or amino acid spacer. In some embodiments, the targeting domain and payload are linked together by chemical conjugation, optionally with a linking group (L). In some embodiments, L is directly conjugated to each of the targeting domain and payload.

[0223] Chemical conjugation can occur by reacting a nucleophilic reactive group of one compound with an electrophilic reactive group of another compound. In some embodiments, when L is a bond, the targeting domain is conjugated to the payload by reacting a nucleophilic reactive moiety on the targeting domain with an electrophilic reactive moiety on the linker, or by reacting an electrophilic reactive moiety on the targeting domain with a nucleophilic reactive moiety on the payload. In embodiments where L is a group that links the targeting domain and the payload together, the targeting domain and / or payload can be conjugated to L by reacting a nucleophilic reactive moiety on the targeting domain and / or payload with an electrophilic reactive moiety on L, or by reacting an electrophilic reactive moiety on the targeting domain and / or payload with a nucleophilic reactive moiety on L. Non-limiting examples of nucleophilic reactive groups include amino, thiol, and hydroxyl. Non-limiting examples of electrophilic reactive groups include carboxyl, acyl chloride, anhydride, ester, succinimide ester, alkyl halide, sulfonate ester, maleimide, haloacetyl, and isocyanate. In embodiments in which the targeting domain and payload are conjugated together by reacting a carboxylic acid with an amine, an activating agent can be used to form an activated ester of the carboxylic acid.

[0224] The activated ester of a carboxylic acid can be, for example, N-hydroxysuccinimide (NHS), tosylate (Tos), mesylate, triflate, carbodiimide, or hexafluorophosphate. In some embodiments, the carbodiimide is 1,3-dicyclohexylcarbodiimide (DCC), 1,1'-carbonyldiimidazole (CDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), or 1,3-diisopropylcarbodiimide (DICD). In some embodiments, the hexafluorophosphate salt is selected from the group consisting of hexafluorophosphate benzotriazole-l-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazole-l-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 2-(1H-7-azabenzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate) (HATU), and o-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluorophosphate (HBTU).

[0225] In some embodiments, the targeting domain (e.g., the first targeting domain and the second targeting domain) comprises a nucleophilic reactive group (e.g., an amino group, a thiol group, or a hydroxyl group on the side chain of a lysine, cysteine, or serine) that can be conjugated to an electrophilic reactive group on the payload or L. In some embodiments, the targeting domain comprises an electrophilic reactive group (e.g., a carboxylate group on the side chain of an Asp or Glu) that can be conjugated to a nucleophilic reactive group on the payload or L. In some embodiments, the targeting domain is chemically modified to include a reactive group that can be directly conjugated to the payload or L. In some embodiments, the targeting domain is modified at the N-terminus or C-terminus to include a natural amino acid with a nucleophilic side chain. In an exemplary embodiment, the N-terminus or C-terminus amino acid of the targeting domain is selected from the group consisting of lysine, ornithine, serine, cysteine, and homocysteine. For example, the N-terminus or C-terminus amino acid of the targeting domain can be modified to include a lysine residue. In some embodiments, the targeting domain is modified at the N- or C-terminal amino acid to include a natural amino acid with an electrophilic side chain, such as, for example, Asp and Glu. In some embodiments, the internal amino acid of the targeting domain is replaced with a natural amino acid with a nucleophilic side chain, as previously described herein. In exemplary embodiments, the internal amino acid of the targeting domain that is replaced is selected from the group consisting of lysine, ornithine, serine, cysteine ​​and homocysteine. For example, the internal amino acid of the targeting domain can be replaced with a lysine residue. In some embodiments, the internal amino acid of the targeting domain is replaced with a natural amino acid with an electrophilic side chain, such as, for example, Asp and Glu.

[0226] In some embodiments, the payload comprises a reactive group that can be conjugated directly to the targeting domain or to L. In some embodiments, the payload comprises a nucleophilic reactive group (e.g., amine, thiol, hydroxyl) that can be conjugated to an electrophilic reactive group on the targeting domain or L. In some embodiments, the payload comprises an electrophilic reactive group (e.g., carboxyl group, activated form of carboxyl group, compound with leaving group) that can be conjugated to a nucleophilic reactive group on the targeting domain or L. In some embodiments, the payload is chemically modified to comprise either a nucleophilic reactive group that can be conjugated to an electrophilic reactive group on the targeting domain or to L. In some embodiments, the payload is chemically modified to comprise an electrophilic reactive group that can be conjugated to a nucleophilic reactive group on the targeting domain or to L.

[0227] In some embodiments, conjugation can be achieved through the use of organosilanes, such as aminosilanes treated with glutaraldehyde, carbonyldiimidazole (CDI) activation of silanol groups, or dendrimers. Various dendrimers are known in the art, including poly(amidoamine) (PAMAM) dendrimers synthesized by a divergent method starting with ammonia or ethylenediamine initiator core reagents, a subclass of PAMAM dendrimers based on a tris-aminoethylene-imine core, radial layered poly(amidoamine-organosilicon) dendrimers (PAMAMOS), which are inverted unimolecular micelles consisting of a hydrophilic nucleophilic polyamidoamine (PAMAM) interior and a hydrophobic organosilicon (OS) exterior, and dendrimer-based ... These include poly(propyleneimine) (PPI) dendrimers, which are generally polyalkylamines with primary amines as terminal groups, while the interior of the dendrimer consists of numerous tertiary tris-propyleneamines, poly(propyleneimine) (POPAM) dendrimers, diaminobutane (DAB) dendrimers, amphiphilic dendrimers, micellar dendrimers which are unimolecular micelles of water-soluble hyperbranched polyphenylenes, polylysine dendrimers, and dendrimers based on poly-benzyl ether hyperbranched backbones.

[0228] In some embodiments, the conjugation can be carried out by olefin metathesis. In some embodiments, the payload and the targeting domain, the payload and L, or the targeting domain and L both contain alkene or alkyne moieties that can undergo metathesis. In some embodiments, a suitable catalyst (e.g., copper, ruthenium) is used to accelerate the metathesis reaction. Suitable methods for carrying out olefin metathesis reactions are described in the art. See, for example, Schafmeister et al., J.Am.Chem.Soc.122:5891-5892(2000), Walensky et al., Science 305:1466-1470(2004), and Blackwell et al., Angew,Chem.,Int.Ed.37:3281-3284(1998).

[0229] In some embodiments, conjugation can be performed by click chemistry, a "click reaction" that is broad in scope, easy to perform, uses only readily available reagents, and is insensitive to oxygen and water. In some embodiments, the click reaction is a cycloaddition reaction between an alkynyl group and an azide group to form a triazolyl group. In some embodiments, the click reaction uses a copper or ruthenium catalyst. Suitable methods for carrying out the click reaction 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), Tomoe 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), See Zhang et al., J. Am. Chem. Soc. 127:15998 (2005); Waser et al., J. Am. Chem. Soc. 127:8294 (2005).

[0230] Indirect conjugation via high affinity specific binding partners, such as streptavidin / biotin, or avidin / biotin, or lectin / carbohydrate, is also contemplated.

[0231] Reactive residues for conjugation In some embodiments, the targeting domain and / or payload are functionalized to include nucleophilic or electrophilic reactive groups using an organic derivatizing agent. The derivatizing agent can react with selected side chains or N- or C-terminal residues of targeted amino acids on the targeting domain and functional groups on the payload. Reactive groups on the targeting domain and / or payload include, for example, aldehyde, amino, ester, thiol, a-haloacetyl, maleimide or hydrazino groups. Derivatizing agents include, for example, maleimidobenzoylsulfosuccinimide ester (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride or other agents known in the art. Alternatively, the targeting domain and / or payload can be indirectly linked to each other through an intermediate carrier such as a polysaccharide or polypeptide carrier. An example of a polysaccharide carrier includes aminodextran. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and mixed polymers of these amino acids with others, such as serine, which impart desirable solubility properties to the resulting loaded carrier.

[0232] Cysteinyl residues most commonly are reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetylphosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0233] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5 to 7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful, and the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

[0234] 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 α-amino-containing residues include transaminase-catalyzed reactions with methylpicolinimide, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, imidoesters such as 2,4-pentanedione, and glyoxylate.

[0235] 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 carried out under alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents can react with lysine groups as well as the arginine ε-amino group.

[0236] The specific modification of tyrosyl residues can be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively.

[0237] Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (RN=C=N-R'), where R and R' are different alkyl groups such as l-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.

[0238] Other modifications include hydroxylation of proline or lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino group of the lysine, arginine, or histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), deamidation of asparagine or glutamine, acetylation of the N-terminal amine, and / or amidation or esterification of the C-terminal carboxylic acid group.

[0239] Another type of covalent modification involves chemically or enzymatically coupling glycosides to peptides. The sugar(s) can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine or hydroxyproline, (e) aromatic residues such as those of tyrosine or tryptophan, or (f) the amide group of glutamine. These methods are described in WO1987 / 05330, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0240] In some embodiments, L is a bond. In these embodiments, the targeting domain and payload are conjugated together by reacting a nucleophilic reactive moiety on the targeting domain with an electrophilic reactive moiety or payload. In alternative embodiments, the targeting domain and payload are conjugated together by reacting an electrophilic reactive moiety on the targeting domain with a nucleophilic moiety on the payload. In an exemplary embodiment, L is an amide bond that forms upon reaction of an amine on the targeting domain (e.g., the ε-amine of a lysine residue) with a carboxyl group on M. In alternative embodiments, the targeting domain and / or payload are derivatized with a derivatizing agent prior to conjugation.

[0241] In some embodiments, L is a linking group. In some embodiments, L is a bifunctional linker and contains only two reactive groups prior to conjugation to the targeting domain and payload. In embodiments where both the targeting domain and the payload have electrophilic reactive groups, L contains two of the same or two different nucleophilic groups (e.g., amine, hydroxyl, thiol) prior to conjugation to the targeting domain and payload. In embodiments where both the targeting domain and the payload have nucleophilic reactive groups, L contains two of the same or two different electrophilic groups (e.g., carboxyl groups, activated forms of carboxyl groups, compounds with leaving groups) prior to conjugation of the payload to the targeting domain. In embodiments where one of the targeting domain or the payload has a nucleophilic reactive group and the other of the targeting domain or the payload has an electrophilic reactive group, L contains one nucleophilic reactive group and one electrophilic group prior to conjugation to the targeting domain and payload.

[0242] L can be any molecule having at least two reactive groups (prior to conjugation to the targeting domain and payload) that can react with the targeting domain and payload, respectively. In some embodiments, L has only two reactive groups and is bifunctional. L (prior to conjugation to the peptide) can be represented by formula VI:ALB, where A and B are independently nucleophilic or electrophilic reactive groups. In some embodiments, A and B are either both nucleophilic groups or both electrophilic groups. In some embodiments, one of A or B is a nucleophilic group and the other of A or B is an electrophilic group.

[0243] In some embodiments, A and B may contain alkene and / or alkyne functional groups suitable for olefin metathesis reactions. In some embodiments, A and B contain moieties suitable for click chemistry (e.g., alkenes, alkynes, nitriles, azides). Other non-limiting examples of reactive groups (A and B) include pyridyldithiols, aryl azides, diazirines, carbodiimides, and hydrazides.

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

[0245] In some embodiments, the hydrophobic linking group comprises either a maleimide group or an iodoacetyl group, and either a carboxylic acid or an activated carboxylic acid (e.g., NHS ester) as the reactive group. In these embodiments, the maleimide or iodoacetyl group can be coupled to a thiol moiety on the targeting domain or payload, and the carboxylic acid or activated carboxylic acid can be coupled to an amine on the targeting domain or payload with or without the use of a coupling reagent. Any coupling agent known to those of skill in the art can be used to couple the carboxylic acid to a free amine, including, for example, DCC, DIC, HATU, HBTU, TBTU, and other activating agents described herein. In certain embodiments, the hydrophilic linking group comprises an aliphatic chain of 2-100 methylene groups, and A and B are carboxyl groups or derivatives thereof (e.g., succinic acid). In other particular embodiments, L is iodoacetic acid.

[0246] In some examples, prior to conjugation of the composition to the peptide, the hydrophobic linking group comprises at least two reactive groups (A and B), as described herein and shown below: A-(hydrophobic linking group)-B. In certain embodiments, the linking group is polyethylene glycol (PEG). In certain embodiments, the PEG 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.

[0247] In some embodiments, the hydrophobic linking group comprises either a maleimide group or an iodoacetyl group and either a carboxylic acid or an activated carboxylic acid (e.g., NHS ester) as the reactive group. In these embodiments, the maleimide or iodoacetyl group can be coupled to a thiol moiety on the targeting domain or payload, and the carboxylic acid or activated carboxylic acid can be coupled to an amine on the targeting domain or payload with or without the use of a coupling reagent. Any suitable coupling agent known to those of skill in the art can be used to couple the carboxylic acid to a free amine, including, for example, DCC, DIC, HATU, HBTU, TBTU, and other activating agents described herein. In some embodiments, the linking group is maleimide-polymer(0.1-2.5 kDa)-COOH, iodoacetyl-polymer(0.1-2.5 kDa)-COOH, maleimide-polymer(0.1-2.5 kDa)-NHS, or iodoacetyl-polymer(0.1-2.5 kDa)-NHS.

[0248] 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, maleimide, disulfide, amide, ester, thioester, alkene, cycloalkene, alkyne, triazole, carbamate, carbonate, cathepsin B-cleavable, and hydrazone.

[0249] In some embodiments, L comprises a chain of atoms from 1 to about 60, or from 1 to 30 atoms or more, from 2 to 5 atoms, from 2 to 10 atoms, from 5 to 10 atoms, or from 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, and the chain atoms and linkers are selected according to their expected solubility (hydrophilicity), in some instances 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 to hydrolysis conditions found in a target tissue or organ or cell. In some embodiments, the length of L is long enough to reduce the possibility of steric hindrance.

[0250] In some embodiments, L is stable in a biological fluid, such as blood or a blood fraction. In some embodiments, L is stable in serum for at least 5 minutes, e.g., less than 25%, 20%, 15%, 10%, or 5% of the conjugate is cleaved when incubated in serum for 5 minutes. In other embodiments, L is stable in serum for at least 10, or 20, or 25, or 30, or 60, or 90, or 120 minutes, or 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, or 24 hours. In these embodiments, L does not contain a functional group capable of undergoing hydrolysis in vivo. In some exemplary embodiments, L is stable in serum for at least about 72 hours. Non-limiting examples of functional groups that cannot undergo significant hydrolysis in vivo include amides, ethers, and thioethers.

[0251] In some embodiments, L is hydrolyzable in vivo. In these embodiments, L comprises a functional group that can undergo hydrolysis in vivo. Non-limiting examples of functional groups that cannot undergo hydrolysis in vivo include esters, anhydrides, and thioesters.

[0252] In some exemplary embodiments, L is labile and undergoes substantial hydrolysis within 3 hours and complete hydrolysis within 6 hours in plasma at 37° C. In some exemplary embodiments, L is not labile.

[0253] In some embodiments, L is metastable in vivo. In these embodiments, L comprises a functional group that can be chemically or enzymatically cleaved in-vivo, optionally over a period of time (e.g., an acid-labile, reduction-labile, or enzyme-labile functional group). In these embodiments, L can comprise, for example, a hydrazone moiety, a disulfide moiety, or a cathepsin-cleavable moiety. When L is metastable, without intending to be bound by any particular theory, the targeting domain-LM conjugate is stable in an extracellular environment, for example, stable in serum over the period of time described above, but is unstable in an intracellular environment or conditions that mimic the intracellular environment, such that it is cleaved upon entering the cell. In some embodiments, when L is metastable, L is stable in serum for at least about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, or 48 hours, e.g., at least about 48, 54, 60, 66, or 72 hours, or for about 24-48, 48-72, 24-60, 36-48, 36-72, or 48-72 hours.

[0254] In some examples, a suitable polymer backbone has the formula X-polymer LY, where the polymer is poly(ethylene glycol), X is a functional group that does not react with azide groups, and Y is a suitable leaving group. Examples of suitable functional groups include, but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl, amine, aminooxy, protected amine, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine, and vinylpyridine, and ketone. Examples of suitable leaving groups include, but are not limited to, chloride, bromide, iodide, mesylate, tresylate, and tosylate.

[0255] Linkers can have a wide range of molecular weights or lengths. Larger or smaller molecular weight linkers can be used to provide a desired spatial relationship or conformation, if any, between the targeting domain and the linked entity, or between the linked entity and its binding partner. Linkers with longer or shorter molecular lengths can also be used to provide a desired space or flexibility between the targeting domain and the linked entity, or between the linked entity and its binding partner.

[0256] In some embodiments, the linker comprises a water-soluble bifunctional linker having a dumbbell structure comprising a) an azide, alkyne, hydrazine, hydrazide, hydroxylamine, or carbonyl-containing moiety on at least a first end of the polymer backbone, and b) at least a second functional group on a second end of the polymer backbone. The second functional group can be the same as or different from the first functional group. The second functional group, in some embodiments, does not react with the first functional group. In some embodiments, the water-soluble compound comprises at least one arm of a branched molecular structure. For example, the branched molecular structure can be dendritic.

[0257] In an exemplary embodiment, the polymer is linked to the targeting domain or modified targeting domain via a linker. For example, the linker can include one or two amino acids, one end of which is attached to the polymer, such as an albumin binding moiety, and the other end of which is attached to any available position on the polypeptide backbone. Further exemplary linkers include hydrophilic linkers, such as chemical moieties that contain at least five non-hydrogen atoms, 30-50% of which are either N or O.

[0258] Optionally, multiple targeting domains or modified targeting domain molecules may be joined by a linker polypeptide, optionally 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12 amino acids in length, and longer, and optionally wherein the N-terminus of one targeting domain 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 domain.

[0259] As used herein, the terms "electrophilic group", "electrophile" and the like refer to an atom or group of atoms that can accept a pair of electrons to form a covalent bond. As used herein, "electrophilic group" includes, but is not limited to, halides, carbonyl and epoxide-containing compounds. Common electrophiles include halides such as thiophosgene, glycerin dichlorohydrin, phthaloyl chloride, succinyl chloride, chloroacetyl chloride, chlorosuccinyl chloride, ketones such as chloroacetone, bromoacetone, aldehydes such as glyoxal, isocyanates such as hexamethylene diisocyanate, tolylene diisocyanate, meta-xylylene diisocyanate, cyclohexylmethane-4,4-diisocyanate, and derivatives of these compounds.

[0260] As used herein, the terms "nucleophilic group", "nucleophile" and the like refer to an atom or group of atoms having a pair of electrons capable of forming a covalent bond. Groups of this type can be ionizable groups that react as anionic groups. As used herein, "nucleophilic groups" include, but are not limited to, hydroxyl, primary amine, secondary amine, tertiary amine, and thiol.

[0261] In general, carbon electrophiles are susceptible to attack by complementary nucleophiles, including carbon nucleophiles, where the attacking nucleophile donates an electron pair to the carbon electrophile to form a new bond between the nucleophile and the carbon electrophile.

[0262] Non-limiting examples of carbon nucleophiles include, but are not limited to, alkyl, alkenyl, aryl and alkynyl Grignards, organolithium, organozinc, alkyl-, alkenyl, aryl- and alkynyl-tin reagents (organostannanes), alkyl-, alkenyl-, aryl- and alkynyl-borane reagents (organoboranes and organoboronates), which have the advantage of being kinetically stable in water or polar organic solvents. Other non-limiting examples of carbon nucleophiles include phosphorus ylides, enol and enolate reagents, which 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, give rise to new carbon-carbon bonds between the carbon nucleophile and the carbon electrophile.

[0263] 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, etc. These non-carbon nucleophiles, when used in conjunction with carbon electrophiles, typically produce heteroatom linkages (CXC), where X is a heteroatom, including, but not limited to, oxygen, sulfur, or nitrogen.

[0264] In some cases, the polymers used herein terminate at 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. Exemplary reactive groups can include reactive groups that are commonly used to react with functional groups found on the 20 common amino acids (including, but not limited to, maleimide groups, activated carbonates (including, but not limited to, p-nitrophenyl esters), activated esters (including, but not limited to, N-hydroxysuccinimide, p-nitrophenyl esters) and aldehydes), as well as functional groups that are inert to the 20 common amino acids but react specifically with complementary functional groups (including, but not limited to, azide groups, alkyne groups). Note that the other end of the polymer, designated Y in the above formula, is directly or indirectly attached to the targeting domain via an amino acid. For example, Y is an amide, carbamate or urea linkage to an amine group of the polypeptide (including but not limited to the epsilon amine or N-terminus of lysine). Alternatively, Y is a maleimide linkage to a thiol group (including but not limited to the thiol group of cysteine). Alternatively, an alkyne group on the polymer can react with an azide group present in the targeting domain to form a similar product. In some embodiments, a strong nucleophile (including but not limited to hydrazine, hydrazide, hydroxylamine, semicarbazide) can react with an aldehyde or ketone group present in the targeting domain to form a hydrazone, oxime or semicarbazone, if applicable, which in some cases can be further reduced by treatment with an appropriate reducing agent. Alternatively, a strong nucleophile can be incorporated into the targeting domain via an amino acid and used to preferentially react with a ketone or aldehyde group present in the water-soluble polymer.

[0265] Any molecular weight of the polymer can be used as practically desired, including but not limited to about 0.1 Daltons (Da) to 2,500 Da or more. The molecular weight of the polymer can be in a wide range, including but not limited to about 100 Da to about 5,000 Da or more. In some examples, the polymer is 50-5000 Da, 50-3000 Da, 50-2500 Da, 100-2500 Da, 250-2500 Da, 250-5000 Da, or 500-5000 Da. Branched polymers include, but are not limited to, polymer molecules in which each chain has a molecular weight in the range of 0.1-5 kDa, 0.1-4 kDa, 0.1-3 kDa, 0.1-2.5 kDa, 0.1-1.5 kDa.

[0266] The polymers may include azide and acetylene-containing polymer derivatives, comprising a water-soluble polymer backbone having an average molecular weight of about 800 Da to about 100,000 Da. The polymer backbone of the water-soluble polymer may 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 intended to encompass and include all such molecules, and the use of the term PEG or poly(ethylene glycol) is intended to encompass and include all such molecules. The term PEG includes, but is not limited to, poly(ethylene glycol) in any of its forms, including bifunctional PEG, multi-arm PEG, derivatized PEG, forked PEG, branched PEG, pendant PEG (i.e., PEG or related polymers having one or more functional groups pendant to the polymer backbone), or PEG having degradable linkages therein.

[0267] In addition to these forms of polymers, polymers can also be prepared with weak or degradable linkages in the backbone. For example, polymers can be prepared with ester bonds in the polymer backbone that undergo hydrolysis. This hydrolysis results in the cleavage of the polymer into lower molecular weight fragments, as shown below:-polymerCO2-polymer+HO-apolymer-CO2H+HO-polymer-.

[0268] The linker may include a polymer, such as one that includes a water-soluble backbone. In some embodiments, the polymer backbone, which is water-soluble, includes from 2 to about 300 termini. Examples of suitable polymers include, but are not limited to, other poly(alkylene glycols), such as poly(propylene glycol) ("PPG"), copolymers thereof (including but not limited to copolymers of ethylene glycol and propylene glycol), terpolymers thereof, mixtures thereof, and the like. The molecular weight of each chain of the polymer backbone can vary, but typically ranges from about 800 Da to about 100,000 Da, often from about 6,000 Da to about 80,000 Da. The molecular weight of each chain of the polymer backbone can be from about 100 Da to about 100,000 Da, including 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, 30,000 Da, 40,000 Da, 45 ... Examples of suitable molecular weights include, but are not limited to, 000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is about 100 Da to about 50,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is about 100 Da to about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is from about 1000 Da to about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is from about 50,000 Da to about 40,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is from about 10,000 Da to about 40,000 Da.

[0269] Suitable physiologically cleavable bonds include, but are not limited to, esters, carbonate esters, carbamates, sulfates, phosphates, acyloxyalkyl ethers, acetals and ketals. Such conjugates must have physiologically cleavable bonds that are stable during storage and administration. For example, the targeting domain or modified targeting domain linked to the polymer must maintain its integrity during preparation of the final pharmaceutical composition, during dissolution in a suitable delivery vehicle (if used), and during administration regardless of route.

[0270] The present invention also includes phosphate-based linkers with tunable stability for intracellular delivery of drug conjugates as disclosed in US2017 / 0182181, which is incorporated herein by reference. Phosphate-based linkers include a monophosphate, diphosphate, triphosphate, or tetraphosphate group (phosphate group) covalently linked to the distal end of a linker arm that includes a tuning element, optionally a spacer element, and a reactive functional group in a distal to proximal direction. The phosphate group of the phosphate-based linker can be conjugated to a payload, and the reactive functional group can be conjugated to a cell-specific targeting ligand, such as an antibody. The general structure of a phosphate-based linker is as follows: The phosphate-based linker conjugated to the payload has the following general structure: It has the general structure Payload-Phosphate group-Tuning element-Optional spacer element-Functional reactive group and when conjugated to a targeting ligand, it has the general structure Payload-Phosphate group-Tuning element-Optional spacer element-Targeting ligand. These phosphate-based linkers have differential and tunable stability in blood and in intracellular environments (e.g., lysosomal compartments). The rate at which the phosphate group is cleaved in the intracellular environment to release the payload in its native or active form can be influenced by the structure of the tuning element, with further effects mediated by the substitution of the phosphate group, as well as whether the phosphate group is a monophosphate, diphosphate, triphosphate, or tetraphosphate. Additionally, these phosphate-based linkers provide the ability to construct conjugates, such as antibody-drug conjugates, that have a reduced tendency to form aggregates as compared to conjugates in which the same payload is conjugated to an antibody or targeting ligand using linkers that are not phosphate-based as disclosed herein.

[0271] In some examples, the targeting domain is linked to the payload via a water-soluble polymer via the methods described herein. In some embodiments, the method includes contacting an isolated targeting domain comprising a reactive amino acid side chain with a linker. In some examples, the conjugate is synthesized by reacting a functional group present on the targeting domain with a reactive group present on the linker. In some examples, the conjugate is synthesized by reacting a functional group present on the linker with a reactive group present on the payload. In some examples, a payload-linker moiety is conjugated to the targeting domain. In some examples, a targeting domain-linker moiety is conjugated to the payload. In some embodiments, the targeting domain is linked to a linker comprising a water-soluble polymer.

[0272] In other embodiments, the targeting domain is conjugated to the payload by a linker. In some examples, the linker comprises a polymer. In some embodiments, the targeting domain is directly or indirectly conjugated to a linker, polymer, or biologically active molecule. In some embodiments, the linker is a cleavable or non-cleavable linker.

[0273] In some embodiments, the linker is between 0.1 kDa and 5 kDa. In other embodiments, the linker is between 0.1 kDa and 2.5 kDa. In other embodiments, the linker or polymer is linear, branched, polymeric, or dendritic. In another embodiment, the linker or polymer is a bifunctional or multifunctional linker or a bifunctional or multifunctional polymer.

[0274] In other embodiments, the 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 of 0.1 kDa to 10 kDa. In other embodiments, the PEG has a molecular weight of 0.1 kDa to 5 kDa. In other embodiments, the PEG has a molecular weight of 0.1 kDa to 4 kDa. In other embodiments, the PEG has a molecular weight of 0.1 kDa to 3 kDa. In other embodiments, the PEG has a molecular weight of 0.1 kDa to 2 kDa. In other embodiments, the PEG has a molecular weight of 0.1 kDa to 2.5 kDa. In some embodiments, the poly(ethylene glycol) has a molecular weight of about 0.1 kDa to 10 kDa. In some embodiments, the poly(ethylene glycol) has a molecular weight of 0.1 kDa to 50 kDa. In some embodiments, the poly(ethylene glycol) has a molecular weight of 0.1 kDa to 2.5 kDa, or 0.2 to 2.2 kDa, or 0.5 kDa to 2 kDa. For example, the molecular weight of the poly(ethylene glycol) polymer is, in some instances, about 0.5 kDa, or about 1 kDa, or about 2 kDa, or about 2.5 kDa. For example, the molecular weight of the poly(ethylene glycol) polymer is, in some instances, 0.1 kDa, or 0.5 kDa, or 1 kDa to 2.5 kDa. In some embodiments, the poly(ethylene glycol) molecule is a branched PEG. In some embodiments, the poly(ethylene glycol) molecule is a branched 1K PEG. In some embodiments, the poly(ethylene glycol) molecule is a branched 2.5K PEG. In some embodiments, the poly(ethylene glycol) molecule is a branched 5K PEG. In some embodiments, the poly(ethylene glycol) molecule is a linear PEG. In some embodiments, the poly(ethylene glycol) molecule is a linear 2.5K 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 2K PEG. In some embodiments, the poly(ethylene glycol) molecule is a linear 0.5K PEG.In some embodiments, the molecular weight of the poly(ethylene glycol) polymer is an average molecular weight. In certain embodiments, the average molecular weight is a number average molecular weight (Mn). The average molecular weight can be determined or measured using GPC or SEC, SDS / PAGE analysis, RP-HPLC, mass spectrometry, or capillary electrophoresis.

[0275] Treatment The conjugates described herein may be used to treat diseases and / or disorders. In some examples, the disease includes a proliferative disease. In some examples, the proliferative disease includes cancer. In some examples, the cancer includes one or more tumors. In some examples, the cancer includes a solid or liquid tumor. In some examples, the conjugates are administered to kill or inhibit the growth of rapidly dividing cells, such as tumor cells. In some examples, a method of treating a proliferative disease or disorder in a subject in need thereof includes administering to the subject a therapeutically effective amount of a conjugate described herein. In some embodiments, the proliferative disease or disorder is cancer. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the solid tumor cancer is bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer. In some examples, the disease includes PCa (prostate cancer), CRPCa (castration-resistant prostate cancer), solid tumor (neovascular), NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), ESCC (esophageal cancer), GC (gastric cancer), CRC (colorectal cancer), SCLC (small cell lung cancer), MPM (mesothelioma), PDAC (pancreatic ductal adenocarcinoma), ALL (acute lymphoblastic leukemia), AML (acute myeloid leukemia), MDS (myelodysplastic syndrome), MSI-high tumor, melanoma, DLBCL (diffuse large B-cell lymphoma), endometrial cancer, cervical cancer, bladder cancer, BrCa (breast cancer), TNBC (triple-negative breast cancer), NE-PCa (neuroendocrine prostate cancer), GBM (glioblastoma), and RCC (renal cell carcinoma).

[0276] In some instances, the tumor cells targeted herein overexpress one or more targets. In some instances, the targets include surface markers or receptors. In some examples, the targets are PSMA, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD166, CLL-1, ADAM9, cKIT, CD36, CD73, I TGaVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, ROR1 , TROP-2, NECTIN4, cMET, CD19, CD22, CD30, CD33, CD123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM.In some examples, the targets are PSMA, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD166, CLL-1, ADAM9, cKIT, CD36, CD73, I TGaVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, ROR1 , TROP-2, NECTIN4, cMET, CD19, CD22, CD30, CD33, CD123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, p-cadherin, CEACAM6, CD47, and EpCAM. In some examples, the conjugate delivers the payload to the tumor cell where the conjugate binds to one or more targets on the tumor cell and the payload kills, inhibits, or slows the growth of the tumor cell.

[0277] In some examples, the conjugates herein are administered to image a particular cell or group of cells. In some cases, the cell or group of cells is a tumor cell or is present in the tumor microenvironment. In some examples, the conjugate binds to one or more targets on a tumor cell, and when the payload is an imaging agent (such as a visual dye or a radioactive label), the conjugate delivers the payload to the tumor cell, and the tumor cell is imaged as a result of the attachment of the conjugate to the tumor cell.

[0278] The pharmaceutical composition is administered in a manner appropriate for the disease to be treated (or prevented). The appropriate dose and the appropriate duration and frequency of administration are determined by factors such as the patient's disease, the type and severity of the patient's disease, the specific form of the active ingredient, and the method of administration. In general, the appropriate dose and treatment regimen provides the composition in an amount sufficient to provide therapeutic and / or prophylactic benefits (e.g., improved clinical outcomes), or a reduction in the severity of symptoms. The optimal dose is generally determined using experimental models and / or clinical trials. The optimal dose depends on the patient's body weight, body weight, or blood volume.

[0279] In one embodiment, the injectable pharmaceutical compositions described herein are used in the preparation of a medicament for the treatment of a disease or condition in a mammal that would benefit from administration of any one of the injectable pharmaceutical compositions of the disclosed conjugates. A method for treating any of the diseases or conditions described herein in a mammal in need of such treatment comprises administering to said mammal a therapeutically effective amount of at least one compound described herein, or a pharma- ceutically acceptable salt, active metabolite, prodrug, or pharma-ceutically acceptable solvate thereof.

[0280] In certain embodiments, the compositions containing the compounds described herein are administered for preventive and / or therapeutic treatment. In certain therapeutic applications, the compositions are administered to patients already suffering from a disease or illness in an amount sufficient to cure or at least partially prevent at least one of the symptoms of the disease or illness. The amount effective for this use depends on the severity and course of the disease or illness, previous treatments, the patient's health status, weight, and response to drugs, and the judgment of the treating physician. The therapeutically effective amount is optionally determined by methods including, but not limited to, dose escalation and / or dose-finding clinical trials.

[0281] In prophylactic applications, compositions containing the compounds described herein are administered to patients susceptible to or at risk of a particular disease, disorder, or condition. Such an amount is defined as a "prophylactically effective amount or dose." In this application, the exact amount also depends on the patient's health, weight, etc. When used in patients, the effective amount for this application will depend on the severity and course of the disease, disorder, or condition, previous treatments, the patient's health and response to the drugs, and the judgment of the treating physician. In one embodiment, prophylactic treatment involves administering a pharmaceutical composition containing a compound described herein or a pharma- ceutically acceptable salt thereof to a mammal that has previously experienced at least one symptom of the disease being treated and is now in remission, to prevent the recurrence of the disease or condition.

[0282] In certain embodiments, administration of the compound is chronic, i.e., administered for an extended period of time, including for the life of the patient, to ameliorate or otherwise control or limit the symptoms of a disease or disorder in a patient.

[0283] Method of production The conjugates described herein can be synthesized using in-vivo or in-vitro methods, or a combination of methods. In some cases, the methods are in-vivo methods. In some cases, the methods are in-vitro methods. In some examples, a targeting domain comprising an unnatural amino acid is synthesized in-vivo and the payload is attached using in-vitro chemical methods.

[0284] In some cases, the method is an ex vivo method. In some cases, the conjugates described herein that include natural or non-natural amino acid mutations are recombinantly produced or chemically synthesized. In some cases, the targeting domains described herein are recombinantly produced, for example, by a host cell system or in a cell-free system.

[0285] In general, methods for producing target polypeptides containing non-standard amino acids are known. In some cases, the amino-acyl-tRNA synthetase / tRNA pair cognate to the unnatural amino acid is orthogonal to the cellular components of the cell in which it is used. The orthogonality (and therefore compatibility) of the exogenous amino-acyl-tRNA synthetase / tRNA pair depends on the type of host organism. There are four main orthogonal aminoacyl-tRNA synthetases: Methanococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) / tRNA CUA vs. Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS) / tRNA CUA The pair, Escherichia coli leucyl-tRNA synthetase (EcLeuRS) / tRNAcuA pair, and the pair pyrrolysyl-tRNA synthetase (PylRS) / tRNAcuA (tRNA pyl ) pair was developed for genetic code expansion. The PylRS / tRNAcuA pair is orthogonal in bacteria, eukaryotic cells, and animals (see, e.g., Chin, Jason W. "Expanding and reprogramming the genetic code of cells and animals", Annual review of biochemistry 83(2014):379-408).

[0286] In some cases, the unnatural amino acids (UAAs) provided herein are synthesized by the enzyme pyrrolysyl-tRNA synthetase (tRNA pyl ) are incorporated onto a transfer RNA molecule (tRNA) so that it can be used for translation. However, the attachment of the unnatural amino acid to the tRNA may not always be accomplished by a natural aminoacyl-tRNA synthetase. Thus, the engineered tRNA pyl Engineered tRNAs prepared and selected from mutant libraries pylEngineered aminoacyl-tRNA synthetases such as can be useful to attach a desired UAA to a tRNA so that the desired UAA can be incorporated into mutagenesis.

[0287] In some cases, the unnatural amino acids (UAAs) provided herein are synthesized by the enzyme pyrrolysyl-tRNA synthetase (tRNA pyl ) Unnatural amino acids (UAAs) are incorporated onto transfer RNA molecules (tRNAs) so that they can be used for translation. However, the attachment of an unnatural amino acid to a tRNA cannot always be achieved by a naturally occurring aminoacyl-tRNA synthetase. pyl Engineered tRNAs prepared and selected from mutant libraries pyl Engineered aminoacyl-tRNA synthetases such as can be useful for attaching a desired UAA to a tRNA so that the desired UAA can be incorporated into mutagenesis.

[0288] In some embodiments, a UAA provided herein (e.g., a UAA of formula (IA)) can be attached to an engineered mutant tRNA to which such a UAA can be attached. pyl The mutants can be used to bind to tRNA. In some embodiments, the mutant tRNA pyl The variant introduces a single amino acid mutation (e.g., incorporates a UAA). pyl The mutants introduce multiple amino acid mutations. pyl A library of mutants is prepared and screened by one skilled in the art to identify tRNAs suitable for attachment of the desired UAA. pyl In some embodiments, the tRNA used to introduce a UAA into the conjugate provided herein is pyl The mutants include tRNAs described in US8,735,093, US9,133,449, and WO2020206341. pylvariants, each of which is incorporated herein by reference.

[0289] In various embodiments, mutant pyrrolysyl-tRNA synthetases can be used. The mutant pyrrolysyl-tRNA synthetase can be derived from or mutants of pyrrolysyl-tRNA synthetases from Methanosarcina barkeri, Methanosarcina mazei, Methanosarcina alvus, or Methanosarcina jannaschii, or chimeras thereof.

[0290] In some embodiments, the mutant pyrrolysyl-tRNA synthetase provided herein comprises at least five amino acid residue substitutions in the substrate binding site of the mutant pyrrolysyl-tRNA synthetase. In some embodiments, the mutant pyrrolysyl-tRNA synthetase comprises at least five amino acid residue substitutions in the amino acid sequence of SEQ ID NO: 90. In some embodiments, the substrate binding site comprises the following residues: alanine at position 302, leucine at position 305, tyrosine at position 306, leucine at position 309, isoleucine at position 322, asparagine at position 346, cysteine ​​at position 348, tyrosine at position 384, valine at position 401, and tryptophan at position 417 shown in the amino acid sequence of SEQ ID NO: 90. In some embodiments, the at least five amino acid residue substitutions are selected from an alanine at position 302, an asparagine at position 346, a cysteine ​​at position 348, a tyrosine at position 384, and a tryptophan at position 417, as set forth in the amino acid sequence of SEQ ID NO: 90. In some embodiments, the at least five amino acid residue substitutions are an isoleucine at alanine at position 302, a threonine at asparagine at position 346, an isoleucine at cysteine ​​at position 348, a leucine at tyrosine at position 384, and a lysine at tryptophan at position 417, as set forth in the amino acid sequence of SEQ ID NO: 90.

[0291] In some embodiments, the mutant pyrrolysyl-tRNA synthetase provided herein has an amino acid sequence of SEQ ID NO: 84. In some embodiments, the mutant pyrrolysyl-tRNA synthetase comprises an amino acid sequence of SEQ ID NO: 84. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 84. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 80% identity to SEQ ID NO: 84. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 85% identity to SEQ ID NO: 84. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 90% identity to SEQ ID NO: 84. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 91% identity to SEQ ID NO: 84. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 92% identity to SEQ ID NO: 84. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 93% identity to SEQ ID NO: 84. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 94% identity to SEQ ID NO: 84. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 95% identity to SEQ ID NO: 84. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 96% identity to SEQ ID NO: 84. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 97% identity to SEQ ID NO: 84. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 98% identity to SEQ ID NO: 84.In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 99% identity to SEQ ID NO:84.

[0292] In some embodiments, the mutant pyrrolysyl-tRNA synthetase provided herein has an amino acid sequence of SEQ ID NO: 87. In some embodiments, the mutant pyrrolysyl-tRNA synthetase comprises an amino acid sequence of SEQ ID NO: 87. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 87. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 80% identity to SEQ ID NO: 87. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 85% identity to SEQ ID NO: 87. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 90% identity to SEQ ID NO: 87. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 91% identity to SEQ ID NO: 87. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 92% identity to SEQ ID NO: 87. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 93% identity to SEQ ID NO: 87. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 94% identity to SEQ ID NO: 87. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 95% identity to SEQ ID NO: 87. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 96% identity to SEQ ID NO: 87. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 97% identity to SEQ ID NO: 87. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 98% identity to SEQ ID NO: 87.In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 99% identity to SEQ ID NO:87.

[0293] In some embodiments, the mutant pyrrolysyl-tRNA synthetase provided herein is encoded by a nucleic acid sequence of SEQ ID NO:85. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO:85. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:85. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 80% identical to SEQ ID NO:85. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 85% identical to SEQ ID NO:85. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 90% identical to SEQ ID NO:85. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 91% identical to SEQ ID NO:85. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 92% identical to SEQ ID NO:85. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 93% identical to SEQ ID NO:85. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 94% identical to SEQ ID NO:85. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 95% identical to SEQ ID NO:85. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 96% identical to SEQ ID NO:85. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 97% identical to SEQ ID NO:85.In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 98% identical to SEQ ID NO: 85. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 85.

[0294] In some embodiments, the mutant pyrrolysyl-tRNA synthetase provided herein is encoded by a nucleic acid sequence of SEQ ID NO:88. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO:88. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:88. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 80% identical to SEQ ID NO:88. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 85% identical to SEQ ID NO:88. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 90% identical to SEQ ID NO:88. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 91% identical to SEQ ID NO:88. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 92% identical to SEQ ID NO:88. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 93% identical to SEQ ID NO:88. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 94% identical to SEQ ID NO:88. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 95% identical to SEQ ID NO:88. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 96% identical to SEQ ID NO:88. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 97% identical to SEQ ID NO:88.In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 98% identical to SEQ ID NO: 88. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 88.

[0295] In some embodiments, the mutant pyrrolysyl-tRNA synthetase provided herein has an amino acid sequence of SEQ ID NO: 92. In some embodiments, the mutant pyrrolysyl-tRNA synthetase comprises an amino acid sequence of SEQ ID NO: 92. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 92. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 80% identity to SEQ ID NO: 92. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 85% identity to SEQ ID NO: 92. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 90% identity to SEQ ID NO: 92. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 91% identity to SEQ ID NO: 92. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 92% identity to SEQ ID NO:92. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 93% identity to SEQ ID NO:92. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 94% identity to SEQ ID NO:92. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 95% identity to SEQ ID NO:92. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 96% identity to SEQ ID NO:92. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 97% identity to SEQ ID NO:92. In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 98% identity to SEQ ID NO:92.In some embodiments, the mutant pyrrolysyl-tRNA synthetase has an amino acid sequence having at least 99% identity to SEQ ID NO:87.

[0296] In some embodiments, the mutant pyrrolysyl-tRNA synthetase provided herein is encoded by a nucleic acid sequence of SEQ ID NO:91. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO:91. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:91. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 80% identical to SEQ ID NO:91. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 85% identical to SEQ ID NO:91. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 90% identical to SEQ ID NO:91. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 91% identical to SEQ ID NO: 91. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 92% identical to SEQ ID NO: 91. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 93% identical to SEQ ID NO: 91. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 94% identical to SEQ ID NO:91. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 95% identical to SEQ ID NO:91. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 96% identical to SEQ ID NO:91. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 97% identical to SEQ ID NO:91. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 98% identical to SEQ ID NO:91. In some embodiments, the mutant pyrrolysyl-tRNA synthetase is encoded by a nucleic acid sequence that is at least 99% identical to SEQ ID NO:91.

[0297] In some examples, sequences associated with mutant pyrrolysyl-tRNA synthetases herein are in Table 1B.

[0298] [Table 3-1]

[0299] [Table 3-2]

[0300] [Table 3-3]

[0301] [Table 3-4]

[0302] [Table 3-5]

[0303] [Table 3-6]

[0304] [Table 3-7]

[0305] [Table 3-8]

[0306] [Table 3-9]

[0307] [Table 3-10]

[0308] [Table 3-11]

[0309] In some cases, the targeting domain is recombinantly produced by a host cell system. In some cases, the host cell is a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell, or a plant cell) or a prokaryotic cell (e.g., a gram-positive or gram-negative bacteria). In some cases, the eukaryotic host cell is a mammalian host cell. In some cases, the mammalian host cell is a stable cell line, or a cell line that has the ability to integrate the genetic material of interest into its genome and express the product of that genetic material after multiple generations of cell division. In other cases, the mammalian host cell is a transient cell line, or a cell line that does not have the ability to integrate the genetic material of interest into its genome and express the product of the genetic material after multiple generations of cell division.

[0310] Exemplary mammalian host cells include 293T cell line, 293A cell line, 293FT cell line, 293F cells, 293H cells, A549 cells, MDCK cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, Expi293F cells (trademark) cells, Flp-In (trademark) T-RExTM293 cell line, Flp-In (trademark)-293 cell line, F lp-In(TM)-3T3 Cell Line, Flp-In(TM) BHK Cell Line, Flp-In(TM)-CHO Cell Line, Flp-In(TM)-CV-1 Cell Line, Flp-In(TM) The Jurkat cell line, FreeStyle(TM) 293-F cells, FreeStyle(TM) CHO-S cells, GripTite(TM) 293MSR cell line, GS-CHO cell line, Heparg(TM) T-REx™ Jurkat cell line, Per.C6 cells, T-REx™-293 cell line, T-REx™-CHO cell line, and T-REx™ HeLa cell line.

[0311] In some embodiments, the eukaryotic host cell is an insect host cell. Exemplary insect host cells include Drosophila S2 cells, Sf9 cells, Sf21 cells, and Cellular High Five™ cells.

[0312] In some embodiments, the eukaryotic host cell is a yeast host cell. Exemplary yeast host cells include Pichia pastoris yeast strains such as GS115, KM71H, SMD1168H, X-33, and Saccharomyces cerevisiae yeast strains such as INVSCl.

[0313] In some embodiments, the eukaryotic host cell is a plant host cell. In some cases, the plant cell comprises a cell derived from algae. Exemplary plant cell lines include strains from Chlamydomonas reinhardtii 137c or Synechococcus elongatus PPC 7942.

[0314] In some embodiments, the host cell is a prokaryotic host cell. Exemplary prokaryotic host cells include BL21, BL21(DE3), Mach1TM, DH10BTM, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F', INVαF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™.

[0315] In some cases, suitable nucleic acid molecules or vectors for producing the targeting domains described herein include any suitable vectors derived from eukaryotic or prokaryotic sources. Exemplary nucleic acid molecules or vectors include vectors from bacterial (e.g., E. coli), insect, yeast (e.g., Pichia pastoris), algae or mammalian sources. Examples of bacterial vectors include pACYC177, pASK75, pBAD series vectors, pBADM series vectors, pET series vectors, pETM series vectors, pGEX series vectors, pHAT2, pMal-C2, pMal-p2, pQE series vectors, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12C, pTAC-MAT-1, pFLAG CTC, and pTAC-MAT-2.

[0316] Insect vectors include, for example, pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBac M30b, pFastBac, M30c, pVL1392, pVL1393M 10, pVL1393M 11, pVL1393M 12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT2, or MAT vectors such as pPolh-MAT1 or pPolh-MAT2.

[0317] Yeast vectors include, for example, a pDEST™14 carrier, a pDEST™15 carrier, a pDEST™17 carrier, a pDEST™24 carrier, a pYES-DEST52 vector, a pBAD-DEST49Target vector, a pAO815 Pichia yeast vector, a pFLD1 Pichia pastoris vector, pGAPZA, a Pichia pastoris C vector, a Pichia pastoris pPIC3.5K vector, pPIC 6A, B and Pichia pastoris C vectors, a pPIC9K vector, pTEF1 / Zeo, a pYES2 yeast vector, a pYES2 / CT yeast vector, pYES2 / NTA, B and C yeast parents, or a pYES3 / CT yeast vector.

[0318] Algal vectors include, for example, the pChlamy-4 vector or the MCS vector.

[0319] Mammalian vectors include, for example, transient expression vectors or stable expression vectors. Exemplary mammalian transient expression vectors include p3xFLAG-CMV8, pFLAG-Myc-CMV19, pFLAG-Myc-CMV23, pFLAG-CMV2, pFLAG-CMV6a, b, c, pFLAG-CMV5.1, pFLAG-CMV 5a, b, c, p3xFLAG-CMV 7.1, pF-CMV20, p3xFLAG-Myc-CMV24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV3, or pBICCMV-4. Exemplary mammalian stable expression vectors include pFLAG-CMV3, p3xFLAG-CMV9, p3xFLAG-CMV13, pFLAG-Myc-CMV21, p3xFLAG-Myc-CMV25, pFLAG-CMV4, p3xFLAG-CMV10, p3xFLAG-CMV14, pFLAG-Myc-CMV22, p3xFLAG-Myc-CMV26, pBICEP-CMV1, or pBICEP-CMV2. In some cases, a cell-free system is used to produce the targeting domains described herein. In some cases, the cell-free system includes a mixture of cytoplasmic and / or nuclear components from a cell and is suitable for in vitro nucleic acid synthesis. In some cases, the cell-free system utilizes prokaryotic cell components. In other cases, the cell-free system utilizes eukaryotic cell components. Nucleic acid synthesis is achieved in cell-free systems based on, for example, Drosophila cells, Xenopus eggs, archaea or HeLa cells. Exemplary cell-free systems include the E. coli S30 extract system, the E. coli T7S30 system or XpressCF and XpressCF+.

[0320] The cell-free translation system includes various components such as plasmids, mRNA, DNA, tRNA, synthetases, release factors, ribosomes, chaperones, translation initiation and elongation factors, natural and / or unnatural amino acids, and / or other components for protein expression. Such components are optionally modified to improve yield, increase synthesis rate, increase fidelity of the protein product, or to incorporate unnatural amino acids. In some embodiments, the unnatural amino acid-containing targeting domains described herein are synthesized using a cell-free translation system described in US8,778,631, US2017 / 0283469, US2018 / 0051065, US2014 / 0315245, or US8,778,631. In some embodiments, the cell-free translation system includes modified release factors, or further, one or more release factors are removed from the system. In some embodiments, the cell-free translation system includes a reduced protease concentration. In some embodiments, the cell-free translation system includes a modified tRNA with a reassigned codon that codes for an unnatural amino acid. In some embodiments, a synthetase described herein for incorporating an unnatural amino acid is used in the cell-free translation system. In some embodiments, the tRNA is preloaded with the unnatural amino acid using an enzymatic or chemical process prior to adding the tRNA to the cell-free translation system. In some embodiments, the components for the cell-free translation system are obtained from engineered organisms, such as engineered bacteria, yeast, or other organisms.

[0321] In some embodiments, the targeting domain is produced in a circular configuration via an expression host system or by a cell-free system.

[0322] Using orthogonal or extended genetic codes to generate the targeting domains described herein, one or more specific codons present in the nucleic acid sequence of the targeting domain are assigned to code for an unnatural amino acid, such that through the use of an orthogonal tRNA synthetase / tRNA pair, the unnatural amino acid can be genetically incorporated into the conjugate (e.g., targeting domain). The orthogonal tRNA synthetase / tRNA pair can charge a tRNA with the unnatural amino acid, which can incorporate the unnatural amino acid into a polypeptide chain in response to a codon.

[0323] In some cases, the codon is an amber codon, an oak codon, an opal codon, or a quadruple codon. In some cases, the codon corresponds to an orthogonal tRNA used to carry an unnatural amino acid. In some cases, the codon is an amber codon. In other cases, the codon is an orthogonal codon.

[0324] In some cases, the codon is a quadruplet codon and can be decoded by the orthogonal ribosome Ribo-Q1. In some cases, the quadruplet codon is as described in Neumann et al., "Encoding multiple unnatural amino acid analysis of a quadruplet-decoding ribosome," Nature, 464(7287):441-444 (2010).

[0325] In some cases, the codons used in this disclosure are re-coded codons, e.g., rare codons replaced with synonymous or replacement codons. In some cases, the re-coded codons are as described in Napolitano et al., "Emergent rules for codon selection isolated by editing of a ray array code in Escherichia coli," PNAS, 113(38):E5588-5597 (2016). In some cases, the re-coded codons are as described in Ostrov et al., "Design, synthesis, and testing translated a 57-code gene," Science 353(6301):819.sub.822 (2016).

[0326] definition The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, g-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbons bonded to hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

[0327] The terms "non-naturally occurring amino acid" and "unnatural amino acid" refer to amino acid residues containing amino acid analogs, synthetic amino acids, and amino acid mimetics not found in nature, such as aryl amides, vinyl sulfonamides, sulfonyl fluorides, arylsulfonyl fluorides, and 4-sulfotetrafluorophenyl (STP) esters.

[0328] Amino acids may be referred to herein by either their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes.

[0329] The term "amino acid side chain" refers to a functional substituent contained on an amino acid. For example, the amino acid side chain can be the side chain of a naturally occurring amino acid. Naturally occurring amino acids are those encoded by the genetic code (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine), as well as amino acids that are later modified, such as hydroxyproline, g-carboxyglutamate, and O-phosphoserine. In embodiments, the amino acid side chain can be a non-naturally occurring amino acid side chain. In embodiments, the side chain of an amino acid is H,

[0330] [ka] In some embodiments, the unnatural amino acid side chain is:

[0331] [ka] In some embodiments, the unnatural amino acid side chain is:

[0332] [ka] It is.

[0333] "Non-natural amino acid side chain" or "unnatural amino acid side chain" or "Uaa" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, allylalanine, and a functional substituent of 2-aminoisobutyric acid. Non-natural amino acids are non-proteinogenic amino acids that are either naturally occurring or chemically synthesized. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Non-limiting examples include exo-cis-3-aminobicyclo[2.2.l]hept-5-ene-2-carboxylic acid hydrochloride, cis-2-aminocycloheptane carboxylic acid hydrochloride, cis-6-amino-3-cyclohexene-l-carboxylic acid hydrochloride, cis-2-amino-2-methylcyclohexane carboxylic acid hydrochloride, cis-2-amino-2-methylcyclopentane carboxylic acid hydrochloride, 2-(Boc aminomethyl), benzoic acid, 2-(Boc-amino)octanedioic acid, Boc-4,5-dehydro-Leu-OH (dicyclohexylammonium), Boc-4-(Fmoc-amino)-L-phenyl Alanine, Boc-P-homopyl-OH, Boc-(2-indanyl)-Gly-OH, 4-Boc-3-morpholineacetic acid, 4-Boc-3-morpholineacetic acid, Boc pentafluoro-D-phenylalanine, Boc-pentafluoro-L-phenylalanine, Boc-Phe(2-Br)-OH, Boc-Phe(4-Br)-OH, Boc-D-Phe(4-Br)-OH, Boc-D-Phe(3-Cl)-OH, Boc-Phe(4-NH2)-OH, Boc-Phe(3-NH2)-OH, Boc-Phe(3,5-F2)-OH, 2-(4-Boc-piperazino)-2-(3,4-dimethoxyphenyl)acetic acid purum, 2-(4-Boc-piperazino)-2-(2-fluorophenyl)acetic acid purum, 2-(4-Boc-piperazino)-2-(3-fluorophenyl)acetic acid purum, 2-(4-Boc-piperazino)-2-(4-fluorophenyl)acetic acid purum, 2-(4-Boc-piperazino)-2-(4-methoxyphenyl)acetic acid purum, 2-(4-Boc-piperazino)-2-phenylacetic acid purum, 2-(4-Boc-piperazino)-2-(3-pyridyl)acetic acid purum, 2-(4-Boc piperazino)-2-[4-(trifluoromethyl)phenyl]acetic acid purum, Boc-P-(2-quinolyl)-Ala-OH, NBoc-1,2,3,6-tetrahydro-2-pyridine carbo and Fmoc-N-(4-Boc-aminobutyl)-Gly-OH, Fmoc-N-(2-Boc-aminoethyl)-Gly-OH, Fmoc-N-(2,4-dimethoxybenzyl)-Gly-OH, Fmoc-(2-indanyl)-Gly-OH, Fmoc-pentafluoro-L-phenylalanine, Fmoc-Pen(Trt)-OH, Fmoc-Phe(2-Br)-OH, Fmoc-Phe(4-Br)-OH, FmocPhe(3,5-F2)-OH, Fmoc-P-(4-thiazolyl)-Ala-OH, Fmoc-P-(2-thienyl)-Ala-OH, and 4-(hydroxymethyl)-D-phenylalanine. , In some embodiments, the unnatural amino acid has the structure of Formula I:

[0334] [ka] Includes. In some embodiments, the unnatural amino acid has the structure of Formula II:

[0335] [ka] In some embodiments, the unnatural amino acid comprises 2-amino-3-(4-((fluorosulfonyl)oxy)phenyl)propanoic acid:

[0336] [ka] In some embodiments, the unnatural amino acid is fluorosulfonyl tyrosine (FSY):

[0337] [ka] In some embodiments, the unnatural amino acid is N6-(4-((fluorosulfonyl)oxy)benzoyl)lysine:

[0338] [ka] In some embodiments, the unnatural amino acid is fluorosulfonyloxybenzoyl-L-lysine (FSK):

[0339] [ka] It is.

[0340] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to nucleic acids that code for identical or essentially identical amino acid sequences. Due to the degeneracy of the genetic code, several nucleic acid sequences code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," one species of conservatively modified variations. Every nucleic acid sequence herein that codes for a polypeptide also describes every possible silent variation of the nucleic acid. One of skill in the art will recognize that each codon in a nucleic acid (except AUG, which is normally the only codon for methionine, and TGG, which is normally the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0341] With respect to amino acid sequences, one of skill in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that change, add, or delete a single amino acid or a small number of amino acids in the encoded sequence are "conservatively modified variants," where the change results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar 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 present disclosure.

[0342] Each of the following eight groups contains amino acids which 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 (1984)).

[0343] In some embodiments, the pyrrolysyl-tRNA synthetase (tRNA pyl ) is an aminoacyl-tRNA synthetase that catalyzes the reactions required to attach the α-amino acid pyrrolysine or a similar unnatural amino acid to a cognate tRNA, thereby allowing the incorporation of pyrrolysine or a similar unnatural amino acid during protein synthesis at an amber stop codon (i.e., UAG). The wild-type tRNA pyl are orthogonal to the endogenous tRNA and aminoacyl-tRNA synthetases of E. coli and eukaryotic cells. Using this pair, and its synthetically evolved derivatives or variants, we and others have directed the efficient incorporation of unnatural amino acids, including post-translationally modified amino acids, chemical handles, and photcaged amino acids, at specific sites in desired proteins in E. coli, yeast, and mammalian cells.

[0344] In some embodiments, the tRNA pyl tRNA pyl Any recombinant or naturally occurring form of pyrrolysyl-tRNA synthetase or a mutant, homolog, or isoform thereof that maintains activity (e.g., wild-type tRNA synthetase, pyl(at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% activity compared to a naturally occurring pyrrolysyl-tRNA synthetase). In some embodiments, a variant, homolog, or isoform has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring pyrrolysyl-tRNA synthetase. In some embodiments, a mutant tRNA pyl catalyzes the attachment of an unnatural amino acid (UAA) (e.g., a UAA of formula (IA), such as fluorosulfate-L-tyrosine (FSY)) to a tRNA pill such that the unnatural amino acid (UAA) is incorporated.

[0345] In some embodiments, the tRNA provided herein pyl can be manipulated by one skilled in the art pyl Derivatives or variants. In some embodiments, the tRNAs provided herein pyl is a single-stranded RNA molecule containing about 70-90 nucleotides, which folds through intrastrand base pairing to form a characteristic cloverleaf structure carrying specific amino acids (e.g., UAA of formula (IA), such as FSY) and matching them to the corresponding codons on mRNA during protein synthesis.

[0346] An "imaging ligand" or "detectable agent" is a composition detectable by appropriate means, such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means. For example, useful detectable agents include: 3 H, 14 C. 18 F, 33 P, 35 S, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu,67 Cu、 67 Ga、 68 Ga、 77 As、 86 Y、 90 Y、 89 Sr、 89 Zr、 94 Tc、 94 Tc、 99 mTc、 99 I、 105 Pd、 105 Rh、 111 Ag、 111 In、 112 In、 123 I、 124 I、 125 I、 131 I、 142 Pr、 143 Pr、 149 Pm、 153 Sm、 154~158 Gd、 161 Tb、 166 DY、 166 YOUR, 169 Er、 175 Ridiculous, 177 Ridiculous, 186 Too, 188 Too, 189 Too, 194 They、 198 I、 199 I、 211 And、 211 Pb、 212 Bi、 212 Pb、 213 Bi、 223 Does, 225 Ac、 153 Sm、 177 Ridiculous, 86 Y、 88 Y、 90 Y、 123 I、 124 I、 125 I、 131 I、 149 Tb、 212 Pb / 212 Bi、および 227 Th, Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, 32P, a fluorophore (e.g., a fluorescent dye), an electron-dense reagent, an enzyme (e.g., as commonly used in ELISA), biotin, digoxigenin, a paramagnetic molecule, a paramagnetic nanoparticle, ultrasmall superparamagnetic iron oxide ("USPIO") nanoparticles, USPIO nanoparticle aggregates, superparamagnetic iron oxide ("SPIO") nanoparticles, SPIO nanoparticle aggregates, single crystal iron oxide nanoparticles, single crystal iron oxide, a nanoparticle contrast agent, a liposome, or a gadolinium chelate ("Gd-chelate") molecule, gadolinium, a radioisotope, a radionuclide (e.g., carbon-11, nitrogen-13, oxygen-15, fluorine-18, rubidium-82), fluorodeoxyglucose (e.g., fluorine-18 labeled), any gamma ray emitting radionuclide, a positron emitting radionuclide, radiolabeled glucose, radiolabeled water, radiolabeled ammonia, a biocolloid, Microbubbles (e.g., microbubble gas cores containing albumin, galactose, lipids, and / or polymers, including air, heavy gas(es), perfluorocarbon, nitrogen, octafluoropropane, perflexan lipid microspheres, perflutren, etc.), iodinated contrast agents (e.g., iohexol, iodixanol, ioversol, iopamidol, ioxilan, iopromide, diatrizoate, metrizoate, ioxaglate), barium sulfate, thorium dioxide, gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores, two-photon fluorophores, or haptens and proteins, or other entities that can be made detectable, such as by incorporating a radioactive label into a peptide or antibody that specifically reacts with a target peptide. The detectable moiety is a monovalent detectable agent, or a detectable agent that can form a bond with another composition.

[0347] Radioactive substances (e.g., radioisotopes) that may be used as imaging and / or labeling agents in accordance with embodiments of the present disclosure include: 3 H, 14 C. 18 F, 33 P, 35 S, 45 Ti, 47 Sc, 52 Fe,59 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 77 As、 86 Y、 90 Y、 89 Sr、 89 Zr、 94 Tc、 94 Tc、 99m Tc、 99 Bride, 105 Pd、 105 Rh、 111 Ag、 111 Through, 112 Through, 123 OF, 124 OF, 125 OF, 131 OF, 142 Pr、 143 Pr、 149 Pm、 153 Sm、 154~158 Gd、 161 Tb、 166 DY、 166 HO、 169 Er、 175 Head, 177 Head, 186 Re、 188 Re、 189 Re、 194 They、 198 Shout, 199 Shout, 211 And、 211 Pb、 212 Bi、 212 Pb、 213 Bi、 223 Ra、 225 Ac、 153 Sm、 177 Head, 86 Y、 88 Y、 90 Y、 123 OF, 124 OF, 125 OF, 131 OF, 149 Tb、 212 Pb / 212 Bi、および 227Paramagnetic ions that may be used as additional contrast agents according to embodiments of the present disclosure include, but are not limited to, ions of transition metals and lanthanide metals (e.g., metals with atomic numbers 21-29, 42, 43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu.

[0348] "Linkage" or "linker", as used herein, refers to a bond or chemical moiety formed from a chemical reaction between a functional group of a linker and another molecule. Such bonds can include, but are not limited to, covalent and non-covalent bonds, while such chemical moieties can include, but are not limited to, esters, carbonates, imines, phosphate esters, hydrazones, acetals, orthoesters, peptide bonds, and oligonucleotide bonds. A hydrolytically stable linkage means that the linkage is substantially stable in water and does not react with water at useful pH values, including, but not limited to, under physiological conditions, over an extended period of time, perhaps even indefinitely. A hydrolytically unstable or degradable linkage means that the linkage is degradable in water or in aqueous solutions, including, for example, blood. An enzymatically unstable or degradable linkage means that the linkage can be degraded by one or more enzymes. By way of example only, PEG and related polymers can 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 bonds formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on the bioactive agent, such ester groups typically hydrolyze under physiological conditions to release the bioactive agent. Other hydrolyzable bonds include, but are not limited to, carbonate bonds, imine bonds resulting from the reaction of an amine with an aldehyde, phosphate ester bonds formed by reacting an alcohol with a phosphate group, hydrazone bonds which are the reaction product of a hydrazide with an aldehyde, acetal bonds which are the reaction product of an aldehyde with an alcohol, orthoester bonds which are the reaction product of a formate with an alcohol, peptide bonds formed by amine groups, including but not limited to, at the terminus of a polymer such as PEG, and carboxyl groups of peptides, and phosphoramidite groups, including but not limited to, at the terminus of a polymer, and oligonucleotide bonds formed by the 5' hydroxyl group of an oligonucleotide.Linkers include, but are not limited to, short linear, branched, multi-armed, or polymeric, such as dendritic molecules. In some embodiments, 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. Mechanisms for release of the drug from these linker groups include, for example, irradiation and acid-catalyzed hydrolysis of photolabile bonds. The length of the linker may be predetermined or selected depending on the desired spatial relationship between the polypeptide and the molecule linked to it.

[0349] The terms "polypeptide," "peptid," and "protein" are used interchangeably herein and refer to a polymer of amino acid residues, which may, in embodiments, be conjugated to a moiety not consisting of amino acids. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers. A "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0350] An amino acid or nucleotide base "position" is indicated by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-terminus). Due to deletions, insertions, truncations, fusions, etc. that must be considered when determining optimal alignment, in general, the number of amino acid residues in a test sequence, determined simply by counting from the N-terminus, is not necessarily the same as the number of its corresponding position in the reference sequence. For example, if a variant has a deletion relative to the aligned reference sequence, there is no amino acid in the variant that corresponds to the position in the reference sequence at the site of the deletion. If there is an insertion in the aligned reference sequence, the insertion does not correspond to a numbered amino acid position in the reference sequence. In the case of a truncation or fusion, there may be a stretch of amino acids in either the reference sequence or the aligned sequence that does not correspond to any amino acid in the corresponding sequence.

[0351] When used in the context of the numbering of a given amino acid or polynucleotide sequence, the term "numbered with reference to" or "corresponding to" refers to the numbering of the residues of a particular reference sequence when a given amino acid or polynucleotide sequence is compared to a reference sequence.

[0352] An amino acid residue in a protein "corresponds" to a given residue if it occupies the same essential structural position in the protein as the given residue. For example, a selected residue in a selected protein corresponds to Ala302 of a PylRS protein if the selected residue occupies the same essential spatial or other structural relationship as Ala302 in the PylRS protein. In an embodiment, the selected protein is aligned for maximum homology with the PylRS protein, and the position in the aligned selected protein that aligns with Ala302 is said to correspond to Ala302. Instead of a primary sequence alignment, a three-dimensional structural alignment can also be used, in which the structure of the selected protein is aligned for maximum correspondence with the PylRS protein, and the overall structures are compared. In this case, an amino acid that occupies the same essential position in the structural model as Ala302 is said to correspond to the Ala302 residue.

[0353] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window can contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where an identical nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0354] The terms "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., about 60% identity over a designated region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity when compared and aligned for maximum correspondence over a comparison window or designated region), as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters, as described below, or by manual alignment and visual inspection (see, e.g., the NCBI web site ncbi.nlm.nih.gov / BLAST / ). Such sequences are said to be "substantially identical." This definition also refers to or can be applied to the complement of a test sequence. This definition also includes sequences that have deletions and / or additions, as well as sequences that have substitutions. As described below, preferred algorithms can take into account gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0355] "Antibodies" are large, complex proteins with complex internal structures. Natural antibody molecules contain two identical pairs of polypeptide chains, each with one light chain and one heavy chain. The light and heavy chains, in turn, consist of two regions, the variable ("V") region, responsible for binding the target antigen, and the constant ("C") region, which interacts with other components of the immune system. The light and heavy chain variable regions come together in three-dimensional space to form the variable region that binds to the antigen (e.g., a receptor on the surface of a cell). Within each light or heavy chain variable region, there are three short segments (average 10 amino acids long) called complementarity determining regions ("CDRs"). The six CDRs in the antibody variable domain (three from the light chain and three from the heavy chain) fold together in three-dimensional space to form the actual antibody binding site that docks onto the target antigen. The positions and lengths of the CDRs are precisely defined in Kabat, E. et al., Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, 1983, 1987. The parts of the variable regions not included in the CDRs are called the framework ("FR") and form the environment of the CDRs.

[0356] The term "antibody" is used according to its commonly known meaning in the art. Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies below the disulfide bond in the hinge region to produce F(ab)'2, a dimer of Fab, itself a light chain linked to VH-CH1 by a disulfide bond. F(ab)'2 can be reduced under mild conditions to break the disulfide bond in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. A Fab' monomer is essentially a Fab with a portion of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993)). Although various antibody fragments are defined with respect to the digestion of intact antibodies, one skilled in the art will understand that such fragments can be synthesized de novo, either chemically or by using recombinant DNA methods. Thus, the term antibody, as used herein, also includes antibody fragments produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies (e.g., single-chain Fvs) or identified using phage display libraries (e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0357] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids that are primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively. The Fc (i.e., fragment crystallizable region) is the "base" or "tail" of the immunoglobulin and is typically composed of two heavy chains that contribute two or three constant domains depending on the class of antibody. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response to a given antigen. The Fc region also binds to various cellular receptors, such as Fc receptors, and other immune molecules, such as complement proteins.

[0358] An antibody "variant" as provided herein refers to a polypeptide that can bind to an antigen and includes one or more structural domains of an antibody or a fragment thereof. Non-limiting examples of antibody variants include single domain antibodies (nanobodies), affibodies (polypeptides smaller than monoclonal antibodies (e.g., 6 kDA)) that can bind antigens with high affinity and can mimic monoclonal antibodies, monospecific Fab2, bispecific Fab2, trispecific Fab3, monovalent IgG, scFv, bispecific diabody, trispecific triabody, scFv-Fc, minibody, IgNAR, V-NAR, hcIgG, VhH, or peptibody. A "peptibody" as provided herein refers to a peptide moiety attached (via a covalent or non-covalent linker) to the Fc domain of an antibody. Further non-limiting examples of antibody variants known in the art include antibodies produced by cartilaginous fish or camelids. Antibodies and variable regions thereof from camelids, as well as methods for their production, isolation, and use, can be found in references WO97 / 49805 and WO97 / 49805, which are incorporated herein by reference in their entirety for all purposes. Similarly, antibodies and variable regions thereof from cartilaginous fish, as well as methods for their production, isolation, and use, can be found in WO2005 / 118629, which is incorporated herein by reference in its entirety for all purposes.

[0359] A "single-domain antibody" or "nanobody" refers to an antibody fragment having a single monomeric variable antibody domain. Like a whole antibody, it is capable of selectively binding to a specific antigen. In some embodiments, a single-domain antibody is a human or humanized single-domain antibody. In some embodiments, a single-domain antibody is a camelid single-domain antibody. A single-domain antibody may be an engineered single-domain antibody and may contain non-naturally occurring amino acids.

[0360] The term "antigen" as provided herein refers to a molecule that can bind to an antibody binding domain as provided herein. An "antigen binding domain" as provided herein is a region of an antibody that binds to an antigen (epitope). As described above, an antigen binding domain may comprise one constant domain and one variable domain of each of the heavy and light chains (VL, VH, CL, and CH1, respectively). In an embodiment, the antigen binding domain comprises a light chain variable domain and a heavy chain variable domain. In an embodiment, the antigen binding domain comprises a light chain variable domain and does not comprise a heavy chain variable domain and / or a heavy chain constant domain. The paratope or antigen binding site is formed at the N-terminus of the antigen binding domain. The two variable domains of the antigen binding domain can bind to an epitope of the antigen. Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies under the disulfide bonds in the hinge region to generate F(ab)'2, a dimer of Fab, itself a light chain linked to VH-CH1 by a disulfide bond. F(ab)'2 can be reduced under mild conditions to break the disulfide bonds in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially the antigen-binding portion with a portion of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993)). Although various antibody fragments have been defined with respect to the digestion of intact antibodies, those skilled in the art will understand that such fragments can be synthesized de novo, either chemically or by using recombinant DNA methods. Thus, the term antibody, as used herein, also includes antibody fragments produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies (e.g., single-chain Fvs) or identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)). EXAMPLES

[0361] Example 1 - Identification of the binding site of PSMA sdAb To evaluate the suitability of PSMA as a target for binding to FSY-containing targeting molecules using single domain antibody (sdAb) C1 (SEQ ID NO:1), FSY sites were screened by replacing each of selected codons in the CDR1, CDR2, and CDR3 regions with a TAG stop codon and producing the encoded protein incorporating FSY at the appropriate location. Approximately 19 potential sites were identified, mostly clustered in the CDR1 and CDR3 regions. These TAG sites are located in CDR1 (sites 26-35: GYTDSNYYMS, SEQ ID NO:5), CDR2 (50-66: VNTGRGSTSYADSVKG, SEQ ID NO:6), CDR3 (99-116, excluding Cys101, Cys104 AACHFCDSLPKTQDEYIL, SEQ ID NO:7). A second sdAb C2 (SEQ ID NO:2) was similarly evaluated for FSY modification sites. Approximately eight sites were found as potential placements of FSY in the C2 CDR, with the majority clustering in CDR2 and CDR3. These TAG sites are located in CDR1 (sites 26-35 RFMISEYSMH, SEQ ID NO:8), CDR2 (50-65: TINPAGTTDYAESVKG, SEQ ID NO:9), CDR3 (96-100 DGYGY, SEQ ID NO:10).

[0362] Example 2 - Expression of FSY modified sdAb The vector pBAD sequence (Invitrogen #43001) lacking the ORF insert was PCR amplified using forward and reverse primers (SEQ ID NO:80) and (SEQ ID NO:81), respectively.

[0363] The resulting PCR amplified vectors were gel extracted and purified using Zymo PCR purification kit (Zymoclean Gel DNA Recovery kit Cat# D4002). The dsDNA sequences of C2 WT His and C1 WT His were ligated with the PCR amplified pBAD backbone and transformed into E. coli DH10b chemically competent cells (Fisher Thermo Scientific™ DH10B Competent Cells, High Efficiency, FEREC0113). Clones were miniprepped and sequences were confirmed using the pBAD forward primer. The expression cassette also incorporated a PelB leader sequence and a His6 tag. The DNA sequences of C1 and C2wt are SEQ ID NO:73 and SEQ ID NO:74, respectively. For constructs incorporating FSY residues, a TAG codon was engineered into the DNA sequence at the desired position for amino acid substitution within the open reading frame of the sdAb or biparatopic construct.

[0364] Construction of the C1 TAG library. Polynucleotides (dsDNA sequences) containing TAG codons in place of each codon within the sites identified in the CDR regions of C1 (see Example 1) were synthesized by IDT. These TAG sites are located in CDR1 (sites 26-35), CDR2 (50-66), and CDR3 (99-116, excluding Cys101 and Cys104). These sites are represented by the following sequences: QVQLQESGGGSVQAGGSLRLSCTAP GYTDSNYYMS WFRQAPGKEREWVA GVNTGRGSTSYADSVKG RFTISQDNAKNTMFLQMNSLKPEDTAIYYCAV AA C HF C DSLPKTQDEYIL The sequence is underlined in WGQGTQVTVSSAAAYPYDVPDYG (SEQ ID NO:1).

[0365] Polynucleotides containing each of these TAG mutants were cloned into the pBAD backbone as described above and the sequences were verified.

[0366] Building a C2 TAG library. Polynucleotide sequences containing a single TAG codon replacing each residue in the CDR regions of C2 identified in Example 1 were synthesized by IDT. These regions in the amino acid sequence of the C2 WT His protein sequence are represented by the following sequences: EVQLVESGGGLVQPGGSLTLSCAAS RFMISEYSMH WVRQAPGKGLEWVS TINPAGTTDYAESVKG RFTISRDNAKNTLYLQMNSLKPEDTAVYYC DGYGY The sequence is underlined in RGQGTQVTVSS (SEQ ID NO:2).

[0367] Polynucleotides containing each of these TAG mutants were cloned into the pBAD backbone as described above and the sequences were verified.

[0368] Library strain generation. The pEVOL-FSYRS plasmid expressing the engineered Mb FSYRS aminoacyl-tRNA synthetase was synthesized by (Order No. U168NGE200; Cat# SC1010) (Ref: J. Am. Chem. Soc. 2018, 140, 15, 4995-4999) and transformed into chemically competent DH10b cells using standard methods to generate the parent strain FSYRS-DH10b. The C1 CDR TAG or C2 CDR Individual plasmids encoding each of the TAG mutants were transformed into chemically competent FSYRS-DH10b using standard methods. Single colonies were inoculated into 2xYT medium (Teknova #Y0166) containing 100 μg / ml ampicillin and 34 μg / ml chloramphenicol and grown overnight at 37°C with shaking at approximately 220 rpm. The overnight cultures were mixed 1:1 with sterile 50% glycerol and stored at -80°C.

[0369] Expression and purification. Strains carrying plasmids encoding sdAbs with individual CDR residues mutated to TAG in the FSYRS-DH10b parent background were grown in 2xYT medium (Teknova #Y0166) in the presence of 100ug / ml ampicillin and 34ug / ml chloramphenicol. Prior to induction, cells were grown at a temperature of 37°C. OD 600 At 0.6, the culture was supplemented with 1 mM FSY and induced with 0.2% arabinose. At induction, the culture was transferred to 25°C and shaken at 220 RPM overnight for a total of 16 hours.

[0370] Expression was harvested by pelleting cells at 2200xg for 30 min at 4°C. The supernatant was removed and the cell pellet was weighed and stored at -80°C. The cell pellet was resuspended for lysis by adding 4mL / g pellet of B-PER Protein Extraction Reagent (ThermoFisher #78243). The resuspended pellet was lysed by placing the sample on an orbital shaker at medium speed for 15 min at room temperature. The lysed cells were then spun down at 2200xg for 30 min. The soluble fraction in the supernatant was removed for further purification.

[0371] To purify the soluble fraction, HisPur Ni-NTA resin (ThermoFisher #88222) was used to capture soluble material from the lysate. The resin storage buffer was removed and equilibrated by batch washing in wash buffer (40 mM sodium phosphate, pH 7.2, 300 mM NaCl, 20 mM imidazole). The batch wash was repeated for a total of 50x resin volumes. The above clarified lysate was added to the washed Ni-NTA resin and allowed to bind for 1 hour at room temperature with constant rotation. After binding, the protein-bound resin was batch washed with 50x resin volumes in wash buffer to remove unbound contaminants. The protein-bound resin was then transferred to a spin column and centrifuged briefly at 700xg to remove residual wash buffer. The target protein was then eluted using a buffer containing elution buffer (40 mM sodium phosphate, pH 7.2, 300 mM NaCl, 500 mM imidazole). Elution buffer was added at 2x resin volume and incubated at room temperature for 5 min. Samples were briefly centrifuged and eluted protein was collected in a new tube. The elution procedure was repeated twice using the same conditions. Elution fractions were pooled, quantified by A280 on a Nanodrop 2000, and blanked with elution buffer. Ni-NTA purified protein was then concentrated using a 0.5 mL, 3 kDa MWCO PES spin filter (ThermoFisher# 88512), repeatedly diluted with 8-10x volumes of sample and concentration, and buffer exchanged into 1x PBS.

[0372] Example 3 - Covalent interaction of FSY modified sdAb with PSMA Semi-purified sdAb products from Example 2 were incubated overnight in 1x PBS containing commercial PSMA (Sino Bio, cat#15877-H07H) at approximately 7:1 molar ratio (sdAb:PSMA, final PSMA concentration 0.125mg / mL, 1.25uM). Crosslinking was analyzed by reducing SDS-PAGE (Figures 1A-1E).

[0373] The sites evaluated in CDR1 and CDR3 of the C1 sdAb exhibited a spectrum of cross-linking capabilities towards PSMA, as shown in Table 3 below.

[0374] [Table 4] A second PSMA specific sdAb, C2, was assessed for cross-linking efficiency using a method similar to that of Example 1, with the change that the sdAb:PSMA molar ratio was approximately 8:1. The cross-linking results are shown in Figures 2A-2C. The sites evaluated in CDR2 and CDR3 of the C2 sdAb showed a spectrum of cross-linking capabilities towards PSMA, as shown in Table 4 below.

[0375] [Table 5]

[0376] Example 4 - Crosslinking kinetics A subset of FSY-modified sdAbs was selected to assess the kinetics of PSMA cross-linking. sdAbs were incubated with PSMA at a 5:1 molar ratio (final PSMA concentration 0.125mg / mL, 1.25uM). Samples were taken from 0 to 180 minutes and the percentage of PSMA cross-linking was assessed by SDS-PAGE. As shown in Figure 3A and Figure 3B, the modified sdAbs exhibited different coupling kinetics. Cross-linking band percentages were calculated by quantifying sdAb-PSMA cross-linked bands and PSMA band intensity was quantified using ImageJ.

[0377] Example 5 - PSMA targeting constructs with modified CDR3 The PSMA-targeting sdAb C1 construct was made by removing two cysteine ​​residues in the CDR3 (SEQ ID NO: 7) of C1 (SEQ ID NO: 1) and modifying these residues to alanines (C101A and C104A) to create C39. This construct was then further modified to introduce FSY at residue 102 (C1-C101A / C104A / H102(FSY), SEQ ID NO: 4, hereafter referred to as C39-102FSY). A diagram of these sdAb constructs is shown in FIG. 4. Constructs were synthesized with the pelB leader sequence (SEQ ID NO: 15) cleaved from the mature protein, and the six C-terminal histidines were used for His-Tag purification. The cross-linking ability of these constructs was assessed using the method of Example 3. SDS-PAGE analysis showed that all constructs cross-linked at comparable levels under these conditions.

[0378] FSY containing PSMA targeting sdAb C39-FSY C1 was cloned and expressed as described in Example 2. A biparatopic construct (C1-C101A / C104A / H102(FSY)-L1-C1-C39, SEQ ID NO: 21, hereafter referred to as C40-FSY) was made by ligating C39-FSY C1 with an additional copy of C1-C101A / C104A / H102H (SEQ ID NO: 71, no FSY). The construct was designed by adding a GGGGSGGGGS linker between the two sdAb amino acid sequences. The biparatopic construct was then cloned into pBAD vector and expressed as described in Example 2. The construct was synthesized with the pelB leader sequence (SEQ ID NO: 15) cleaved from the mature protein and the six C-terminal histidines were used for His-Tag purification.

[0379] Monoparatopic sdAb and biparatopic constructs were assessed for crosslinking to PSMA over time by SDS-PAGE. SDS-PAGE analysis showed that the dimeric form C40-102FSY crosslinked 2-fold faster than the monomeric C39-102FSY (t1 / 2 max crosslinking for dimer ∼30 min vs. t1 / 2 max crosslinking for monomer ∼60 min) (Figures 5A-5B).

[0380] C39-102FSY amino acid sequence (* is 102FSY position, SEQ ID NO:4, alanine modifications are underlined): QVQLQESGGGSVQAGGSLRLSCTAPGYTDSNYYMSWFRQAPGKEREWVAGVNTGRGSTSYADSVKGRFTISQDNAKNTMFLQMNSLKPEDTAIYYCAVAA A *F A DSLPKTQDEYILWGQGTQVTVSSAAAAYPYDVPDYGSCHHHHHHH

[0381] Example 6 - Synthesis of constructs Site-specific conjugation via the C-terminal cysteine ​​residue. An additional Cys residue was engineered near the C-terminus of the construct to allow for site-specific bioconjugation to sdAb compounds. sdAb-Cys compounds were prepared and formulated at 1 mg / ml in 1x PBS, pH 7.4, 5 mM EDTA. TCEP was added to 10 mM for mild reduction and incubated at room temperature for 15 minutes. Samples were then buffer exchanged using Zeba Spin columns to remove TCEP and exchange into 1x PBS, pH 7.4, 1 mM EDTA. Maleimide-reactive payload compounds (such as AZDye 647 Maleimide cat#:1122-5) were then added in 10x molar excess and incubated at 37°C for 2 hours or 4°C for 16 hours. Unreacted maleimide compounds were removed from the samples using size exclusion chromatography, desalting columns, dialysis or TFF. Samples were subjected to SDS-PAGE under reducing and non-reducing conditions to observe the labeling efficiency and analyzed using LC-MS to quantify the intact mass and relative proportions of labeled and unlabeled species.

[0382] Example 7 - Cross-linking activity assay Molecules are assayed for specific binding and cross-linking to target tumor antigens. To assess the cell binding potency of PSMA-targeting constructs, specific FSY or Tyr-containing sdAbs are used to test the effect of covalent cross-linking to specific antigens of interest. These test substances are serially diluted and associated with in vitro cultured prostate cancer cell lines LnCAP (PSMA+) and PC3 (PSMA-). After incubation at 37°C for different times, cells are washed and medium is replaced to remove unbound substances. Samples are analyzed by flow cytometry using anti-His tag antibody or anti-VHH to measure the bound population as a function of input concentration. To measure cross-linking efficiency and specificity, cells are harvested, lysed, and Western blotted using anti-his or anti-VHH antibodies to measure the covalently bound population via gel shift, and compared to gel shift Western blot using anti-PSMA antibody to measure the total target antigen cross-linked to sdAb test substances.

[0383] The conjugated molecules are further assayed for functional activity, in this case the ability to deliver a toxic payload in vitro specifically to tumor cells expressing the target antigen. Constructs containing engineered C-terminal or proximal Cys residues are coupled to a cytotoxic payload, such as MMAE or other classes of highly cytotoxic compounds, using the general methods described in Example 8. Dose-response curves are generated and samples are incubated in vitro with LnCAP and PC3 cells. After various periods of incubation, cells are washed to remove free compound and cell viability is measured using an assay such as Promega CytoxGreen or other assays that measure cell viability via reporter compound detection, detection of viable cells with Presto Blue, CCK-8 or similar reagents, and / or assays that measure apoptosis via Annexin V-FITC, propidium iodide staining or similar reagents.

[0384] Example 8 - In-Vivo Delivery To measure the ability of the constructs to specifically deliver payload to tumor tissue in vivo and compare tumor exposure between the constructs and non-covalent sdAb conjugates, fluorophore labels are used as proxy payloads to allow tracking / biodistribution measurements over time. sdAb constructs (e.g., C1, C2, or C3) with a single residue substituted with either Tyr or FSY are conjugated to a chemical fluorophore (Alexa647 or similar) via maleimide chemistry through an engineered cysteine ​​residue and purified as described in Example 6. Fluorophore-labeled sdADC conjugate molecules are administered by tail vein IV injection to male NSG mice bearing PSMA+ or - tumors and the biodistribution of the test article is observed over time by whole animal imaging using an AmiX imager or similar. The tumor-specific and peripheral exposures will be quantified by image densitometry and compared between the FSY and Tyr versions of the sdAb.

[0385] Example 9: Design of additional FSY-modified SdAbs The C3wt anti-PSMA sdAb was constructed by cloning the C3wt sequence (synthesized from IDT, SEQ ID NO: 3) into a digested vector (synthesized by Genscript) with NdeI and HindIII restriction enzymes. The expression cassette also incorporated a PelB leader sequence and a His6 tag. The C3 wt DNA sequence is shown as SEQ ID NO: 75.

[0386] The amino acid sequences in the CDR regions were changed to TAG codons in the plasmid at the sites selected for FSY modification. TAG mutants in these ORFs were prepared by Genscript via site-directed mutagenesis.

[0387] C3 CDR1(26-35: GWPYSTYSMN , SEQ ID NO: 11), CDR2 (50-65: GISSTMSGIIFAESKAG, SEQ ID NO: 12), CDR3 (99-113: RRDYSLSSSSDDFDY , sequence number 13).

[0388] Example 10 - Screening of FSY modified sdAb The C3 construct from Example 9 was co-transformed with pEVOL-FSYRS into DH10b competent cells as described in Example 2. A single colony from the transformation was picked and inoculated into 1 mL of 2xYT in a 24-deep well plate supplemented with 100 μg / mL Amp + 34 μg / mL Cm with shaking at 220 rpm at 37°C. When the OD reached 0.5, the temperature was reduced to 25°C. Expression in each well was induced by adding 0.2% arabinose 1 mM + FSY. The products were expressed overnight at 25°C. After overnight, the cells were transferred to a 1.5 mL Eppendorf tube and spun in a benchtop centrifuge. The supernatant was removed. For cell lysis, the pellet was treated with 50 μL of B-per (ThermoFisher #78243) for 15 minutes. The cell lysate was then spun down at maximum speed in a benchtop centrifuge at 4°C.

[0389] The cross-linking reaction was initiated by incubating 3 μL of the supernatant with 3 μL of PBS or 3 μL of 0.25 mg / mL PSMAO. The reactions were incubated at 37° C. for a shorter time window of 3 hr. Afterwards, the incubation mixture was treated with 2X SDS loading dye. Cross-linking was examined by performing SDS-PAGE staining with Coomassie blue. The results are shown in Figures 6A and 6B and summarized in Table 5 below.

[0390] [Table 6]

[0391] Example 11 - Identification and kinetics of FSK modified FAP sdAb Sequence analysis (abYsis) was used to define the complementarity determining region (CDR) loop positions in the Fap antibody sequence. Libraries were constructed to individually replace each CDR residue with a TAG codon, and the resulting proteins were expressed under conditions that incorporated the unnatural amino acid FSK at each position.

[0392] C8 FAP Library-Generation. To evaluate different FSK incorporation sites in sdAb, vector pBAD-C8 WT (SEQ ID NO: 82) was constructed as follows: C8 WT sequence (SEQ ID NO: 23) was codon-optimized for E. coli expression and synthesized by IDT company. pBAD-C8 WT vector was constructed by cloning C8 WT gblock sequence (SEQ ID NO: 78) into pBAD vector (Genscript) digested with NdeI and HindIII restriction enzymes.

[0393] Library-strain generation. For expression of the corresponding synthetases, pEVOL-FSKRS plasmid (Genscript) encoding the engineered Ma FSKRS aminoacyl-tRNA synthetase (SEQ ID NO: 87) was synthesized and pEVOL-FSKRS (SEQ ID NO: 89) was transformed into chemically competent DH10b competent cells to generate the parent strain FSKRS-DH10b. Individual plasmids encoding each C8 CDR TAG mutant were transformed into chemically competent FSKRS-DH10b cells. Single colonies were inoculated into Superbroth medium (Teknova#S1530) containing 100 μg / ml ampicillin and 34 μg / ml chloramphenicol and grown overnight at 37° C. with shaking at approximately 220 rpm.

[0394] Expression and purification. Strains carrying plasmids encoding sdAbs with individual CDR residues mutated to TAG in the FSKRS-DH10b parent background were cultured in Superbroth medium in the presence of 100 μg / ml ampicillin and 34 μg / ml chloramphenicol. A single colony from each pool was inoculated into 5 mL of Superbroth medium containing the relevant antibiotic and grown overnight at 37°C. The next day, cells were diluted at a 1:10 ratio into 30 mL (diluted 3 mL to 30 mL) Superbroth medium containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol, 1 mM FSK, 0.2% arabinose. Cells were induced for FSK uptake for 6 hours at 30°C and 220 rpm. After expression, FSK-modified sdAbs (C8-FSK) were harvested and purified according to the method described in Example 2.

[0395] Crosslinking kinetics. FAP receptor (Acrobiosystem #AP-H5263-100ug) was incubated with the pooled antibody mixture (Table 6) at a molar ratio of approximately 8:1 (antibody:receptor) at 37°C in 1XPBS, pH 7.4, for 1 hour, 2 hours, or overnight. After incubation, the crosslinking reaction was stopped by adding 1X Laemmli Sample Buffer containing 100mM DTT. Samples were heated at 95°C for 5 minutes, followed by Tris-glucyine SDS-PAGE (4-20% Mini-PROTEAN® TGX™). Relative band intensities were then quantified using ImageJ software. FSK pool shift results demonstrated FAP receptor shift via crosslinking activity within pools 2 and 3 (Figure 7A).

[0396] [Table 7]

[0397] Identification of a single FSK site involved in cross-linking with FAP. To identify the individual FSK sites involved in cross-linking, each TAG mutant was cotransformed with pEVOL-FSKRS into DH10b cells and expressed and purified as described above. Each individual FSK mutant from the candidate pool was incubated with FAP receptor for gel shift studies (Figure 7B). Nine FSK mutants in CDR2 were found to have cross-linking with FAP (52, 53, 54, 55, 56, 58, 60, 62, 64). One site (56) was found to have the highest cross-linking yield of all cross-linking sites within 1 h.

[0398] Measurement of C8-FSK cross-linking kinetics to FAP receptor. To evaluate FSK cross-linking kinetics, C8-54FSK and C8-56FSK were incubated with FAP receptor for 15, 30, 60, and 120 min and examined for cross-linking yield. The 56FSK site was found to have the highest cross-linking kinetics compared to the other sites, indicating that C8-56FSK has the fastest cross-linking kinetics (>50% cross-linked within 2 h) (Figure 7C).

[0399] Example 12 - Identification and kinetics of FSY modified Her3SdAb C9 Library Generation. Sequence analysis was used (abYsis) to define the complementarity determining region (CDR) loop positions in the Her-3 antibody sequence. Libraries were constructed to individually replace each CDR residue with a TAG codon and expressed under conditions that incorporated the unnatural amino acid FSY at each position.

[0400] The vector pBAD-C9 WT was constructed. The C9 WT sequence (SEQ ID NO:24) was codon optimized for E. coli expression. The pBAD-C9 WT vector (SEQ ID NO:83) was constructed by cloning the C9 gblock sequence (SEQ ID NO:79) into digested pBAD vector (Genescript cat# SC1010) with NdeI and HindIII restriction enzymes. A library of C9-FSY mutants was generated as described in Example 2, except that the pEVOL-FSYRS plasmid and individual mutant pBAD-C9 mutants were transformed and expressed in BL21 cells instead of DH10b cells. Expression of the FSY modified sdAb (C9-FSY) followed the method described in Example 2, except that the FSY modified C9 sdAb was expressed in and purified from the parental strain FSYRS-BL21.

[0401] Identification of Pools Involved in Cross-Linking. To initially identify regions of the CDRs that contain cross-linking compatible FSY sites, strains containing plasmids with TAG sites in multiple CDR regions were first pooled (Table 7). Pooled strains were expressed, purified via Ni-NTA affinity, normalized for concentration, and assessed for cross-linking via SDS-PAGE gel shift as described in Example 2. The C9-FSY pool mixture was incubated with Her3 (Acrobiosystem #ER3-H5223-100ug) at a 3:1 ratio (3μg of C9 FSY to 1ug of Her3 receptor) at 37°C, 1xPBS, pH 7.4, overnight. 4 Overnight. Reactions were run in 4-20% Mini-PROTEAN® TGX™ and then stained with Coomassie blue. Pooled shift results showed Her3 receptor cross-linking activity within pools 2, 3, and 4 (Figure 8A).

[0402] [Table 8]

[0403] Identification of a single FSY site involved in cross-linking with Her3. To identify each individual FSY site involved in crosslinking, each TAG mutant was cotransformed with pEVOL-FSYRS into BL21 cells and expressed and purified as described above. Each of the individual FSY mutants from the candidate pool was incubated with Her3 receptor overnight at 37°C. Crosslinking was assessed by running the reaction on 4-20% Mini-PROTEAN® TGX™ with Coomassie blue staining (Figure 8B). Eight C9-FSY mutants in CDR2 were found to support crosslinking with Her3 (53, 55, 56, 57, 58, 60, 64, 67). Two sites (55, 57) were found to have the highest crosslinking yields of all crosslinking sites.

[0404] Identification of the C9-FSY crosslinking site with the fastest rate. To identify the FSY crosslinking site with the fastest rate, C9 with the FSY site identified above was incubated with Her3 receptor (Acrobiosystem) and the crosslinking yield was determined. The C9-FSY variant was incubated with Her3 receptor at a molar ratio of 8:1 in 1xPBS, pH 7.4. The incubation was stopped after 1 hour by adding a final 1X SDS loading dye containing 100 mM DTT, and the reaction was analyzed by 4-20% Mini-PROTEAN® TGX™ with Coomassie blue staining.

[0405] The unique 55 FSY site was found to have the highest cross-linking yield (>50% within 1 h) compared to other sites, indicating that 55 FSY has the fastest cross-linking rate (Figure 8C). C9-55FSY was further analyzed by incubating with Her3 receptor and quenching the reaction over a time course of 0, 15, 30, 60, 120, and 180 min. As shown in Figure 8D, cross-linking proceeded rapidly, detected within 15 min, with over 50% cross-linking within 1 h.

[0406] Example 13 - Binding affinity of FSY modified and unmodified C2 sdAb C2 sdAb with FSY at position 54 (C2-54FSY) was selected to compare binding affinity with C2 sdAb with tyrosine at the same position (C2-54TYR). Proteins were prepared as in Example 2 and then further purified by FPLC size exclusion chromatography (HiLoad 16 / 600 Superdex 200pg size exclusion colum Cytiva#28989335). Proteins were collected by isocratic elution using 1xDPBS as running buffer. For each C2 sdAb, monomer peak fractions were analyzed by reducing SDS-PAGE, pooled and dialyzed overnight at 4°C against anion exchange running buffer (20mM Tris, 7.5 and 20mM NaC). To remove endotoxin, the dialyzed sample pool was run through a HiTrap Q XL, 1 ml column (Cytiva #17515801) to allow endotoxin to bind to the column. The flow-through was collected and checked for endotoxin levels. Finally, the fully purified sample was dialyzed into 1xDPBS as the final formulation buffer. Samples were aliquoted and stored at -80°C.

[0407] Binding kinetics were measured using biolayer interferometry with an AHC sensor (Sartorius Item #18-5060). PSMA (50nM) with an Fc tag (Acrobiosystem PSA-H5264-100ug) with either C2-54FSY or C2-54TYR sdAb (protein concentrations 400nM, 200nM, 100nM, 50nM). OCTET steps measured were baseline: 60sec, receptor loading: 300sec, washout of unbound receptor: 300sec, loading of sdAb for association: 100sec, dissociation: 600sec. C2-54TYR had a KD of 9.1nM and C2-54FSY had a KD of 10.9nM, indicating that incorporation of FSY in place of tyrosine at position 54 did not alter binding affinity.

[0408] Example 14 - Cell Binding Assay C2-54FSY and C2-54TYR sdAbs were evaluated for binding to human prostate tumor cell lines LNCaP (PSMA+) and PC3 (PSMA-) using flow cytometry. Protein from Example 13 was formulated to 3 μM (1000 μL) with FACS buffer (1×PBS+2% HI-FBS) and then serially diluted 5× (200 μL sdAb to 800 μL FACS buffer) to generate 8 concentration points for the assay, with the lowest concentration being 0.0000384 μM. A control sdAb (human PSMA Alexa Fluor Alexa Fluor® 647-conjugated antibody) was formulated to 1 μM (450 μL) in FACS buffer (1× PBS + 2% HI-FBS) and then serially diluted 3× (150 μL test article vs. 300 μL FACS buffer) with the lowest concentration being 0.00045 μM.

[0409] Binding assay. LNCaP and PC3 cell lines (ATCC) were maintained in RPMI-1640 and F12K medium supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher) at 37°C in a humidified environment containing 5% CO2. Cells were harvested in exponential growth phase, counted, and aliquoted into v-bottom 96-well plates at 100 μL cells / well. Cells were pelleted by centrifugation. C2 sdAb and control sdAb samples were added to the cells and incubated on ice for 1 hour. For cells treated with C2 sdAb, after incubation, cells were washed twice with FACS buffer and incubated with 100 μL ice-cold FACS buffer containing 5 μg / mL secondary antibody (Alexa Fluor 647 AffiniPure Goat Anti-Alpaca IgG, VHH domain) for 30 minutes on ice. Cells were then washed twice with FACS buffer and fixed with fixation buffer (Thermofisher, Cat. No. 0082249) at 4° C. For cells treated with control sdAb, after incubation, cells were washed twice with FACS buffer and fixed with fixation buffer (Thermofisher, Cat. No. 0082249) at 4° C. After fixation, cells were washed twice and analyzed by Attune flow cytometry (ThermoFisher).

[0410] Raw data (FCS files) were analyzed (Flowjo 10.7.2). Total cells were separated using FSC-A and SSC-A, and intact cells were gated on FSC-A and SSC-A. Singlets were then gated on FSC-A versus FSC-H. Singlets were separated with Alexa Fluor-647. The geometric mean intensity of the Alexa Fluor-647 signal intensity of the samples was used for further analysis and plotting. The mean and STDEV values ​​of the geometric mean intensity of Alexa Fluor-647 of the samples were calculated (Microsoft® Excel® for Microsoft 365 MSO Version 2202 Build 16.0.14931.20128 64-bit). Curve fitting and plotting were performed using "log(agonist) vs response-variable slope (4 parameters)" (GraphPad Prism 9.2.0).

[0411] C2-54TYR and C2-54FSY bound to LNCaP cells in a dose-dependent manner, but showed no binding to PSMA-negative PC3 cells (Figure 9). The binding affinities for C2-54TYR and C2-54FSY were determined from EC50 values ​​after flow staining of LNCaP cells (Table 8).

[0412] [Table 9]

[0413] Example 15 - Crosslinking of FSY modified sdAb to target cells Crosslinking of C2-54TYR, C2-54FSY and C3-101FSY were compared for cell binding. LNCaP and PC3 cells were cultured as described in Example 14. Cells were seeded in their growth medium in 6-well plates at a density of 500,000 cells / well. 48 hours after seeding, the culture medium was removed and 0.8 mL of culture medium containing sdAb was added at the indicated concentrations and time points (see Tables 9 and 10). Cells were rinsed twice (1 mL / well of 1×PBS) and 0.2 mL / well of 0.25% EDTA-trypsin (Gibco, Cat. No. 25200-056) was added. After 5 min of incubation in the incubator, 1 ml of complete medium was added to neutralize the trypsin. Cells were rinsed and pelleted.

[0414] Cells were lysed in RIPA buffer supplemented with a protease inhibitor cocktail (Santa Cruz Biotechnology, Cat. No. SC-24948A). Denatured samples were analyzed by electrophoresis on 4-20% Criterion TGX Gels (Bio-Rad, Cat. No. 5671095), followed by Western Blot Kits (Bio-Rad, Cat. No. 1704271) and Pierce Fast Western Blot Kits (Thermo Scientific, Cat. Nos. 35060 and 35061) using primary antibodies anti-human PSMA (Invitrogen, Cat. No. 37-3900) or anti-alpaca VHH (Jackson ImmunoResearch, Cat. No. 128-035-232), and internal standard anti-GAPDH (Cell Signaling Technology, Cat. No. CST-2118).

[0415] Images were acquired by Azure Biosystem C600. Images were processed using GIMP 2.10.28. Band density of Western blots was measured using ImageJ 1.51j8 according to the instructions (imagej.nih.gov / ij / docs / guide / 146-30.html#infobox:Densitometry, Section 30.13). The percentage of cross-linked PSMA to total PSMA was calculated by the following formula: density of cross-linked PSMA / (density of cross-linked PSMA+density of non-cross-linked PSMA)×100%.

[0416] In western blots from LNCaP cells, a predominant band (~100 kD) detected with anti-PSMA antibody was observed (Figure 10A), whereas in PC3 cells, no band with a similar molecular weight was detected (Figure 10B), demonstrating the specificity of the anti-PSMA antibody and confirming the specificity of PSMA expression in LNCaP cells. In LNCaP cells treated with C2-54FSY, an additional band above PSMA was detected. The intensity of the band increased in a time- and concentration-dependent manner, indicating specific cross-linking. However, in LNCaP cells treated with various concentrations of C2-54TYR for various times, a non-cross-linked PSMA band of similar intensity, rather than a cross-linked band, was observed, indicating that the sdAb without FSY did not cross-link to PSMA in LNCaP cells (Figure 10B). Quantification of cross-linking showed that C2-54TYR cross-linked to PSMA in a time- and dose-dependent manner in LNCaP cells. (Table 9 and Figure 10C)

[0417] [Table 10]

[0418] A similar pattern was observed in LNCaP cells treated with C3-101FSY, where the intensity of the crosslinked band (PSMA-sdAb) increased in a time- and concentration-dependent manner. Because the molecular weight of C3-101FSY is higher than that of C2-54FSY, the crosslinked band migrated at a higher apparent molecular weight than that of C2-54FSY (Figure 11A). A comparison of the crosslinking kinetics of C2-54FSY and C3-101FSY is shown in Table 10 and Figure 11B.

[0419] [Table 11]

[0420] Example 16 - In vivo cross-linking assay This study evaluated the in vivo crosslinking, systemic, and intratumoral exposure of C2-54TYR and C2-54FSY with PSMA in two mouse xenograft tumor models, LNCaP and PC3. LNCaP and PC3 cell lines (ATCC) were maintained in RPMI-1640 and F12K medium supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher) at 37°C in a humidified environment containing 5% CO2, respectively. Cells were harvested in exponential growth phase, centrifuged at 335xg in a refrigerated centrifuge, and the medium was aspirated. For cell inoculation, cell pellets were resuspended in 100μl of serum-free F-12K medium for PC3 or RPMI + 100μL Matrigel for LNCaP. 200 μL containing 5 million PC3 cells or 3 million LNCaP cells were implanted into the flank of male NSG mice (Jackson Labs). Tumor size was approximately 200 mm 3 When the mice reached 100 mg / kg / day, C2-54TYR and C2-54FSY were injected via the tail vein. Peripheral blood samples were collected by cheek bleeding. Six hours later, the mice were sacrificed and both peripheral blood samples and tumors were collected, weighed, and frozen at -80°C.

[0421] Tumor samples (~30-40 mg) were added to 1 ml of RIPA buffer containing a protease inhibitor cocktail (Santa Cruz Biotechnology, Cat. No. SC-24948A) and then homogenized with a Qiagen Tissuelyser II Sample Disruptor. After homogenization, samples were centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was retained and the pellet was discarded. The centrifugation process was repeated once (total of 2 times).

[0422] Protein concentrations of tumor lysates were quantified using a BCA protein assay kit (Thermo Scientific, Cat. No. 23225). Samples were formulated to the same concentration in RIPA buffer and then heated at 100°C for 10 min after adding 6x reduced loading buffer (Alfa Aesar, Cat. No. J61337). Denatured samples were analyzed by electrophoresis on 4-20% Criterion TGX gels (Bio-Rad, Cat. No. 5671095) followed by Western blot (Bio-Rad, Cat. No. 1704271 and Thermo Scientific, Cat. No. 35060, 35061) using anti-PSMA and anti-VHH antibodies to detect crosslinking, and anti-GAPDH antibody as a loading control. Images were acquired by Azure Biosystem C600. Images were processed using GIMP 2.10.28. Plasma was subjected to ELISA and data was processed and plotted (GraphPad Prism 9.2.0). Means and STDEV were calculated and the significance of plasma concentrations was assessed using unpaired two-tailed Student's t-test.

[0423] Samples from LNCaP and PC3 tumors derived from animals receiving C2-FSY or C2-TYR were prepared as above and Western blotted with anti-VHH antibodies to detect uncrosslinked C2 compounds (15 kD region of the blot) and crosslinked C2 compounds (~100 kD region of PSMA above). In LNCaP tumor samples from animals receiving C2-54FSY, anti-VHH Western blots detected bands above the PSMA region (~100 kD) in all samples, but no crosslinked bands were observed in C2-54TYR or vehicle samples (Figure 12A), indicating specific and reproducible crosslinking by C2-54FSY. No anti-VHH signal was observed in PC3 tumor samples, but a different pattern of bands was observed in LNCaP samples treated with C2-54TYR and C2-54FSY. In the ∼15 kD region of the blot representing uncrosslinked / free sdAb, both C2-54TYR and C2-54FSY treated LNCaP samples showed bands of similar intensity, demonstrating the presence of similar levels of uncrosslinked sdAb. An additional band migrating at ∼100 kD was observed specifically in C2-54FSY treated LNCaP tumor tissue, whereas no band was observed in this region in PC3 or LNCaP samples from C2-54TYR treated animals, indicating crosslinking of C2-54FSY to PSMA.

[0424] To estimate and compare systemic exposure of the sdAbs, plasma concentrations of C2-54TYR and C2-54FSY were measured 30 min and 6 h after dosing using ELISA (Table 11 and Figure 12B).

[0425] [Table 12]

[0426] The results show that after a single IV administration, C2-54TYR and C2-54FSY gain access to tumors and accumulate in a PSMA-dependent manner, but only C2-54FSY specifically crosslinks to PSMA in tumors.

[0427] Example 17 Construction of FSK- and FSY-modified sdAbs for target-specific cross-linking Libraries and library strains were constructed, expressed, and purified generally according to the methods of Examples 2 and 11. To initially identify regions of the CDRs that contain sites suitable for insertion of FSY or FSK, strains were constructed that contain plasmids with a TAG codon within the coding region of the CDR region. Table 12 shows the sdAb and CDR sequences used for screening.

[0428] [Table 13]

[0429] Screening. Strains containing plasmids with a TAG codon within the coding region of the CDR region were screened in pools and assayed for cross-linking generally according to the methods of Examples 11 and 12. Pools showing cross-linking activity to the target were further analyzed by evaluating individual FSY or FSK mutants for cross-linking. Individual mutants showing cross-linking are shown in Table 13.

[0430] [Table 14]

[0431] Reagents used for crosslinking assay targets. CEACAM5 was purchased from Acrobiosystem (#CE5-HF255-25μg) and Sinobiological (#11077-H02H-100μg). FAP receptor was purchased from Acrobiosystem (#AP-H5263-100ug). Human FolRa (Folate Receptor alpha) receptor was purchased from Acrobiosystem (#FO1-H5253-100ug). MSLN (Mesothelin) was purchased from Sinobiological (Cat:13128-HNCH, Cat:13128-H01H-B) and Acrobiosystem (#MSN-H526x-100ug). Human MSLN extracellular domain was purchased from Acrobiosystem (#MSN-H5253-100ug, #MSN-HF223-25ug). Her3 extracellular domain was purchased from Acrobiosystem (#ER3-H5223-100ug). Human CD123 protein extracellular domain was purchased from SinoBiological (#10518-H02H-50ug). Human 5T4 extracellular domain was purchased from Acrobiosystem (#TPG-H5253-100ug).

[0432] For the C8 FSY sdAb, the crosslinking kinetics was analyzed according to the method of Example 11. Figure 13 shows the crosslinking from 0 to 180 minutes. The C8-54 FSY site was found to have the highest crosslinking rate compared to the other sites (approximately 50% crosslinked within 2 hours). Similarly, the crosslinking rates of C18-FSY and C18-FSK sdAb to the HER3 receptor were compared. FSY at position 101 was observed to achieve the highest crosslinking efficiency and crosslinking rate, as well as between positions 33, 101, and 103. For the C18 sdAb FSK variants at positions 30, 32, 35, FSK at position 35 was observed to achieve the highest crosslinking efficiency and crosslinking rate to Her3. Evaluation of the three sites in the C20 FSY sdAb showed that position 57 achieved the highest crosslinking efficiency and crosslinking rate to CD123.

[0433] Example 18. Identification and kinetics of FSY-modified DARPins A Her2-binding DARPin (C15) was screened for its ability to crosslink via the insertion of the FSY amino acid. Libraries and library strains were constructed, expressed, and purified according to the general methods of Example 2. The C15 WT sequence (SEQ ID NO: 29) is shown in Table 14, with the region screened for FSY insertion in bold and the amino acid position of the FSY substitution indicated in the second row of the table.

[0434] [Table 15]

[0435] Each individual C15-FSY mutant was incubated with Her2 receptor for cross-linking efficiency, generally following the method of Example 11. Sites 9, 12, 37, 66, 68 in C15 showed cross-linking with Her2 receptor, as shown in FIG. 14. To identify the FSY cross-linking site with the fastest rate, DARPins with the FSY sites identified above were incubated with Fc-tagged HER2 (Acrobiosystem:Acrobiosystem:#HER2-H5253-100ug) and examined for cross-linking at 15 and 30 minutes. The C15-66 FSY site was found to have a higher cross-linking yield compared to the other sites.

[0436] A biparatopic DARPin (C15-66FSY-C16, hereafter referred to as C38-FSY) was constructed by fusing one copy of the DARPin without the FSY substitution C16 (SEQ ID NO: 30) to the C-terminus of C15-66FSY with a 5 amino acid GGGGS linker to generate a fusion protein. The C38-FSY protein was incubated with Fc-tagged Her2 and cross-linking was examined generally according to the method of Example 11. As shown in Figure 15, biparatopic C38-FSY cross-linked Her2 in a time-dependent manner, with 50% cross-linking within 2.5 hours.

[0437] Example 19. Cellular binding with targeted imaging payloads C22-TYR and C22-FSY (SEQ ID NOs: 39 and 40, respectively) were expressed and purified generally according to the method of Example 2, except that after elution from the Ni-NTA resin, the purified proteins were then buffer exchanged into anion exchange running buffer (20 mM Tris, 7.5 and 20 mM NaCl) overnight at 4° C. using a dilution factor of 1:100. The dialyzed sample pool was passed through a HiTrap Q XL, 5 ml column (Cytiva#17515801) at flow-through, allowing endotoxin to bind to the column. The sdAb monomer flow-through fractions were analyzed by reducing SDS-PAGE and pooled. The purified C22-TYR and C22-FSY were then conjugated to AZ Dye 680 (Click Chemistry Tools, Catalog#1578-25).

[0438] AZ Dye680 (Click Chemistry Tools, Catalog#1578-25) was conjugated to C22-FSY and C22-TYR using sortase-mediated ligation to generate C23-TYR and C23-FSY. The sortase g-block sequence was codon-optimized, sequenced by IDT, cloned into pBAD vector and prepared as described (PubMed:21697512). To conjugate the sdAb construct to AZ Dye 680, 20 mg of protein purified above in PBS pH 7.4 was incubated with 1 mM CaCl2, 0.5 mM AZ Dye 680, 2 mg sortase in a shaker at 30°C (final concentration of sdAb was approximately 0.7 mg / mL). After 2 hours, the reaction was quenched by adding 2 mM MTSET. The quenched reaction was batch bound to 2 mL of Ni resin (PBS equilibrated) to remove sortase and unconjugated material, nutating at room temperature for 30 min. The beads were spun to facilitate separation of the resin from the unbound fraction (700 g x 10 min, 20°C), packed into a glass column, and the flow-through was collected by washing the beads with PBS to collect the remaining protein. The flow-through and wash were combined and concentrated (5K MWCO, 4k rcf, 10°C, 1.5 h) and loaded onto a HiLoad 26 / 600 Superdex 75 pg (Cytiva, #28-9893-34) to remove unconjugated dye. Fractions were collected, analyzed by SDS-PAGE, pooled, and stored at -80°C.

[0439] The resulting conjugates C23-TYR and C23-FSY were assessed for binding to the human epidermal squamous cell carcinoma line A431 (EGFR+) and the human colorectal carcinoma cell line COLO320DM (EGFR-) by flow cytometry. Proteins were formulated to 30 μM (10× final) with FACS buffer (1× PBS + 2% HI-FBS + 5 mM EDTA) and then serially diluted 5× (20 μL sdAb vs. 80 μL FACS buffer) to generate eight concentration points for the assay, with the lowest concentration being 0.000384 μM (10× final). Both A431 and COLO320DM cell lines (ATCC) were maintained in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher) at 37° C. in a humidified environment of 5% CO2. Cells were harvested in exponential growth phase, counted, and diluted to 1.1 x 10 in ice-cold FACS buffer. 6 The cells were resuspended at 10 μL / mL and dispensed at 90 μL / well into a v-bottom 96-well plate (Corning 3897). C23-TYR and C23-FSY proteins were added to A431 cells at 10 μL / well to a final concentration of 1x. To control for specificity of binding to EGFR+ cells, the same dilution range of test substances was added to COLO320DM cells. After 2 h incubation on ice, cells were pelleted at 2000 rpm for 2 min, then washed twice with 200 μL FACS buffer, then resuspended in 100 μL ice-cold FACS buffer and analyzed on a NovoCyte flow cytometer (ACEA / Aglient).

[0440] Raw data (FCS files) were analyzed with FlowJo 10.7.2. Total intact cells were identified using FSC-A and SSC-A, and singlets were then gated out using FSC-A versus FSC-H. The geometric mean fluorescence intensity of AZ680 of the samples was used for further analysis and plotting. The geometric mean fluorescence intensity (GeoMean or GeoMFI) of the AZ680 channel was used to calculate the EC50 using "log(agonist) vs log(agonist) vs response-variable slope (four parameters)" in GraphPad Prism (V9.3.1).

[0441] As shown in Figure 16, C23-FSY and C23-TYR bound to A431 cells in a dose-dependent manner, but did not show binding to EGFR-negative COLO320DM cells. As shown in Table 15, the binding affinity for C23-FSY and C23-TYR was measured from the EC50 values ​​after staining of A431 cells and flow cytometry.

[0442] [Table 16]

[0443] Example 20. In vivo tumor locking using targeted imaging payloads In this study, in vivo crosslinking of fluorescently labeled sdAbs was evaluated. C22-FSY and C22-TYR were modified to generate AZ680 conjugates C23-FSY and C23-TYR (SEQ ID NO: 42, 41, respectively) according to the method of Example 19. In vivo crosslinking of C23-FSY and C23-TYR to EGFR was evaluated in A431 epidermoid squamous carcinoma xenograft tumor model. Human colorectal cancer xenograft model COLO320DM was used as EGFR-control. Both cell lines were cultured according to the supplier's protocol (ATCC). On the day of injection, cells were harvested, washed in serum-free medium, counted, and resuspended in cold serum-free medium. Resuspend the A431 cell pellet in 100 mL of serum-free DMEM and 100 µL of RPMI1640, then mix the cells with 100 mL of Matrigel and solubilize at 5 × 10 6 A final concentration of 10 viable cells / 100 μL was obtained. Six to eight week old female Balb / c nude mice (Charles River) were implanted with 200 mL of 10 million A431 or COLO 320DM cells in the upper right flank. Tumor size was approximately 200 mm. 3When the rats reached 10 mg / kg, 5 mg / kg of C23-FSY, C23-TYR or vehicle control (PBS) was injected via the tail vein in a volume of 10 mL / kg. To confirm dosing accuracy, peripheral blood samples were collected by buccal bleeding 0.5 hours after dosing.

[0444] The biodistribution of the test articles was studied at two different time points as shown in Table 16. Animals were euthanized and tumors were harvested, weighed, placed on ice for ex vivo IVIS imaging, and then flash frozen for Western blot analysis.

[0445] [Table 17]

[0446] Tissue processing. Tumor pieces (approximately 30-50 mg) were weighed and minced into small pieces with a razor blade. The minced tumor tissue was placed in a CK28-R tube (Bertin Instruments, Cat. P000916LYSK0-A) and 0.5 ml of T-PER buffer (Thermo Scientific Cat. 78510) with the addition of Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Scientific Cat. 78446). The tissue was then homogenized in a Precellys 24 tissue homogenizer. After homogenization, the samples were centrifuged at 12,000 × rpm for 10 min at 4 °C. The supernatant was then retained and the pellet was discarded. The centrifugation process was repeated once more (for a total of 2 times).

[0447] Gel imaging. Protein concentrations of tumor lysates were quantified using the Pierce Rapid Gold BCA Protein Assay Kit (Thermo Scientific Cat. A53225) according to the manufacturer's instructions. Seven-point two-fold dilutions of BSA starting at 1 mg / mL BSA from the kit were used as standards. Samples were prepared to the same concentration (3.6 mg / mL) in T-PER buffer and then heated at 95 °C for 10 min in 1x reducing loading buffer to obtain a final concentration of 3 mg / mL. Then, 30 μg of total protein from each sample was loaded onto a 4–20% Criterion TGX Gel (Bio-Rad, Cat. 5671085) for electrophoresis. Images were acquired by Azure Biosystem C600 using the NIR-700 channel. Images were processed using GIMP 2.10.28.

[0448] Figure 17 shows intratumoral free and EGFR crosslinking sdAb in a time-dependent manner in A431 (EGFR+) and COLO320DM (EGFR-) tumors. Free sdAb was detected in the region of -15 kD (lower panel, labeled free VHH) and EGFR crosslinking sdAb bands were detected in the region of -175 kD (upper panel, labeled VHH-EGFR crosslink). In EGFR+ A431 tumors, time-dependent crosslinking of EGFR was observed with C23-FSY but not with C23-TYR. Neither free sdAb retention nor crosslinking was observed in EGFR- COLO320DM tumors.

[0449] Tumors were excised and imaged on an AMI HTX, and imaging data were analyzed with Aura software (Spectral Instruments Imaging, Version 4.0.7). Average radiant efficiency values ​​were obtained by creating ROIs around individual tumors (Ellipse ROI tool in Aura). Total luminescence (photons / sec) was measured and recorded. Total luminescence values ​​were normalized to tissue weight (photons / sec / g). Statistical significance was determined using a paired t-test in Prism (v9.1.0(221)) (*=p≦0.05, **=p≦0.005). Figure 18 shows ex vivo imaging of A431 and COLO320DM tumor tissues 8 and 24 hours after administration of C23-FSY and C23-TYR.

[0450] Figure 18A shows photons / sec / g tumor tissue across three biological replicates. Values ​​from individual animals are shown, with the center bar showing the mean intensity and the error bars showing SEM (*p=0.024, **p=0.002 by paired t-test). C23-FSY was present at significantly higher levels in A431 tumors compared to C23-TYR protein at both the 8 and 24 hour time points. After 24 hours, C23-TYR was observed at very low levels in A431 tumor tissue, whereas C23-FSY, especially the EGFR-VHH cross-linked species, was prominent (Figures 17, 18A). As the test articles have similar affinities (Figure 16), these results together indicate that the tumor AUC of FSY-containing sdAb can be increased by covalent binding.

[0451] FIG. 18B shows quantitative ex vivo fluorescence intensity of A431 and COLO320DM tumors from animals administered C23-FSY at 8 and 24 hours post-dose. Plots show photons / sec / g tumor tissue across triplicate biological replicates. Values ​​from individual animals are shown, with the center bar showing the mean intensity and the error bars showing SEM. C23-FSY was present at significantly higher levels in A431 tumors compared to the amount present in EGFR-COLO320DM tumors at both the 8 and 24 hour time points, demonstrating the specificity of tumor locking of the C23-FSY protein.

[0452] These results demonstrate that targeting domains such as sdAbs with cross-linking compatible unnatural amino acids can deliver payloads (here, imaging dyes) to tumors with target specificity and can improve retention of the payload at the target site compared to targeting domains without cross-linking compatible unnatural amino acids.

[0453] Example 21. PSMA Tumor Locking In Vivo Using Targeted Imaging Payloads In this study, we evaluated the in vivo PSMA cross-linking, systemic, and intratumoral exposure of a fluorescently labeled sdAb in an LNCaP mouse prostate cancer xenograft tumor model.

[0454] PSMA-targeted sdAbs conjugated to AZ Dye 680 were prepared using C29-101TYR and C29-101FSY (SEQ ID NO: 53 or 54) with either TYR or FSY at position 101 according to the method of Example 19 to generate C30-TYR and C30-FSY (SEQ ID NO: 55, 56, respectively). LNCaP cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a humidified environment of 5% CO2. On the day of implantation, cells were harvested, washed with serum-free medium, counted, and resuspended in cold serum-free medium. Cell pellets were resuspended in 50 μL serum-free RPMI, mixed with 50 μL matrigel, and diluted at 5×10 6A volume of 100 μL containing 5 million LNCaP cells was implanted into the right upper flank of male NU / J mice (Jackson Labs). Tumor size was approximately 200 mm. 3 When the tumor reached 100 mg / kg, either C30-TYR or C30-FSY was injected via the tail vein at a volume of 10 mL / kg. Peripheral blood samples were collected by buccal bleeding 0.5 hours after dosing to confirm dosing accuracy. Biodistribution of test articles was studied at eight different time points (pre-dose, 15 min, 1 h, 6 h, 24 h, 48 h, 72 h, and 96 h after dosing) as shown in Table 17. After euthanasia, tumor tissues were harvested, weighed, ex vivo IVIS imaged, and then flash frozen for Western blot analysis.

[0455] [Table 18]

[0456] Tissue processing, protein quantification and normalization were performed using the methods of Example 20. For quantification of free and PSMA cross-linked sdAb test articles, a 2x 16-point standard curve of C30-FSY was prepared in T-PER buffer starting at 500ng / mL. Standards were heated in 1x reduced loading buffer at 95°C for 10 minutes. Different ranges of standards were used for different gels based on the estimate of test article concentration in the samples. Standard curves were generated using standard band areas versus standard concentration using a linear fit function in Excel. The density of the bands in the images was determined using ImageJ 1.51j8 according to the instructions (https: / / imagej.nih.gov / ij / docs / guide / 146-30.html#infobox:Densitometry, Section 30.13). After obtaining the area of ​​the sample bands using ImageJ, the amount of TA in the lane was calculated using the formula subtracted from the linear fit of the standard curve. 30μg of total protein was loaded, and the amount of TA in the lane is also the amount of TA in 30μg of total protein. Tumor sdAb concentration (pg sdAb / mg tumor tissue) = amount of sdAb in lane (pg) * (Protein concentration (mg / ml) * (0.5ml * 1000 / 30μg) / tumor tissue weight (mg). The standard curve obtained contained at least 5 points and had an R2>0.99. Area under the curve (AUC) was calculated in GraphPad Prism using the trapezoidal rule, and the area between two adjacent points was calculated as ΔX*([(Y1+Y2) / 2]-baseline](https: / / www.graphpad.com / guides / prism / latest / statistics / stat_area_under_the_curve.htm). The AUC for C30-TYR was 1676 and the AUC for C30-FSY was 4902.

[0457] Figure 19 shows fluorescent images of SDS PAGE gels showing tumor-associated free and PSMA cross-linked sdAb in a time-dependent manner following administration of C30-TYR and C30-FSY. At the indicated time points after treatment, tumors were harvested and processed for gel electrophoresis to detect fluorophore-conjugated sdAb test articles. Free (uncross-linked) sdAb-AF680 was detected in the approx. 20 kD region (lower panel), whereas the PSMA cross-linked sdAb-AF680 species of C30-FSY migrated in the 100 kD region (upper panel). * The lanes marked with a represent vehicle samples.

[0458] Figure 20 shows quantitative analysis of tumor-associated free and PSMA crosslinked test article. Fluorescent band intensity of free and PSMA crosslinked species bands from the gels above was quantified by densitometry and comparison to a standard curve. Total intratumor test article concentration (free and PSMA crosslinked, pg / mg tumor tissue) is plotted against time (h). * Data points denoted by * indicate samples that were below the limits of detection and quantitation. Tumor exposure of C30-FSY was increased approximately 3-fold relative to non-covalent C30-TYR.

[0459] Example 22. Delivery of cytotoxic payloads C25 and C27 sdAbs were prepared with and without FSY substitutions to generate C25-54TYR, C25-54FSY, C27-101TYR and C27-101FSY (SEQ ID NOs: 45, 46, 49, 50, respectively). Proteins were expressed and purified according to the method of Example 2, except that after elution from the Ni-NTA resin, the purified proteins were then buffer exchanged into anion exchange running buffer (20 mM Tris, 7.5 and 20 mM NaCl) overnight at 4° C. using a dilution factor of 1:100. The dialyzed sample pool was passed through a HiTrap Q XL, 5 ml column (Cytiva #17515801) in flow-through mode with endotoxin bound to the column. The sdAb monomer flow-through fractions were analyzed by reducing SDS-PAGE and pooled.

[0460] The sdAb constructs were conjugated to MC-PEG8-VC-PABC-MMAE (structure shown below) to generate C26-54 TYR, C26-54 FSY, C28-101TYR and C28-101FSY. Samples were first reduced with 1 mM EDTA and 1.5 equivalents of TCEP (10 mM in deoxygenated water) and incubated at room temperature for 1 hour. After reduction, any residual TCEP was removed using a Zeba Spin desalting column (Thermo P / N 89891, 7K MWCO) with 1 mM EDTA in 1×PBS as the equilibration buffer. To improve the solubility of the linker payload, propylene glycol (PG) and dimethylacetamide (DMA) were added to the reaction mixture (Cf=10% v / v) and 1.5 equivalents of linker payload were added. The reaction was gently vortexed and incubated overnight at 4°C. The conjugation mixture was added to a pre-hydrated dialysis cassette (approximately 1 mL sample: 3500 mL buffer volume) and dialyzed for a minimum of 2 hours. The dialysis buffer was exchanged and the sample was dialyzed overnight at 4°C before being removed for analysis by A280 and analytical SEC. Unconjugated linker payload was removed using a Cytiva PD-10 column (4.3 mL Sephadex G-25 sorbent per cartridge) following a standard PD-10 spin protocol (equilibration buffer: 1X PBS, pH 7.4). After collection of the eluate, the column was rinsed with an additional 1 mL of 1X PBS, pH 7.4. After purification on the PD-10, the sample was dialyzed a final time against 50 mL of 1X PBS using a Thermo Scientific Slide-A-Lyzer mini dialysis device (3.5k MWCO). All samples were passed through a 0.2 μm sterile PES filter and stored at -20°C.

[0461] [ka]

[0462] To determine the ability of sdAb conjugates containing FSY to specifically deliver cytotoxic payloads to immortalized PSMA-expressing tumor cell lines in vitro compared to non-covalent sdAb conjugates, the cell-permeable payload monomethyl auristatin was conjugated to sdAbs with a drug-to-antibody ratio (DAR) of 1. PC3pip cell lines engineered to express PSMA, and PC3flu PSMA-negative cell lines (kindly provided by Prof. Xinning Wang and Prof. Warren D. Heston, Case Western Reserve University, Cleveland, OH) were maintained in growth medium consisting of RPMI-1640 (Thermo Scientific, 11875-903) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher, FB-12) at 37°C in a humidified atmosphere of 5% CO2. Cells were harvested in exponential growth phase using TrypLE Express (Thermo Scientific, 14175-095) and seeded at 800 cells / well in 100 μL growth medium in black 96-well flat clear bottom plates (Costar, 3603). Cells were then incubated overnight at 37°C in a humidified environment of 5% CO2 to allow cells to adhere to the plate. sdAb-MMAE conjugates listed in the table below were diluted in growth medium to 50x final concentration and diluted in 3-fold increments to generate a 10-point dilution series. A volume of 2 μL of each 50x test article dilution series was added to triplicate wells to achieve a final concentration of 1x (the highest concentration in each series shown in Table 18).

[0463] [Table 19]

[0464] Plates were returned to the incubator for 4 days, after which cell viability was assessed. CellTiter-Glo (Promega, G5737) was added to all plates at 50 μL / well, and the plates were then shaken at 1000 rpm for 1 minute at room temperature. Luminescence was then read on a Victor X5 plate reader (PerkinElmer) and reported as relative luminescence units (RLU). Viability was calculated using Excel (Microsoft® Excel® for Microsoft 365 MSO, Version 2202 Build 16.0.14931.20128, 64-bit), with % viability = 100. * RLU was calculated as (treated RLU / untreated RLU). Data was plotted as % viability versus non-test substance concentration in GraphPad Prism (V9.3.1) and IC50 was calculated using log[inhibitor] versus response-variable slope (4 parameters).

[0465] Very low levels of cytotoxic activity were observed for the sdAb-PEG8-VC-PABC-MMAE conjugate against the PC3flu cell line, consistent with the lack of PSMA target expression in this cell line. C26-54TYR had an estimated affinity of 14.9 nM for PMSA and showed a dose-dependent increase in cytotoxicity in PC3pip cells (as shown in Example 14 for the unconjugated compound in LNCaP cells), consistent with targeted delivery of the payload via PSMA binding and internalization. As shown in Table 19, C26-54FSY delivered the payload 3.57-fold more potently than C26-54TYR, demonstrating that covalent conjugation enhances the cellular potency of single domain antibody drug conjugates. In a second example, C28-101TYR, which has an affinity for PSMA >500 nM, had low levels of cytotoxic activity against PC3pip cells, whereas the crosslinking activity of C28-101FSY shifted potency by over 400-fold, confirming the effect of covalent binding on cellular potency. Comparison of the cytotoxicity of the test articles showing the concentration vs. cell viability curves of sdAb conjugated to MMAE is shown in Figure 21A (C26 construct) and Figure 21B (C28 construct). After 4 days of incubation with sdAb-PEG8-VC-PABC-MMAE conjugates, the viability of PSMA+PC3pip and PSMA-PC3flu cells was assessed by CellTiter-Glo assay. Dose-response analysis was performed in triplicate, symbols represent the mean, and errors represent STDEV.

[0466] [Table 20]

[0467] Example 23. Delivery of cytotoxic payloads to HER2 targets A HER2-targeting sdAb (C17; SEQ ID NO:31) was evaluated to identify the individual positions of FSY insertion that resulted in cross-linking to the HER2 target. The sdAbs were cloned, expressed and purified generally according to the methods of Example 2, except that specific single sites were selected for modification and testing rather than using a pooled screen. The sites selected for FSY insertion are shown in Table 20.

[0468] [Table 21]

[0469] Positions 52...

Claims

1. A conjugate comprising a targeting domain and a payload, the targeting domain comprises an antibody, antibody fragment, or antigen-binding domain having complementarity-determining regions (CDRs); the targeting domain comprises at least one unnatural amino acid (UAA) residue in or near the CDR; the targeting domain is configured to bind to a target; The conjugate, wherein the at least one UAA residue is sufficiently close to form a covalent bond with the target when the targeting domain is attached.

2. The conjugate described in claim 1, wherein the targeting domain binds to a cell surface molecule.

3. The conjugate described in claim 2, wherein the cell surface molecule is a tumor-associated antigen.

4. The conjugate described in claim 1, wherein the at least one UAA residue is configured to form a covalent bond with a histidine, lysine, or tyrosine residue of the target.

5. The conjugate described in claim 1, wherein at least one UAA residue comprises a fluorosulfate moiety.

6. The method of claim 1, wherein the at least one UAA residue is a group represented by formula I: 【Chemistry 1】 6. The conjugate of claim 5, comprising:

7. The method of claim 1, wherein the at least one UAA residue has the structure: 【Chemistry 2】 6. The conjugate of claim 5, having the formula:

8. The method of claim 7, wherein the at least one UAA residue is a residue of formula II: 【Transformation 3】 6. The conjugate of claim 5, comprising:

9. The method of claim 1, wherein the at least one UAA residue has the structure: 【Chemistry 4】 6. The conjugate of claim 5, having the formula:

10. The conjugate of claim 1, wherein the payload comprises an imaging agent, a radioligand agent, or a cytotoxic agent.

11. The conjugate described in claim 10, wherein the at least one UAA residue is contained within the CDR region.

12. The conjugate of claim 1, wherein the targeting domain comprises a single domain antibody (sdAb).

13. The conjugate of claim 12, wherein the sdAb comprises any one of SEQ ID NOs: 1-4 or 16-64.

14. A conjugate according to any one of claims 1 to 9, wherein the payload is not linked via a UAA side chain.

15. A conjugate described in any one of claims 1 to 9, further comprising a second targeting domain.

Images