DLL3-binding molecules and uses thereof

Abstract

Described herein are heavy chain antibodies that bind to DLL3, and immunoconjugates of DLL3 heavy chain antibodies, that are useful in cancer treatment.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of priority to U.S. Provisional Application No. 63 / 373,184, filed August 22, 2022, and U.S. Provisional Application No. 63,477,261, filed December 27, 2022, both of which are incorporated herein by reference in their entireties.

[0002] The excellent antigen-specificity of antibodies such as IgG makes them a highly targeted platform for therapeutics. However, the typical serum half-life of at least three weeks for IgG is unfavorable for the delivery of radioisotopes, including α-emitting isotopes such as Ac-225 and β-emitting isotopes such as Lu-177 and Y-90, particularly due to prolonged exposure and chronic off-target toxicities. 225-Ac is the most cytotoxic of α-emitting radioisotopes, and a single decay event can effectively destroy cancer cells by causing double-stranded DNA breaks and subsequent cell death. The potency of α-emitting radioisotopes makes them attractive as cell killing agents, potentially overcoming acquired resistance observed in response to other therapies.

[0003] Furthermore, there are additional issues with targeted radioscope delivering platforms, including alpha and beta-emitting isotopes, that require simultaneous optimization when designing the platform, such as immunogenicity, specificity, tissue penetration, stability, ease of manufacture, and acceptable therapeutic window. Summary of the Invention

[0004] The present disclosure relates to DLL3-binding molecules (e.g., VHHs). The disclosure further relates to immunoconjugates or radioimmunoconjugates, compositions comprising DLL3-binding regions, and methods of using such immunoconjugates and compositions. These DLL3-binding molecules and antigen-binding regions can be advantageously formed as VHH-Fc containing molecules that 1) have a reduced size, allowing for greater tissue penetration, and 2) have altered FcRn binding, allowing for a reduced serum half-life, which reduces radiation toxicity.

[0005] The present disclosure addresses several challenges inherent in targeted delivery of alpha particle emitters in vivo through the selection and specific combination of specific delivery platform components. The alpha particle-emitting radioisotope delivery platform of the present disclosure offers a shorter half-life compared to conventional IgG, but a longer half-life than smaller monomeric antibody fragment formats. Such a half-life allows for reduced toxicity from the alpha emitter while preserving the antibody fragment long enough to exert therapeutic activity in the body. For example, the alpha particle-emitting radioisotope delivery platform of the present disclosure exhibits enhanced tumor targeting and reduced accumulation in radiation-sensitive tissues such as bone marrow and kidney. Furthermore, surprisingly, the alpha particle-emitting radioisotope delivery platform of the present disclosure exhibits excellent tumor binding and labeling properties for tumors with different antigen densities, which can be a limitation for some uses of some immunoconjugates.

[0006] In one aspect, the present specification describes a polypeptide comprising an antigen-binding region that binds to DLL3, wherein the antigen-binding region is selected from the group consisting of (a) a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of any one of SEQ ID NOs: 107 to 109, 207 to 209, 307 to 309, 407 to 409, and 507 to 509; (b) a heavy chain complementarity-determining region 2 (HCDR3) comprising the amino acid sequence of any one of SEQ ID NOs: 110 to 112, 210 to 212, 310 to 312, 313 to 314, 315 to 316, 317 to 318, 319 to 320, 321 to 322, 322 to 323, 323 to 324, 324 to 325, 325 to 326, 326 to 327, 327 to 328, 328 to 329, 330 to 331, 332 to 333, 334 to 335, 336 to 337, 338 to 339, 339 to 340, 341 to 342, 342 to 343, 343 to 344, 345 to 346, 347 to 348, 348 to 349, 350 to 351, 352 to 353, 353 to 354, 354 to 355, 355 to 356, 357 to 357, 358 to 359, 360 to 361, and / or (c) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 410 to 412, or 510 to 512, and / or (d) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 113 to 115, 213 to 215, 313 to 315, 413 to 415, 513 to 515, SEQ ID NO: 131, SEQ ID NO: 231, SEQ ID NO: 431, or SEQ ID NO: 531. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to that set forth in any one of SEQ ID NOs: 101-106, 201-206, 301-306, 401-306, and 501-506. In certain embodiments, the antigen-binding region comprises: (a) a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 107-109; (b) a heavy chain complementarity-determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 110-112; and (c) a heavy chain complementarity-determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 113-115 or 131. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 101 to 106. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein:The heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NOs: 101 to 106. In certain embodiments, the antigen-binding region comprises (a) heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of any one of SEQ ID NOs: 207 to 209, (b) heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence of any one of SEQ ID NOs: 210 to 212, and (c) heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence of any one of SEQ ID NOs: 213 to 215 or 231. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 201 to 206. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 201 to 206. In certain embodiments, the antigen-binding region comprises (a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 307 to 309, (b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 310 to 312, and (c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 313 to 315. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 301 to 306. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 301 to 306. In certain embodiments, the antigen-binding region comprises (a) a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 407 to 409, (b) a heavy chain complementarity-determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 410 to 412,(c) comprises a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 413 to 415 or 431. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to that set forth in any one of SEQ ID NOs: 401 to 406. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 401 to 406. In certain embodiments, the antigen-binding region comprises: (a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 507 to 509; (b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 510 to 512; and (c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 513 to 515 or 531. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 501 to 506. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 501 to 506. In certain embodiments, the antigen-binding region is humanized. In certain embodiments, the antigen-binding region does not comprise an immunoglobulin light chain. In certain embodiments, the antigen-binding region comprises a VHH. In certain embodiments, the polypeptide comprises an immunoglobulin heavy chain constant region. In certain embodiments, the immunoglobulin heavy chain constant region comprises an immunoglobulin CH2 domain, an immunoglobulin CH3 domain, or an immunoglobulin CH2 and CH3 domains. In certain embodiments, the immunoglobulin heavy chain constant region comprises an immunoglobulin CH2 and CH3 domains. In certain embodiments, the immunoglobulin heavy chain constant region is an IgA, IgG1, IgG2, IgG3, or IgG4 isotype. In certain embodiments,The immunoglobulin heavy chain constant region is of the IgG1 isotype. In certain embodiments, the immunoglobulin heavy chain constant region is of the IgG4 isotype. In certain embodiments, the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region or alters binding of the polypeptide to the neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region and alters binding of the polypeptide to the neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region. In certain embodiments, the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that alters binding of the polypeptide to the neonatal Fc receptor (FcRn). In certain embodiments, the changes to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region are changes that reduce complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or a combination thereof. In certain embodiments, the changes to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region are (a) 297A, 297Q, 297G, or 297D, (b) 279F, 279K, or 279L, (c) 228P, (d) 235A, 235E, 235G, 235Q, 235R, or 235S, (e) 237A, 237E, 237K, 237N, or 237R, (f) 234A, 234V, or 234F, (g) 233P, (h) 328A, (i) 327Q, or 327T, (j) 328A, (k) 328B, (j) 328C, or 328D, (k) 328E, (k) 328F, (k) 328G, or (k) 328H, (k) 328H, or ... j) 329A, 329G, 329Y or 329R (k) 331S, (l) 236F or 236R, (m) 238A, 238E, 238G, 238H, 238I, 238V, 238W or 238Y, (n) 248A, (o) 254D, 254E, 254G, 254H, 254I, 254N, 254P, 254Q, 254T or 254V, (p) 255N, (q) 256H, 256K, 256R or 256V, (r) 264S, (s) 265H, 265K, 265S, 265Y or 265A,(t) 267G, 267H, 267I or 267K, (u) 268K, (v) 269N or 269Q, (w) 270A, 270G, 270M or 270N, (x) 271T, (y) 272N, (z) 292E, 292F, 292G or 292I, (aa) 293S, (bb) 301W, (cc) 304E, (dd) 311E, 311G or 311S, (ee) 316F, (ff) 328V, (gg) 330R, (hh) 339E or 339L, (ii) 343I or 343 V, (jj) 373A, 373G, or 373S, (kk) 376E, 376W, or 376Y, (ll) 380D, (mm) 382D or 382P, (nn) 385P, (oo) 424H, 424M, or 424V, (pp) 434I, (qq) 438G, (rr) 439E, 439H, or 439Q, (ss) 440A, 440D, 440E, 440F, 440M, 440T, or 440V, (tt) K322A, (uu) L235E, (vv) L234A and L235A , (ww) L234A, L235A and G237A, (xx) L234A, L235A and P329G, (yy) L234F, L235E and P331S, (zz) L234A, L235E and G237A, (aaa) L234A, L235E, G237A and P331S (bbb) L234A, L235A, G237A, P238S, H268A, A330S and P331S, (ccc) L234A, L235A and P329A, (ddd) G236R and L328R, ( (eee) G237A, (fff) F241A, (ggg) V264A, (hhh) D265A, (iii) D265A and N297A, (jjj) D265A and N297G, (kkk) D270A, (ll) A330L, (mmm) P331A or P331S, or (nnn) E233P, (ooo) L234A, L235E, G237A, A330S, and P331S, or (ppp) any combination of (a)-(ppp) according to EU numbering. In certain embodiments, the changes to one or more amino acid residues that reduce an effector function of an immunoglobulin heavy chain constant region comprise L234A, L235E, G237A, A330S, and P331S according to EU numbering. In certain embodiments,The amino acid changes to one or more amino acid residues that alter binding of the polypeptide to the neonatal Fc receptor (FcRn) decrease the serum half-life of the polypeptide. In certain embodiments, the changes to one or more amino acid residues that alter binding of the polypeptide to the neonatal Fc receptor (FcRn) are 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 28 In certain embodiments, the alteration to one or more amino acid residues that alters binding of the polypeptide to a neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of 253, 254, 310, 435, 436, 439, 447, and combinations thereof, according to EU numbering. In certain embodiments, the changes to one or more amino acid residues that alter binding of the polypeptide to the neonatal Fc receptor (FcRn) are to amino acid residues selected from the list consisting of I253A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof, according to EU numbering. In certain embodiments, the changes to one or more amino acid residues that alter binding of the polypeptide to the neonatal Fc receptor (FcRn) are to amino acid residues selected from the list consisting of I253A, S254A, H310A, H435Q, Y436A, and combinations thereof, according to EU numbering. In certain embodiments, the changes to one or more amino acid residues that alter binding of the polypeptide to the neonatal Fc receptor (FcRn) are to amino acid residues selected from the list consisting of I253A, H310A, H435Q, and combinations thereof, according to EU numbering. In certain embodiments, the alteration to one or more amino acid residues that alters binding of the polypeptide to the neonatal Fc receptor (FcRn) comprises I253A according to EU numbering. In certain embodiments, the alteration to one or more amino acid residues that alters binding of the polypeptide to the neonatal Fc receptor (FcRn) comprises H310A according to EU numbering. In certain embodiments, the alteration to one or more amino acid residues that alters binding of the polypeptide to the neonatal Fc receptor (FcRn) comprises H435Q according to EU numbering. In certain embodiments, the antigen-binding region comprises a linker amino acid sequence or a human IgG hinge region, in certain embodiments, the antigen-binding region is connected to the immunoglobulin heavy chain constant region by a human IgG hinge region.In certain embodiments, the human IgG hinge region comprises the amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 116-120, 216-220, 316-320, 416-420, and 516-520. In certain embodiments, the polypeptide comprises an amino acid sequence that is identical to any one of SEQ ID NOs: 116-120, 216-220, 316-320, 416-420, and 516-520. In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NOs: 116-120. In certain embodiments, the polypeptide comprises an amino acid sequence identical to SEQ ID NO:116-120. In certain embodiments, the polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:216-220. In certain embodiments, the polypeptide comprises an amino acid sequence identical to SEQ ID NO:216-220. In certain embodiments, the polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:316-320. In certain embodiments, the polypeptide comprises an amino acid sequence identical to SEQ ID NO:316-. In embodiments, the polypeptide comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:416-420. In certain embodiments, the polypeptide comprises an amino acid sequence identical to SEQ ID NO: 416-420. In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 516-520.In certain embodiments, the polypeptide comprises an amino acid sequence identical to SEQ ID NO:516-520. In certain embodiments, the polypeptide has a KD of 10 nanomolar or less. In certain embodiments, the polypeptide has a KD of 5 nanomolar or less. In certain embodiments, the polypeptide has a KD of 2 nanomolar or less. In certain embodiments, the polypeptide has a KD of 1 nanomolar or less. In certain embodiments, the polypeptide has a KD of 1 nanomolar or less. In certain embodiments, the immunoconjugate described herein is an immunoconjugate comprising a polypeptide and a chelating agent. In certain embodiments, the molecular weight of the immunoconjugate is 60-110 kDa. In certain embodiments, the immunoconjugate has a serum half-life of less than 15 days. In certain embodiments, the immunoconjugate has a serum half-life of less than 10 days. In certain embodiments, the immunoconjugate has a serum half-life of less than 120 hours. In certain embodiments, the immunoconjugate has a serum half-life of less than 72 hours. In certain embodiments, the chelating agent is a radioisotope chelating agent. In certain embodiments, the chelating agent is an alpha-emitter chelating agent. In certain embodiments, the chelating agent is a beta-emitter or a gamma-emitter. In certain embodiments, the chelating agent is selected from the list consisting of DOTA, DO3A, DOTAGA, DOTAGA anhydride, Py4Pa, Py4Pa-NCS, Crown, Macropa, Macropa-NCS, HEHA, CHX Octapa, Bispa, Noneunpa, and combinations thereof. In certain embodiments, the chelating agent is DOTMA, DOTPA, DO3AM-acetate, DOTP, DOTMP, DOTA-4AMP, CB-TE2A, NOTA, NOTP, TETPA, TETA, PEPA, H4 Octapa, HDedpa, DO2P, EDTA, DTPA-BMA, 3,2,3-LI(HOPO), 3,2-HOPO, Neunpa, Neunpa-NCS, Octapa, PyPa, porphyrin, deferoxamine, or DFO. *and combinations thereof. In certain embodiments, the chelator is DOTA. In certain embodiments, the chelator is DOTAGA. In certain embodiments, the chelator is Py4Pa. In certain embodiments, the chelator is directly attached to the antigen binding region and / or the immunoglobulin heavy chain constant region. In certain embodiments, the chelator is attached to the antigen binding region and / or the immunoglobulin heavy chain constant region by a linker. In certain embodiments, the linker is selected from those resulting from conjugation with 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and the linker reagents: N-succinimidyl 4-(2-pyridylthio)pentanoate forming linker moiety 4-mercaptopentanoic acid (SPP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB), polyethylene glycol (PEG), polyethylene glycol polymer (PEGn), and S-2-(4-isothiocyanatobenzyl) (SCN). In certain embodiments, the linker is selected from polyethylene glycol (PEG), polyethylene glycol polymer (PEG), and S-2-(4-isothiocyanatobenzyl) (SCN). In certain embodiments, the linker is PEG5. In certain embodiments, the linker is SCN. In certain embodiments, the chelator is a linker-chelator selected from the list consisting of TFP-Ad-PEG5-DOTAGA, p-SCN-Bn-DOTA, p-SCN-Ph-Et-Py4Pa, and TFP-Ad-PEG5-Ac-Py4Pa. In certain embodiments, the chelator is TFP-Ad-PEG5-DOTAGA. In certain embodiments, the chelator is p-SCN-Bn-DOTA. In certain embodiments, the chelator is p-SCN-Ph-Et-Py4Pa.In certain embodiments, the chelating agent is TFP-Ad-PEG5-Ac-Py4Pa. In certain embodiments, the chelating agent is conjugated to the antigen-binding region and / or the immunoglobulin heavy chain constant region in a ratio of 1:1 to 8:1. In certain embodiments, the chelating agent is conjugated to the antigen-binding region and / or the immunoglobulin heavy chain constant region in a ratio of 1:1 to 6:1. In certain embodiments, the chelating agent is conjugated to the antigen-binding region and / or the immunoglobulin heavy chain constant region in a ratio of 2:1 to 6:1. In certain embodiments, the immunoconjugate further comprises a radioisotope. In certain embodiments, the radioisotope is an alpha emitter. In certain embodiments, the radioisotope is an alpha emitter selected from the list consisting of 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi. In certain embodiments, the radioisotope is 225-Ac. In certain embodiments, the radioisotope is a β-emitter. In certain embodiments, the radioisotope is a β-emitter selected from 177-Lu, 90-Y, 67-Cu, and 153-Sm. In certain embodiments, the radioisotope is a gamma-emitter. In certain embodiments, the radioisotope is a γ-emitter selected from 111-In, 89-Zn, 123-I, 99m-Tc, and 68-Ga. In certain embodiments, the molecular weight of the immunoconjugate is 60-100 kDa. In certain embodiments, the molecular weight of the immunoconjugate is 60-90 kDa. In certain embodiments, the molecular weight of the immunoconjugate is 65-90 kDa. In certain embodiments, the molecular weight of the immunoconjugate is 70-90 kDa. In certain embodiments, the immunoconjugate forms a dimer with another immunoconjugate. In certain embodiments, the immunoconjugate further comprises a pharmaceutically acceptable excipient or carrier. In certain embodiments, the immunoconjugate is formulated for intravenous administration.

[0007] Also described herein are methods for producing immunoconjugates, in which the immunoconjugate is loaded with a radioisotope. In certain embodiments, the radioisotope is an alpha emitter. In certain embodiments, the radioisotope is an alpha emitter selected from the list consisting of 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi. In certain embodiments, the radioisotope is 225-Ac. In certain embodiments, the radioisotope is a beta emitter. In certain embodiments, the radioisotope is a beta emitter selected from 177-Lu, 90-Y, 67-Cu, and 153-Sm. In certain embodiments, the radioisotope is 177-Lu. In certain embodiments, the radioisotope is a gamma ray emitter. In certain embodiments, the radioisotope is a gamma emitter selected from 111-In, 89-Zn, 123-I, 99m-Tc, and 68-Ga.

[0008] Also described herein are methods of treating cancer or tumor in an individual, the method comprising administering an immunoconjugate to the individual, thereby treating the cancer or tumor. In certain embodiments, the individual is a human. In certain embodiments, the cancer or tumor is a solid cancer or tumor. In certain embodiments, the cancer or tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer. In certain embodiments, the method further comprises administering 0.5 μCi to 30.0 μCi per kilogram to the individual. In certain embodiments, the cancer or tumor expresses an antigen that is specifically bound by the immunoconjugate.

[0009] Also described herein are immunoconjugates for use in methods of treating cancer or tumors in an individual. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer or tumor is a solid cancer or tumor. In certain embodiments, the cancer or tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer. In certain embodiments, 0.5 μCi to 30.0 μCi per kilogram is administered to the individual. In certain embodiments, the cancer or tumor expresses an antigen that is specifically bound by the immunoconjugate.

[0010] Also described herein are methods of killing cancer cells in an individual, the method comprising administering an immunoconjugate to the individual, thereby killing the cancer cells. In certain embodiments, the individual is a human. In certain embodiments, the cancer cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells. In certain embodiments, the method comprises administering to the individual between 0.1 μCi and 30.0 μCi per kilogram. In certain embodiments, the method comprises administering to the individual between 10 mCi and 75 mCi per square meter of body surface area. In certain embodiments, the cancer cells express an antigen that is specifically bound by the immunoconjugate.

[0011] Also described herein is the use of an immunoconjugate in a method for killing cancer cells in an individual. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells. In certain embodiments, the method comprises administering 0.5 μCi to 30.0 μCi per kilogram to the individual. In certain embodiments, the cancer cells express an antigen that is specifically bound by the immunoconjugate.

[0012] Also described herein are methods for delivering a radioisotope to cancer or tumor cells in an individual, the method comprising administering an immunoconjugate to the individual, thereby delivering the radioisotope to the cancer or tumor cells. In certain embodiments, the individual is a human. In certain embodiments, the cancer or tumor cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells. In certain embodiments, the method comprises administering 0.5 μCi to 30.0 μCi per kilogram to the individual. In certain embodiments, the cancer or tumor cells express an antigen that is specifically bound by the immunoconjugate.

[0013] Also described herein are immunoconjugates for use in delivering radioisotopes to cancer or tumor cells in an individual. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer or tumor cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells. In certain embodiments, the cancer or tumor cells express an antigen that is specifically bound by the immunoconjugate.

[0014] Also described herein are methods for imaging a tumor in an individual, comprising administering an immunoconjugate to the individual. In certain embodiments, the individual is a human. In certain embodiments, the cancer or tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer. In certain embodiments, the tumor expresses an antigen specifically bound by the immunoconjugate.

[0015] Also described herein are immunoconjugates for use in methods for imaging tumors in individuals. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer or tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer. In certain embodiments, the tumor expresses an antigen specifically bound by the immunoconjugate.

[0016] Also described are nucleic acids encoding the immunoconjugates. In certain embodiments, an expression vector comprises the nucleic acid. In certain embodiments, an A cell comprises the nucleic acid or the expression vector. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a CHO cell.

[0017] In some embodiments, the subject radioisotope delivery platforms have a molecular size large enough (e.g., 60 kDa-110 kDa) to substantially reduce off-target toxicities, particularly nephrotoxicity (e.g., from the α-radioisotope cargo), and a small enough size to maintain target specificity and increase tissue penetration compared to conventional IgG, increasing the probability of a first decay event in the target tissue. Such a size allows for preferential clearance by the liver, as opposed to the kidney, protecting the kidney from radiation toxicity.

[0018] In some embodiments, the subject radioisotope delivery platforms are useful for the safe and effective targeted delivery of alpha emitters in vivo, in part by reducing certain adverse effects caused by platforms having half-lives over 5 days and / or molecular weights less than 60 kDa.

[0019] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description and appended claims. The foregoing elements of the present disclosure may be individually combined or freely removed to create other embodiments of the present disclosure, without any reference hereinafter to such combinations or removals. [Brief explanation of the drawings]

[0020] [Figure 1A] Binding of anti-HER2 and anti-DLL3 VHH-Fc constructs is shown. [Figure 1B] Binding of anti-HER2 and anti-DLL3 VHH-Fc constructs is shown. [Figure 2A]Binding of anti-HER2 and anti-DLL3 VHH-Fc constructs to cells expressing HER2 and / or DLL3 is shown. [Figure 2B] Binding of anti-HER2 and anti-DLL3 VHH-Fc constructs to cells expressing HER2 and / or DLL3 is shown. [Figure 2C] Binding of anti-HER2 and anti-DLL3 VHH-Fc constructs to cells expressing HER2 and / or DLL3 is shown. [Figure 3A] 1 shows the internalization of anti-HER2 and anti-DLL3 VHH-Fc constructs in cells expressing HER2 and DLL3. [Figure 3B] 1 shows the internalization of anti-HER2 and anti-DLL3 VHH-Fc constructs in cells expressing HER2 and DLL3. [Figure 4] Self-interaction data for anti-HER2 and anti-DLL3 VHH-Fc constructs are shown. [Figure 5] 1 shows a diagram of the chemical synthesis of the linker molecule. [Figure 6] 1 shows a diagram of the chemical synthesis of the linker molecule. [Figure 7A] The immunoreactive fractions of the different VHH-Fc constructs are shown. [Figure 7B] The immunoreactive fractions of the different VHH-Fc constructs are shown. [Figure 7C] The immunoreactive fractions of the different VHH-Fc constructs are shown. [Figure 8] A comparison of imaging with 111In-labeled VHH-Fc compared to the biodistribution of 225Ac-labeled VHH-Fc is shown. [Figure 9A] 1 shows the biodistribution of labeled anti-HER2 VHH-Fc constructs over time. [Figure 9B] 1 shows the biodistribution of labeled anti-HER2 VHH-Fc constructs over time. [Figure 9C] 1 shows the biodistribution of labeled anti-HER2 VHH-Fc constructs over time. [Figure 9D]1 shows the biodistribution of labeled anti-HER2 VHH-Fc constructs over time. [Figure 10A] Tumor:non-tumor tissue ratios are shown for labeled anti-HER2 VHH-Fc constructs. [Figure 10B] Tumor:non-tumor tissue ratios are shown for labeled anti-HER2 VHH-Fc constructs. [Figure 10C] Tumor:non-tumor tissue ratios are shown for labeled anti-HER2 VHH-Fc constructs. [Figure 11] 1 shows the biodistribution of labeled anti-HER2 VHH-Fc constructs. [Figure 12] Figure 1 shows the systemic clearance of 111In-labeled VHH-Fc (H101) and VHH-Fc variants (H105, H107, and H108). [Figure 13] 1 shows the biodistribution of labeled anti-DLL3 VHH-Fc constructs over time. [Figure 14] 1 shows the biodistribution of labeled anti-DLL3 VHH-Fc constructs. [Figure 15A] Figure 15 shows the biodistribution of 225Ac-labeled anti-HER2 (15A) and anti-DLL3 (15B) VHH-Fc constructs. [Figure 15B] Figure 15 shows the biodistribution of 225Ac-labeled anti-HER2 (15A) and anti-DLL3 (15B) VHH-Fc constructs. [Figure 16A] 1 shows the results of a toxicity study performed with 225Ac-labeled anti-HER2 VHH-Fc constructs. [Figure 16B] 1 shows the results of a toxicity study performed with 225Ac-labeled anti-HER2 VHH-Fc constructs. [Figure 16C] 1 shows the results of a toxicity study performed with 225Ac-labeled anti-HER2 VHH-Fc constructs. [Figure 17] Immunoreactive fractions of different anti-DDL3 VHH-Fc constructs loaded with 177Lu are shown. [Figure 18] 1 shows the chemical structures of certain linker-chelators described herein. [Figure 19]1 shows the immunogenicity scores for selected clones described herein. [Figure 20A] Figure 1 shows SHP-77 cell binding of humanized VHHFc. [Figure 20B] Figure 1 shows SHP-77 cell internalization of humanized VHHFc. [Figure 21] Figure 1 shows humanized VHHFc conjugate SHP-77 cell binding. [Figure 22] Figure 1 shows humanized VHHFc conjugate SHP-77 cell internalization. [Figure 23] Results from a membrane protein array are shown. [Figure 24] 1 shows the biodistribution of In-111 radiolabeled humanized VHHFc in non-tumor-bearing mice. [Figure 25] Biodistribution of In-111 radiolabeled humanized VHHFc in NCI-H82 tumor-bearing mice. [Figure 26] 1 shows a comparison of In-111 radiolabeled humanized VHHFc tissue activity concentrations in NCI-H82 tumor-bearing mice. [Figure 27] 1 shows the biodistribution of Acc-225 radiolabeled humanized VHHFc in NCI-H82 tumor-bearing nude mice. [Figure 28] 1 shows the biodistribution of Ind-111 labeled VHHFc in SHP-77 tumor-bearing SCID beige mice. [Figure 29] 1 shows tumor growth and survival with a single dose of Lu-177 labeled 126_zu2 in NCI-H82 SCLC tumor-bearing mice. [Figure 30] 1 shows tumor growth and survival in SHP-77SCLC tumor-bearing mice with a single dose of Ac-225 labeled 126_zu2. [Figure 31] 1 shows the structure of hDLL3 (SEQ ID NO: 532). The N-terminal domain is represented by aa 27-175 (italics), the DSL domain is aa 176-215 (bold underlined), and EGF1-6 is aa 216-492 (normal text). [Figure 32]Epitope mapping of different VHH binding clones is shown. Detailed Description of the Invention

[0021] The present invention will now be described in more detail using illustrative, non-limiting embodiments. However, the present invention may be embodied in many different forms and should not be construed as being limited to the embodiments set forth below. Rather, these embodiments are provided so that this disclosure will be thorough and will convey the scope of the invention to those skilled in the art. In order that the present invention may be more readily understood, certain terms are defined below. Further definitions can be found in the detailed description of the invention.

[0022] In one embodiment, the present specification describes binding molecules and binding regions that specifically bind to DLL3. These binding regions can further be a) an immunoglobulin hinge region, an immunoglobulin Fc region, or both (e.g., VHH-Fc). The VHH-Fc of the present disclosure can dimerize (via their respective Fc regions) to form bivalent binding molecules. These bivalent VHH-Fc can be further conjugated with a cytotoxic moiety (e.g., a radionuclide) via a chelator bound to the bivalent VHH-Fc. Certain radionuclides, such as α- or β-emitters, can be loaded onto the chelator so that the bivalent VHH-Fc can be used to target tumors for imaging or therapeutic purposes.

[0023] In particular, in embodiments, the present disclosure addresses several challenges inherent in targeted delivery of radioisotopes in vivo through the selection and specific assembly of specific immunoconjugate and radioimmunoconjugate components. The radioisotope delivery platform of the present disclosure provides a shorter half-life compared to traditional IgG, but a longer half-life than smaller monomeric antibody fragment formats. In some embodiments, the subject radioisotope delivery platform has a molecular size (e.g., 60 kDa-110 kDa) large enough to substantially reduce off-target toxicity, particularly renal damage (e.g., from α- or β-emitting isotope cargoes), while maintaining target specificity and increasing the probability of a first decay event in the target tissue, and a size small enough to increase tissue penetration compared to traditional IgG. In some embodiments, the subject radioisotope delivery platforms are useful for safe and effective targeted in vivo delivery of radioisotopes (such as α-emitters or β-emitters), in part by reducing certain adverse effects caused by platforms with half-lives greater than 5 days and / or molecular weights less than 60 kDa. In some embodiments, the subject radioisotope delivery platforms are useful for safe and effective targeted in vivo delivery of radioisotopes (such as α-emitters or β-emitters), in part by exhibiting reduced loss of targeting ability due to radiolysis, compared to other possible delivery platforms. In some embodiments, the subject radioisotope delivery platforms are useful for safe and effective targeted in vivo delivery of radioisotopes (e.g., α-emitters or β-emitters), in part by exhibiting increased stability in manufacturing under temperatures required for certain radiolabeling processes (e.g., high-temperature chelation with certain chelators), compared to other possible delivery platforms that use antibody fragments.

[0024] Immunoconjugates In one aspect, the present disclosure provides immunoconjugates that specifically bind to target antigens with high affinity. In some embodiments, the present disclosure provides immunoconjugates that specifically bind to cell surface antigens of cancer cells. In some embodiments, the immunoconjugates comprise three, four, five, six, or more CDRs or HVRs (Kabat). In some embodiments, the immunoconjugates have a K of ≦1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM. D binds to a specific antigen and / or epitope with an affinity characterized by (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 M). In certain embodiments, the polypeptide or immunoconjugate has a KD of 10 nanomolar or less. In certain embodiments, the polypeptide or immunoconjugate has a KD of 5 nanomolar or less. In certain embodiments, the polypeptide or immunoconjugate has a KD of 2 nanomolar or less. In certain embodiments, the polypeptide or immunoconjugate has a KD of 1 nanomolar or less. In certain embodiments, the polypeptide or immunoconjugate has a KD of 0.1 nanomolar or greater. In certain embodiments, the polypeptide or immunoconjugate has a KD of 0.5 nanomolar or greater.

[0025] In one embodiment, an immunoconjugate of the present disclosure comprises a) a DLL3 antigen-binding region and b) an immunoglobulin heavy chain constant region. In one embodiment, an immunoconjugate of the present disclosure comprises a) a DLL3 antigen-binding region, b) an immunoglobulin heavy chain constant region, and c) a chelating agent. In one embodiment, an immunoconjugate of the present disclosure comprises a) a DLL3 antigen-binding region, b) an immunoglobulin heavy chain constant region, and c) a radioisotope chelating agent. In one embodiment, an immunoconjugate of the present disclosure comprises a) an antigen-binding region, b) an immunoglobulin heavy chain constant region, and c) a radioisotope chelating agent, wherein the molecular weight of the immunoconjugate is 60 to 110 kDa.

[0026] In one embodiment, an immunoconjugate of the present disclosure comprises a) a VHH antigen-binding region that specifically binds to DLL3, and b) an immunoglobulin heavy chain constant region. In one embodiment, an immunoconjugate of the present disclosure comprises a) a VHH antigen-binding region that specifically binds to DLL3, b) an immunoglobulin heavy chain constant region, and c) a chelating agent. In one embodiment, an immunoconjugate of the present disclosure comprises a) a VHH antigen-binding region that specifically binds to DLL3, b) an immunoglobulin heavy chain constant region, and c) a radioisotope chelating agent. In one embodiment, an immunoconjugate of the present disclosure comprises a) a VHH antigen-binding region that specifically binds to DLL3, b) an immunoglobulin heavy chain constant region, and c) a radioisotope chelating agent, wherein the molecular weight of the immunoconjugate is 60 to 110 kDa.

[0027] In one embodiment, an immunoconjugate of the present disclosure comprises a) a VHH antigen-binding region and b) a variant immunoglobulin Fc region. In one embodiment, an immunoconjugate of the present disclosure comprises a) a VHH antigen-binding region that specifically binds to DLL3, b) a variant immunoglobulin Fc region, and c) a chelator. In one embodiment, an immunoconjugate of the present disclosure comprises a) a VHH antigen-binding region that specifically binds to DLL3, b) a variant immunoglobulin Fc region, and c) a radioisotope chelator. In one embodiment, an immunoconjugate of the present disclosure comprises a) a VHH antigen-binding region that specifically binds to DLL3, b) a variant immunoglobulin Fc region, and c) a radioisotope chelator, wherein the molecular weight of the immunoconjugate is 60 to 110 kDa. In certain embodiments, the variant immunoglobulin Fc region comprises one or more amino acid changes to decrease the serum half-life or plasma half-life of the immunoconjugate. In certain embodiments, the variant immunoglobulin Fc region comprises one or more amino acid changes to decrease the serum or plasma half-life of the immunoconjugate and decrease an Fc effector function (e.g., ADCC, CDC, or ADCP).

[0028] In some embodiments, the radioisotope delivery platform has a size greater than about 60 kDa to avoid certain toxicities, such as off-target nephrotoxicity, from the α-radioisotope cargo. In some embodiments, the radioisotope delivery platform has a size less than about 110 kDa to improve tumor penetration. In some embodiments, the radioisotope delivery platform has a size of 60-110 kDa due to its dimeric structure of two individual antigen-binding arms, each with a VHH polypeptide fused to a hinge region and a wild-type or mutant constant region. In some embodiments, the mutant constant region has specific amino acid substitutions relative to the wild-type Fc region to reduce half-life and / or eliminate Fc effector function.

[0029] In one embodiment, the antibody construct of the immunoconjugate consists of two antigen-binding arms covalently linked to one another (e.g., via disulfide bonds between associated heavy chain constant regions or immunoglobulin hinge regions). Each antigen-binding arm independently consists of an antigen-binding region, a hinge region, and a variant constant region. Within each antigen-binding arm, the antigen-binding region of the arm is covalently linked to the hinge region of the arm, which in turn is covalently linked to the variant constant region of the arm, such that the hinge region is interposed between the antigen-binding region and the variant constant region in the antigen-binding arm, thereby linking the antigen-binding region and the variant constant region. In certain embodiments, the variant constant region comprises one or more amino acid changes to decrease the serum half-life or plasma half-life of the immunoconjugate and decrease an Fc effector function (e.g., ADCC, CDC, or ADCP).

[0030] DLL3 antigen binding region Described herein are polypeptides that bind to Delta-like protein 3 (DLL3). In certain embodiments, the polypeptide binds to human DLL3. In certain embodiments, the polypeptide binds to cynomolgus monkey DLL3. The sequence of the human DLL3 protein is available, for example, at www.uniprot.org entry Q9NYJ7. In certain embodiments, the DLL3-binding polypeptide comprises a heavy chain binding region, such as a VHH. In certain embodiments, the DLL3-binding polypeptide does not comprise an immunoglobulin light chain.

[0031] In certain embodiments, the polypeptides described herein comprise an antigen-binding region that binds to DLL3, and the antigen-binding region comprises (a) a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 107 to 109, 207 to 209, 307 to 309, 407 to 409, or 507 to 509; (b) a heavy chain complementarity-determining region 2 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 110 to 112, 210 to 212, 310 to 312, 313 to 314, 315 to 316, 317 to 318, 319 to 320, 321 to 322, 322 to 323, 323 to 324, 324 to 325, 325 to 326, 326 to 327, 327 to 328, 328 to 329, 330 to 331, 332 to 333, 334 to 335, 336 to 337, 338 to 339, 339 to 340, 341 to 342, 342 to 343, 343 to 344, 345 to 346, 347 to 348, 348 to 349, 350 to 351, 352 to 353, 353 to 354, 354 to 355, 355 to 356, 357 to 357, 358 to 359, 359 to 360, 361 and / or (c) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 410 to 412, or 510 to 512, and / or (d) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 113 to 115, 213 to 215, 313 to 315, 413 to 415, 513 to 515, SEQ ID NO: 131, SEQ ID NO: 231, SEQ ID NO: 431, or SEQ ID NO: 531. In certain embodiments, the polypeptides described herein comprise an antigen-binding region that binds to DLL3, and the antigen-binding region comprises (a) a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 107 to 109, 207 to 209, 307 to 309, 407 to 409, or 507 to 509; (b) a heavy chain complementarity-determining region 2 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 110 to 112, 210 to 212, 213 to 214, 215 to 216, 217 to 218, 219 to 220, 221 to 222, 222 to 223, 223 to 224, 224 to 225, 225 to 226, 226 to 227, 227 to 228, 228 to 229, 230 to 231, 232 to 233, 234 to 235, 236 to 237, 238 to 239, 240 to 241, 242 to 243, 243 to 244, 245 to 246, 247 to 248, 248 to 249, 250 to 251, 252 to 253, 253 to 254, 255 to 256, 257 to 258, 259 to 260, 261 to 262, 262 to 263, 263 to 264, 264 to 265, 265 and / or (c) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 310 to 312, 410 to 412, or 510 to 512, and / or (b) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 113 to 115, 213 to 215, 313 to 315, 413 to 415, or 513 to 515.In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 101-106, 201-206, 301-306, 401-306, and 501-506.

[0032] In certain embodiments, the polypeptides described herein comprise an antigen-binding region that binds to DLL3, wherein the antigen-binding region comprises: (a) a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 107 to 109; (b) a heavy chain complementarity-determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 110 to 112; and (c) a heavy chain complementarity-determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 113 to 115. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 101 to 106. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 101 to 106.

[0033] In certain embodiments, the polypeptides described herein comprise an antigen-binding region that binds to DLL3, wherein the antigen-binding region comprises: (a) a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 207-209; (b) a heavy chain complementarity-determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 210-212; and (c) a heavy chain complementarity-determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 213-215. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 201-206. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 201-206.

[0034] In certain embodiments, the polypeptides described herein comprise an antigen-binding region that binds to DLL3, wherein the antigen-binding region comprises: (a) a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 307-309; (b) a heavy chain complementarity-determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 310-312; and (c) a heavy chain complementarity-determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 313-315. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 301-306. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 301-306.

[0035] In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:303.

[0036] 10. The polypeptide of any one of claims 1, 2 or 9, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 303.

[0037] In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:304.

[0038] In a specific embodiment, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:304.

[0039] In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:305.

[0040] In certain embodiments, the polypeptides described herein comprise an antigen-binding region that binds to DLL3, wherein the antigen-binding region comprises: (a) a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 407-409; (b) a heavy chain complementarity-determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 410-412; and (c) a heavy chain complementarity-determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 413-415. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 401-406. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 401-406.

[0041] In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:403.

[0042] In a specific embodiment, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:403.

[0043] In certain embodiments, the polypeptides described herein comprise an antigen-binding region that binds to DLL3, wherein the antigen-binding region comprises: (a) a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 507-509; (b) a heavy chain complementarity-determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 510-512; and (c) a heavy chain complementarity-determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 513-515. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 501-506. In certain embodiments, the antigen-binding region comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 501-506.

[0044] The DLL3 antigen-binding region confers specificity to the immunoconjugate and may suitably comprise a small antigen-binding polypeptide. Such small antigen-binding polypeptides confer advantages such as a reduced overall size of the immunoconjugate molecule, enabling tumor penetration and labeling. The small antigen-binding polypeptide may lack certain dispensable regions, such as the light chain constant region, heavy chain constant region, CH1 region, or hinge region, that are not required for binding. In certain embodiments, the antigen-binding region may lack the light chain variable region. In certain embodiments, the small antigen-binding region may have a molecular weight of 10 kDa to 40 kDa.

[0045] In some embodiments, the small antigen-binding region has a molecular weight of about 10 kDa to about 40 kDa. In some embodiments, the small antigen-binding region has a molecular weight of about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, about 10 kDa to about 30 kDa, about 10 kDa to about 35 kDa, about 10 kDa to about 40 kDa, about 15 kDa to about 20 kDa, about 15 kDa to about 25 kDa, about 15 kDa to about 30 kDa, about 15 ... The small antigen-binding region has a molecular weight of about 5 kDa to about 40 kDa, about 20 kDa to about 25 kDa, about 20 kDa to about 30 kDa, about 20 kDa to about 35 kDa, about 20 kDa to about 40 kDa, about 25 kDa to about 30 kDa, about 25 kDa to about 35 kDa, about 25 kDa to about 40 kDa, about 30 kDa to about 35 kDa, about 30 kDa to about 40 kDa, or about 35 kDa to about 40 kDa. In some embodiments, the small antigen-binding region has a molecular weight of about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, or about 40 kDa. In some embodiments, the small antigen-binding region has a molecular weight of at least about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, or about 35 kDa, hi some embodiments, the small antigen-binding region has a molecular weight of at most about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, or about 40 kDa.

[0046] The antigen-binding region may comprise a VHH polypeptide, an scFv polypeptide, or a VNAR polypeptide. In certain embodiments, the antigen-binding region comprises a VHH polypeptide. In certain embodiments, the antigen-binding region comprises an ScFv polypeptide. In certain embodiments, the antigen-binding region comprises a VNAR polypeptide. In certain embodiments, the antigen-binding region is humanized. In certain embodiments, the antigen-binding region does not comprise an immunoglobulin light chain.

[0047] In some embodiments, the immunoconjugates of the present disclosure comprise, for example, an autonomous V Hdomains (autonomous VH domains) (e.g., from camelid, murine, or human sources), single domain antibody domains (sdAb), camelid-derived heavy chain antibody domains (V H H fragment or V H domain fragment), camelid V H H fragment or V H Heavy chain antibody domain derived from domain fragment, heavy chain antibody domain derived from cartilaginous fish, immunoglobulin novel antigen receptor (IgNAR), V NAR fragments, single-chain variable (scFv) fragments, nanobodies, V H "Camelized" or "camelized" scaffold containing domains, heavy chain and C H Single-domain Fd fragments, single-chain Fv-CH3 minibodies, Fc antigen-binding domains (Fcabs), scFv-Fc fusions, multimerized scFv fragments (diabodies, triabodies, tetrabodies), disulfide-stabilized antibody variable (Fv) fragments (dsFv), V L , V H , C L and C H Disulfide-stabilized antigen-binding (Fab) fragments consisting of one domain, scFvs containing disulfide-stabilized heavy and light chains (sc-dsFvs), bivalent nanobodies, bivalent minibodies, bivalent F(ab')2 fragments (Fab dimers), and bispecific tandem V H Includes synthetically engineered antibody derivatives such as proteins or polypeptides, including H fragments, bispecific tandem scFv fragments, bispecific nanobodies, bispecific minibodies, and genetically engineered counterparts of any of the above that retain paratope and target antigen binding function.

[0048] In some embodiments, the immunoconjugate is monovalent. In other embodiments, the immunoconjugate is multivalent, e.g., bivalent. In some further embodiments, the immunoconjugate is bivalent and dimeric. In some further embodiments, the bivalent immunoconjugate is homodimeric.

[0049] In one aspect, the present disclosure provides an antibody construct (alone or in the context of an immunoconjugate, radioimmunoconjugate, or targeted imaging complex, respectively, of the present disclosure) comprising a VHH fragment comprising a heavy chain variable region comprising three heavy chain CDRs derived from a camelid, which binds to an antigen with specificity and high affinity.

[0050] In some embodiments, the antibody construct, immunoconjugate, radioimmunoconjugate, or targeted imaging conjugate specifically binds to at least one extracellular portion of an antigen expressed on the cell surface, hi some embodiments, the immunoconjugate specifically binds to at least one extracellular portion of an antigen expressed by a target cell, e.g., a tumor cell.

[0051] In some embodiments, the present disclosure provides an immunoconjugate that specifically binds to an antigen. In some embodiments, the immunoconjugate comprises an antibody construct comprising a heavy chain variable region (HVR-H) comprising three CDRs: hCDR1, hCDR2, and hCDR3, such as those derived from a camelid antibody or IgNAR. In some embodiments, the immunoconjugate comprises (a) a light chain variable region (HVR-L) comprising three CDRs: hCDR1, hCDR2, and hCDR3, and (b) a heavy chain variable region (HVR-H) comprising three CDRs: hCDR1, hCDR2, and hCDR3. In some embodiments, the antibody construct is chimeric or humanized.

[0052] In some embodiments, the immunoconjugates of the present disclosure may be, for example, Fv, Fab, Fab', scFv, HcAb fragments, V Hand antibody constructs comprising an antigen-binding domain that is an antibody fragment, including, but not limited to, an H fragment, an sdAb fragment, a diabody, or an F(ab')2 fragment. In some further embodiments, the immunoconjugates of the present disclosure comprise multimers of two or more antibody fragments, e.g., homodimers or heterodimers, each comprising two antibody fragments that are capable of binding to an antigen with specificity and high affinity, and each comprising a heavy chain variable region (HVR-H) comprising three CDRs: hCDR1, hCDR2, and hCDR3.

[0053] Heavy Chain Constant Regions The antigen-binding region of the immunoconjugates described herein may comprise an Fc or heavy chain constant region. The antigen-binding molecule may be linked to the Fc or heavy chain constant region directly, via an appropriate linker, or via an IgG hinge region. The inclusion of a heavy chain constant region or Fc region confers advantages such as allowing for optimization and adjustment of serum half-life, the addition of additional sites for conjugating chelators or cytotoxic agents, and the purification of the immunoconjugate using standard processes and methods. The addition of a heavy chain constant region also increases size, which may shift catabolism and elimination of the immunoconjugate from the kidney to the liver. This may provide a safety advantage, particularly for radioimmunoconjugates, because the kidney is more sensitive to radiation than the liver. Changes affecting effector function or serum half-life may be made to residues present in the heavy chain constant region involved in neonatal Fc receptor (FcRn) binding. Binding to FcRn generally contributes to an increase in the half-life of immunoglobulin Fc-containing molecules, and therefore, reduced binding to FcRn can reduce the half-life of Fc-containing molecules. Reduced FcRn binding can provide benefits such as reduced half-life of immunoconjugates and, therefore, subsequent toxicity due to cytotoxic agents or radioisotopes. In certain embodiments, the immunoglobulin constant region comprises or consists of an Fc region. In certain embodiments, the immunoglobulin heavy chain constant region comprises an immunoglobulin CH2 domain, an immunoglobulin CH3 domain, or an immunoglobulin CH2 and CH3 domains. In certain embodiments, the immunoglobulin heavy chain constant region comprises an immunoglobulin CH2 and CH3 domains. For treatment or imaging of human individuals, the immunoglobulin heavy chain constant region can be human to prevent or reduce endogenous immune responses to the immunoconjugate. In certain embodiments, the immunoglobulin heavy chain constant region is a human immunoglobulin heavy chain constant region. In certain embodiments, the immunoglobulin heavy chain constant region is an IgA, IgG1, IgG2, IgG3, or IgG4 isotype. In certain embodiments, the immunoglobulin heavy chain constant region is an IgG1 isotype.In certain embodiments, the immunoglobulin heavy chain constant region is of the IgG4 isotype.

[0054] In some embodiments, the present disclosure contemplates variants of the immunoconjugates of the present disclosure that include an Fc region, which possess some but not all effector functions, making them desirable candidates for applications in which the half-life of the immunoconjugate in vivo is important, yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the immunoconjugate lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in US 5,500,362 (see, e.g., Hellstrom, I. et al. Proc Natl Acad Sci USA 83:7059-7063 (1986), and Hellstrom, I. et al., Proc Natl Acad Sci USA 82:1499-1502 (1985)), 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay (EllTechnology, Inc. Mountain View, CA), and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI) for flow cytometry). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc Natl Acad Sci USA 95:652-656 (1998). To confirm that the immunoconjugate is unable to bind C1q and thus lacks CDC activity, a C1q binding assay may be performed (see, e.g., C1q and C3c binding ELISAs in WO2006 / 029879 and WO 2005 / 100402). To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0055] The immunoglobulin heavy chain constant region may be a variant constant region comprising one or more changes to amino acid residues that confer additional utility and advantageous properties to the immunoconjugates described herein. In certain embodiments, the immunoglobulin heavy chain constant region comprises changes to one or more amino acid residues that reduce an effector function of the immunoglobulin heavy chain constant region or alter binding of the immunoconjugate to neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises changes to one or more amino acid residues that reduce an effector function of the immunoglobulin heavy chain constant region or reduce binding of the immunoconjugate to neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises changes to one or more amino acid residues that reduce an effector function of the immunoglobulin heavy chain constant region or reduce binding of the immunoconjugate to neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises changes to one or more amino acid residues that reduce an effector function of the immunoglobulin heavy chain constant region or reduce binding of the immunoconjugate to neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn).

[0056] Changes to the heavy chain constant region of the immunoconjugate may reduce an effector function associated with the heavy chain constant region, such as, for example, the ability to fix complement, promote phagocytosis, or recruit other immune effector cells (e.g., NK cells) to the heavy chain constant region. In certain embodiments, changes to one or more amino acid residues that reduce an effector function of an immunoglobulin heavy chain constant region are changes that reduce complement dependent cytotoxicity (CDC), antibody-dependent cell-cytotoxicity (ADCC), antibody-dependent cell-phagocytosis (ADCP), or a combination thereof. In certain embodiments, the changes to one or more amino acid residues that reduce an effector function of an immunoglobulin heavy chain constant region are: (a) 297A, 297Q, 297G, or 297D; (b) 279F, 279K, or 279L; (c) 228P; (d) 235A, 235E, 235G, 235Q, 235R, or 235S; (e) 237A, 237E, 237K; , 237N or 237R, (f) 234A, 234V or 234F, (g) 233P, (h) 328A, (i) 327Q or 327T, (j) 329A, 329G, 329Y or 329R (k) 331S, (l) 236F or 236R, (m) 238A, 238E, 238G, 238H, 238I, 238V, 238W or 238Y, (n) 2 48A, (o) 254D, 254E, 254G, 254H, 254I, 254N, 254P, 254Q, 254T or 254V, (p) 255N, (q) 256H, 256K, 256R or 256V, (r) 264S, (s) 265H, 265K, 265S, 265Y or 265A, (t) 267G, 267H, 267I or 267K, (u) 26 8K, (v) 269N or 269Q, (w) 270A, 270G, 270M or 270N, (x) 271T, (y) 272N, (z) 292E, 292F, 292G, or 292I, (aa) 293S, (bb) 301W, (cc) 304E, (dd) 311E, 311G, or 311S, (ee) 316F, (ff) 328V, (gg) 330R,(hh) 339E or 339L, (ii) 343I or 343V, (jj) 373A, 373G, or 373S, (kk) 376E, 376W, or 376Y, (ll) 380D, (mm) 382D or 382P, (nn) 385P, (oo) 424H, 424M, or 424V, (pp) 434I, (qq) 438G, (rr) 439E, 439H, or 439Q, ( ss) 440A, 440D, 440E, 440F, 440M, 440T, or 440V, (tt) K322A, (uu) L235E, (vv) L234A and L235A, (ww) L234A, L235A and G237A, (xx) L234A, L235A and P329G, (yy) L234F, L235E and P331S, (zz) L234A, L235E and G237A , (aaa) L234A, L235E, G237A and P331S (bbb) L234A, L235A, G237A, P238S, H268A, A330S and P331S, (ccc) L234A, L235A and P329A, (ddd) G236R and L328R, (eee) G237A, (fff) F241A, (ggg) V264A, (hhh) D265A, (iii) In certain embodiments, the changes to one or more amino acid residues that reduce an effector function of an immunoglobulin heavy chain constant region are selected from the list consisting of D265A and N297A, (jjj) D265A and N297G, (kkk) D270A, (II1) A330L, (mmm) P331A or P331S, or (nnn) E233P, (ooo) L234A, L235E, G237A, A330S, and P331S, or (ppp) any combination of (a) through (ooo) according to EU numbering. In certain embodiments, the changes to one or more amino acid residues that reduce an effector function of an immunoglobulin heavy chain constant region include L234A, L235E, G237A, A330S, and P331S according to EU numbering.

[0057] Changes to the heavy chain constant region of the immunoconjugate can decrease the serum half-life of the immunoconjugate. In certain embodiments, amino acid changes that alter or reduce binding of the immunoconjugate to neonatal Fc receptor (FcRn) decrease the serum half-life of the immunoconjugate. In certain embodiments, the changes that alter or reduce binding of the immunoconjugate to neonatal Fc receptor (FcRn) are to amino acid residues selected from the list consisting of 251, 252, 253, 254, 255, 288, 309, 310, 312, 385, 386, 388, 400, 415, 433, 435, 436, 439, 447, according to EU numbering, and combinations thereof. In certain embodiments, the alteration that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of 253, 254, 310, 435, 436, and combinations thereof, according to EU numbering. In certain embodiments, the alteration that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of I253A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof, according to EU numbering. In certain embodiments, the change that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 1253A, S254A, H310A, H435Q, Y436A, and combinations thereof, according to EU numbering. In certain embodiments, the change that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 1253A, H310A, H435Q, and combinations thereof, according to EU numbering. In certain embodiments, the change that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: H310A, H435Q, and combinations thereof, according to EU numbering.

[0058] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 1. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 1, wherein the heavy chain constant region comprises a I253A substitution according to EU numbering.

[0059] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 2. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is identical to SEQ ID NO: 2. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 2, wherein the heavy chain constant region comprises a S254A substitution according to EU numbering.

[0060] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 3. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 3, wherein the heavy chain constant region comprises an H310A substitution according to EU numbering.

[0061] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 4. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence identical to SEQ ID NO: 4. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 4, wherein the heavy chain constant region comprises an H435Q substitution according to EU numbering.

[0062] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 5. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is identical to SEQ ID NO: 5. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 5, wherein the heavy chain constant region comprises a Y436A substitution according to EU numbering.

[0063] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 6. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 6, wherein the heavy chain constant region comprises H310A / H435Q substitutions according to EU numbering.

[0064] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 7. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 7, wherein the heavy chain constant region comprises L234A, L235E, G237A, A330S, and P331S substitutions according to EU numbering.

[0065] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 8. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO: 8, wherein the heavy chain constant region comprises L234A, L235E, G237A, H310A, A330S, and P331S substitutions according to EU numbering.

[0066] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 9. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence identical to SEQ ID NO: 9, wherein the heavy chain constant region comprises L234A, L235E, G237A, H435Q, A330S, and P331S substitutions according to EU numbering.

[0067] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 10. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence identical to SEQ ID NO: 10 according to EU numbering.

[0068] In one embodiment, each of the two mutant constant regions has at least one FcRn-binding mutation. In one embodiment, each of the two mutant constant regions has the same FcRn-binding mutation. In one embodiment, each of the two mutant constant regions has a different FcRn-binding mutation.

[0069] In one embodiment, at least one of the mutated constant regions in the immunoconjugate has at least one FcRn-binding mutation, hi certain embodiments, each of the two mutated constant regions of the immunoconjugate has at least one FcRn-binding mutation, wherein the FcRn-binding mutations are the same or different.

[0070] Changes that affect FcRn binding can decrease the serum half-life of the immunoconjugate, thus allowing one of skill in the art to select a half-life appropriate for a particular imaging or therapeutic goal. In certain embodiments, the immunoconjugate has a serum half-life of about 12 hours to about 120 hours. In certain embodiments, the immunoconjugate is administered for about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 12 hours to about 48 hours, about 12 hours to about 60 hours, about 12 hours to about 72 hours, about 12 hours to about 84 hours, about 12 hours to about 96 hours, about 12 hours to about 108 hours, about 12 hours to about 120 hours, about 24 hours to about 36 hours, about 24 hours to about 48 hours, about 24 hours to about 60 hours, about 24 hours to about 72 hours, about 24 hours to about 84 hours, about 24 hours to about 96 hours, about 24 hours to about 108 hours, about 24 hours to about 120 hours, about 36 hours to about 48 hours, about 36 hours to about 60 hours, about 36 hours to about 72 hours, about 36 hours to about 84 hours, about 36 hours to about 96 hours, about 36 hours to about The serum half-life is 108 hours, about 36 hours to about 120 hours, about 48 hours to about 60 hours, about 48 hours to about 72 hours, about 48 hours to about 84 hours, about 48 hours to about 96 hours, about 48 hours to about 108 hours, about 48 hours to about 120 hours, about 60 hours to about 72 hours, about 60 hours to about 84 hours, about 60 hours to about 96 hours, about 60 hours to about 108 hours, about 60 hours to about 120 hours, about 72 hours to about 84 hours, about 72 hours to about 96 hours, about 72 hours to about 108 hours, about 72 hours to about 120 hours, about 84 hours to about 96 hours, about 84 hours to about 108 hours, about 84 hours to about 120 hours, about 96 hours to about 108 hours, about 96 hours to about 120 hours, or about 108 hours to about 120 hours. In certain embodiments, the immunoconjugate has a serum half-life of about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours, or about 120 hours. In certain embodiments, the immunoconjugate has a serum half-life of at least about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, or about 108 hours.In certain embodiments, the immunoconjugate has a serum half-life of at most about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours, or about 120 hours.

[0071] In certain embodiments, the immunoconjugate has a serum half-life of about 1 to about 10 days. In certain embodiments, the immunoconjugate has a serum half-life of about 1 to about 2 days, about 1 to about 3 days, about 1 to about 4 days, about 1 to about 5 days, about 1 to about 6 days, about 1 to about 7 days, about 1 to about 8 days, about 1 to about 9 days, about 1 to about 10 days, about 2 to about 3 days, about 2 to about 4 days, about 2 to about 5 days, about 2 to about 6 days, about 2 to about 7 days, about 2 to about 8 days, about 2 to about 9 days, about 2 to about 10 days, about 3 to about 4 days, about 3 to about 5 days, about 3 to about 6 days, about 3 to about 7 days, about 3 to about 8 days, about 3 to about 8 days, about 3 to about 3 days. The serum half-life is from about 3 to about 9 days, from about 3 to about 10 days, from about 4 to about 5 days, from about 4 to about 6 days, from about 4 to about 7 days, from about 4 to about 8 days, from about 4 to about 9 days, from about 4 to about 10 days, from about 5 to about 6 days, from about 5 to about 7 days, from about 5 to about 8 days, from about 5 to about 9 days, from about 5 to about 10 days, from about 6 to about 7 days, from about 6 to about 8 days, from about 6 to about 9 days, from about 6 to about 10 days, from about 7 to about 8 days, from about 7 to about 9 days, from about 7 to about 10 days, from about 8 to about 9 days, from about 8 to about 10 days, or from about 9 to about 10 days. In certain embodiments, the immunoconjugate has a serum half-life of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days. In certain embodiments, the immunoconjugate has a serum half-life of at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, or about 9 days. In certain embodiments, the immunoconjugate has a serum half-life of at most about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

[0072] In certain embodiments, the heavy chain constant region has a molecular weight of about 10 kDa to about 25 kDa. In certain embodiments, the heavy chain constant region has a molecular weight of about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, about 15 kDa to about 20 kDa, about 15 kDa to about 25 kDa, or about 20 kDa to about 25 kDa. In certain embodiments, the heavy chain constant region has a molecular weight of about 10 kDa, about 15 kDa, about 20 kDa, or about 25 kDa. In certain embodiments, the heavy chain constant region has a molecular weight of at least about 10 kDa, about 15 kDa, or about 20 kDa. In certain embodiments, the heavy chain constant region has a molecular weight of at most about 15 kDa, about 20 kDa, or about 25 kDa.

[0073] In some embodiments, the immunoconjugate of the present disclosure comprises a linker or hinge region, which is a polypeptide that connects the antigen-binding region to the heavy chain constant region or variant constant region of the present disclosure.Naturally occurring and synthetic hinge regions that connect immunoglobulin components are well known in the art and can be used in the present disclosure.See, for example, US8,067,548 and references therein.

[0074] In one embodiment, the hinge regions of the immunoconjugates are identical. In one embodiment, the hinge regions of the immunoconjugates are different.

[0075] The antigen-binding region and the heavy chain constant region (with or without an altered amino acid sequence) may be linked by a suitable hinge or linker sequence. In certain embodiments, the antigen-binding region is linked to the immunoglobulin heavy chain constant region by a linker amino acid sequence or a human IgG hinge region. Suitable IgG hinge regions comprise or include IgG1 or IgG4 hinge regions. In certain embodiments, the hinge region is an IgG1 hinge region. In certain embodiments, the hinge region is an IgG1 hinge region with a C220S substitution according to EU numbering. In certain embodiments, the hinge region is an IgG1 hinge region with a C220P substitution according to EU numbering. Suitable hinge regions include those described in Wu et al., "Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange," Protein Engineering, Design and Selection, Volume 14, Issue 12, December 2001, Pages 1025-1033; Shu et al., "Secretion of a single-gene-encoded immunoglobulin from myeloma cells," Proceedings of the National Academy of Sciences, September 1993, 90(17)7995-7999; and Davis et al., "Abatacept binds to the Fc receptor CD64 but does not mediate complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity," J Rheumatol. November 2007;34(11):2204-10. Suitable hinges may also include non-IgG-based polypeptide linkers. The linker amino acid sequence may include primarily the following amino acid residues: Gly, Ser, Ala, or Thr.The linker peptide should be of an appropriate length to link the two molecules in a manner that allows them to assume the correct conformation relative to one another and retain the desired activity. In one embodiment, the linker is about 1 to 50 amino acids in length, or about 1 to 30 amino acids in length. In one embodiment, linkers of 1 to 20 amino acids in length can be used. Useful linkers include, for example, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers, including (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least 1. Exemplary linkers for linking antibody fragments or single-chain variable fragments include AAEPKSS, AAEPKSSSDKTHTCPPCP, GGGG, or GGGGDKTHTCPPCP. Alternatively, various nonproteinaceous polymers can be used as linkers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. In certain embodiments, the human IgG hinge region comprises the amino acid sequence set forth in SEQ ID NO: 40 or 41. In certain embodiments, the human IgG hinge region comprises the amino acid sequence set forth in SEQ ID NO: 40. In certain embodiments, the human IgG hinge region comprises the amino acid sequence set forth in SEQ ID NO: 41.

[0076] The overall size of the immunoconjugate can be such that it facilitates tissue penetration, stability, and / or clearance. In certain embodiments, the immunoconjugate has a molecular weight of about 60 kDa to about 120 kDa. In certain embodiments, the immunoconjugate has a molecular weight of about 60 kDa to about 65 kDa, about 60 kDa to about 70 kDa, about 60 kDa to about 75 kDa, about 60 kDa to about 80 kDa, about 60 kDa to about 90 kDa, about 60 kDa to about 100 kDa, about 60 kDa to about 110 kDa, about 60 kDa to about 120 kDa, about 65 kDa to about 120 kDa, or about 65 kDa to about 120 kDa. Da to about 70kDa, about 65kDa to about 75kDa, about 65kDa to about 80kDa, about 65kDa to about 90kDa, about 65kDa to about 100kDa, about 65kDa to about 110kDa, about 65kDa to about 120kDa, about 70kDa to about 75kDa, about 70kDa to about 80kDa, about 70kDa to about 90kDa, about 70kDa to about 100kDa, about 70kDa to about 110kDa, about 70kDa to about 120kDa, about 75kDa to about 80kDa, about 75kDa to about 90kDa, about 75kDa to about 100kDa, about 75kDa to about 110kDa, about 75kDa to about 120kDa, about 80kDa to about 90kDa, about 80kDa to about 1 In certain embodiments, the immunoconjugate has a molecular weight of about 60 kDa, about 65 kDa, about 70 kDa, about 75 kDa, about 80 kDa to about 110 kDa, about 80 kDa to about 120 kDa, about 90 kDa to about 100 kDa, about 90 kDa to about 110 kDa, about 90 kDa to about 120 kDa, about 100 kDa to about 110 kDa, about 100 kDa to about 120 kDa, or about 110 kDa to about 120 kDa. In certain embodiments, the immunoconjugate has a molecular weight of at least about 60 kDa, about 65 kDa, about 70 kDa, about 75 kDa, about 80 kDa, about 90 kDa, about 100 kDa, or about 110 kDa, hi certain embodiments, the immunoconjugate has a molecular weight of at most about 65 kDa, about 70 kDa, about 75 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 110 kDa, or about 120 kDa.

[0077] In some embodiments, the immunoconjugate has a molecular weight greater than 60, 70, 75, 80, 82, 83, 85, 86, 87, 88, or 89 kDa. In some embodiments, the immunoconjugate has a molecular weight less than 110, 100, 95, 93, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, or 80 kDa. In some embodiments, the immunoconjugate has a molecular weight greater than 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, or 79 kDa and less than 110, 100, 95, 93, 91, or 90 kDa.

[0078] The size of the immunoconjugates and / or heavy chain constant region variants described herein allows for an increased safety profile or therapeutic index of the immunoconjugates contained herein. Such a safety profile may be reflected in reduced accumulation of radiation in key radiosensitive tissues such as the kidney and bone marrow, and / or increased accumulation of radiation in target tissues (i.e., tumor or cancerous tissue) or more radioresistant organs such as the liver.

[0079] In certain embodiments, the immunoconjugates of the present disclosure provide a total radiation exposure per treatment measured in Gray (Gy). In certain embodiments, the kidneys are exposed to 20 Gy or less per treatment. In certain embodiments, the kidneys are exposed to 19 Gy or less per treatment. In certain embodiments, the kidneys are exposed to 18 Gy or less per treatment. In certain embodiments, the kidneys are exposed to 17 Gy or less per treatment. In certain embodiments, the kidneys are exposed to 16 Gy or less per treatment. In certain embodiments, the kidneys are exposed to 15 Gy or less per treatment. In certain embodiments, the kidneys are exposed to 14 Gy or less per treatment. In certain embodiments, the kidneys are exposed to 13 Gy or less per treatment. In certain embodiments, the kidneys are exposed to 12 Gy or less per treatment. In certain embodiments, the kidneys are exposed to 11 Gy or less per treatment. In certain embodiments, the kidneys are exposed to 10 Gy or less per treatment. In certain embodiments, the kidneys are exposed to 9 Gy or less per treatment. In certain embodiments, the kidneys are exposed to 8 Gy or less per treatment. In certain embodiments, the kidneys are exposed to 5 Gy or less per treatment.

[0080] In certain embodiments, the immunoconjugates of the present disclosure provide a total radiation exposure per treatment measured in Gray (Gy). In certain embodiments, bone marrow is exposed to 4 Gy or less per treatment. In certain embodiments, bone marrow is exposed to 3 Gy or less per treatment. In certain embodiments, bone marrow is exposed to 2 Gy or less per treatment. In certain embodiments, bone marrow is exposed to 1.5 Gy or less per treatment. In certain embodiments, bone marrow is exposed to 1.0 Gy or less per treatment. In certain embodiments, bone marrow is exposed to 0.5 Gy or less per treatment.

[0081] In certain embodiments, the immunoconjugates of the present disclosure provide an increased radiation dose in the tumor relative to the kidney, measured as percent injected dose per gram, hi certain embodiments, the ratio of percent tumor injected dose per gram to percent kidney injected dose per gram is greater than 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0082] In certain embodiments, the immunoconjugates of the present disclosure provide an increased radiation dose in the tumor compared to the blood when measured as percent injected dose per gram, hi certain embodiments, the ratio of percent tumor injected dose per gram to percent blood injected dose per gram is greater than 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0083] In certain embodiments, the immunoconjugates of the present disclosure provide an increased radiation dose in the tumor relative to the bone marrow when measured as percent injected dose per gram, hi certain embodiments, the ratio of percent tumor injected dose per gram to percent bone marrow injected dose per gram is greater than 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0084] In certain embodiments, immunoconjugates of the present disclosure result in an increased radiation dose in the liver compared to the kidney, measured as injected dose per gram, hi certain embodiments, the ratio of percent tumor injected dose per gram to percent bone marrow injected dose per gram is greater than 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0085] As will be recognized by those skilled in the art, certain teachings herein apply to the antibody constructs, targeted imaging conjugates, immunoconjugates, and radioimmunoconjugates of the present disclosure, although reference is made herein to only one or two such compositions (e.g., immunoconjugates) as non-limiting examples. All such uses are encompassed by the present disclosure.

[0086] An immunoconjugate herein can comprise a polypeptide comprising a VHH region, a hinge region, a CH2 region, and a CH3 region. In certain embodiments, the polypeptide can comprise a homodimer with another polypeptide to create a dimeric bivalent polypeptide. In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 116-120, 216-220, 316-320, 416-420, and 516-520. In certain embodiments, the polypeptide comprises an amino acid sequence identical to any one of SEQ ID NOs: 116-120, 216-220, 316-320, 416-420, and 516-520. In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NOs: 116-120. In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NOs: 216-220. In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NOs: 216-220. In certain embodiments, the polypeptide comprises an amino acid sequence that is identical to SEQ ID NOs: 216-220. In certain embodiments, a polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NOs: 316-320. In certain embodiments, a polypeptide comprises an amino acid sequence that is identical to SEQ ID NOs: 316-320. In certain embodiments, a polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NOs: 416-420. In certain embodiments, a polypeptide comprises an amino acid sequence that is identical to SEQ ID NOs: 416-420.In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NOs: 516-520. In certain embodiments, the polypeptide comprises an amino acid sequence that is identical to SEQ ID NOs: 516-520.

[0087] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:317.

[0088] In certain embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:317.

[0089] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:318.

[0090] In certain embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:318.

[0091] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:319.

[0092] In certain embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:319.

[0093] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:417.

[0094] In certain embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:417.

[0095] chelating agents As described herein, a chelating agent can be attached to an immunoconjugate, an antigen-binding region / immunoglobulin heavy chain constant region molecule, a VHH antigen-binding region / immunoglobulin heavy chain constant region molecule (wild-type or mutant), or a VHH antigen-binding region / immunoglobulin Fc molecule (wild-type or mutant). The chelating agent allows the immunoconjugate to be loaded with an appropriate radioisotope, such as a beta-emitter or an alpha-emitter. The chelating agent can be attached to the immunoconjugate via the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof. Such attachment can suitably be via covalent bonding to one or more amino acids of the immunoconjugate, the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof.

[0096] In one embodiment, the chelator of the immunoconjugate is covalently bound to the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof. In one embodiment, the chelator is covalently bound directly (e.g., without a spacer, stretcher, or linker) to the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof. In one embodiment, the chelator is covalently bound to the antigen-binding arm via a linker that is covalently bound to the chelator and to the antigen-binding arm. In one embodiment, the linker is hydrophilic (e.g., a PEG chain). In one embodiment, the linker is hydrophobic (e.g., an alkyl or alkene chain). The chelator can be linked or attached to the immunoconjugate as described in Sadiki, A. et al. "Site-specific conjugation of native antibody." Antibody Therapeutics 2020, 3, 271-284.

[0097] In some embodiments, immunoconjugates are formed by site-specific binding of a chelator-linker to a specific amino acid or glycan residue. In some embodiments, the site-specific conjugate involves the directed functionalization of a specific lysine residue in the framework region with a chelator-linker. In other embodiments, this residue can be functionalized with a different reactive functional group, which is then reacted with the chelator-linker in a second step to provide the immunoconjugate. In some embodiments, the reactive functional group is thiopropionate.

[0098] In some embodiments, non-naturally occurring cysteine ​​residues are engineered into the antibody framework as sites for thiol-directed conjugation to provide immunoconjugates, hi some embodiments, other non-naturally occurring amino acids or amino acid sequences are engineered into the framework to serve as attachment sites for chelator-linkers or secondary reactive groups to which chelator-linkers are conjugated to provide immunoconjugates.

[0099] In some embodiments, a non-natural amino acid containing a bridging group is engineered into the framework for chelator-linker attachment. In some embodiments, the non-natural amino acid contains an azide.

[0100] In some embodiments, the chelator-linker is attached to the glutamine residue via the action of a transglutaminase enzyme, while in other embodiments, a secondary reactive group is attached by transglutaminase, onto which the chelator-linker is added to provide the immunoconjugate.

[0101] In some embodiments, the chelator-linker is attached by modifying one or more N-glycans with reactive functional groups by the action of a glycosidase, and then conjugating the chelator-linker to the site. In some embodiments, the glycan is modified by the action of β-galactosidase. In some embodiments, the glycan is modified with an azide-containing glycoside for attachment of an appropriately functionalized chelator-linker.

[0102] In one embodiment, the immunoconjugate comprises more than one chelating agent, which may be the same or different.

[0103] In one embodiment, an immunoconjugate having more than one chelator has more than one chelator attached to the same antigen-binding arm.

[0104] In one embodiment, an immunoconjugate having more than 1 chelator and less than 11 chelators has more than 2 chelators, more than 3 chelators, more than 4 chelators, more than 5 chelators, more than 6 chelators, more than 7 chelators, more than 8 chelators, or more than 9 chelators. In one embodiment, the chelators are the same. In one embodiment, each antigen-binding arm is linked directly or indirectly to multiple chelators.

[0105] In one embodiment, the chelator comprises a radioisotope chelating moiety and a functional group that allows for covalent attachment to the antigen-binding arm. In one embodiment, the functional group is directly attached to the radioisotope chelating moiety. In one embodiment, the chelator further comprises a linker between the functional group and the radioisotope chelating moiety.

[0106] In one embodiment, the radioisotope chelating moiety comprises DOTA or a DOTA derivative. In one embodiment, the radioisotope chelating moiety comprises DOTAGA. In one embodiment, the radioisotope chelating moiety comprises Macropa or a Macropa derivative. In one embodiment, the radioisotope chelating moiety comprises Py4Pa or a Py4Pa derivative.

[0107] In certain embodiments, the chelator of the immunoconjugate does not bind to the antigen-binding region in the antigen-binding arm of the immunoconjugate.

[0108] In one embodiment, the chelator of the immunoconjugate is non-covalently associated with the antigen-binding arm, hi certain embodiments, the chelator is not associated with the antigen-binding region in the antigen-binding arm of the immunoconjugate.

[0109] In one embodiment, the chelating agent comprises DOTA or a DOTA derivative. In one embodiment, the chelating agent comprises DOTAGA. In one embodiment, the chelating agent comprises Macropa or a Macropa derivative. In one embodiment, the chelating agent comprises Py4Pa or a Py4Pa derivative. In one embodiment, the chelating agent comprises siderocalin or a siderocalin derivative.

[0110] In certain embodiments, the present specification describes an immunoconjugate conjugated to a chelating agent. In certain embodiments, the chelating agent is a radioisotope chelating agent. In certain embodiments, the radioisotope chelating agent is selected from the list consisting of tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), α-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTAGA), or (Py4Pa). In certain embodiments, the radioisotope chelating agent is DOTA. In certain embodiments, the radioisotope chelating agent is DOTAGA. In certain embodiments, the radioisotope chelating agent is Py4Pa. In certain embodiments, the radioisotope chelating agent is directly attached to the antigen-binding region and / or the immunoglobulin heavy chain constant region. In certain embodiments, the radioisotope chelator is attached to the antigen binding region or the immunoglobulin heavy chain constant region by a linker. In certain embodiments, the linker is selected from those derived from conjugates with 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and the linker reagents: N-succinimidyl 4-(2-pyridylthio)pentanoate forming linker moiety 4-mercaptopentanoic acid (SPP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB), polyethylene glycol (PEG), polyethylene glycol polymer (PEGn), and S-2-(4-isothiocyanatobenzyl) (SCN). In certain embodiments, the linker is selected from polyethylene glycol (PEG), polyethylene glycol polymer (PEG), and S-2-(4-isothiocyanatobenzyl) (SCN). In certain embodiments, the linker is PEG5. In certain embodiments, the linker is SCN.In certain embodiments, the radioisotope chelator is a linker-chelator selected from the list consisting of TFP-Ad-PEG5-DOTAGA, p-SCN-Bn-DOTA, p-SCN-Ph-Et-Py4Pa, and TFP-Ad-PEG5-Ac-Py4Pa.

[0111] The chelating agent may be conjugated to the protein or antigen-binding region and / or immunoglobulin heavy chain constant region in a ratio of 1:1 to 8:1. In certain embodiments, the radioisotope chelating agent is conjugated to the antigen-binding region and / or immunoglobulin heavy chain constant region in a ratio of 1:1 to 6:1. In certain embodiments, the radioisotope chelating agent is conjugated to the antigen-binding region and / or immunoglobulin heavy chain constant region in a ratio of 2:1 to 6:1.

[0112] In some embodiments, immunoconjugates of the present disclosure include a linker for linking, for example, the antigen-binding arm to a chelator (interchangeably "chelator"), or to a radioisotope, or to a cargo (e.g., a cytotoxin). The linker may include one or more linker moieties. In some embodiments, immunoconjugates of the present disclosure are engineered to have a terminal lysine available for conjugation with a chelator or linker.

[0113] For example, a bifunctional chelator is used to conjugate a radioisotope to the radioisotope delivery platform of the present disclosure to produce an immunoconjugate of the present disclosure. (See, e.g., Scheinberg D, McDevitt M, Curr Radiopharm 4:306-20 (2011)). Examples of bifunctional chelators known in the art include DOTA, DTPA, DO3A-NHS, DOTAGA-NHS, DOTAGA-anhydride DOTAGA-TFP, p-SCN-Bn-DOTA, p-SCN-Bn-DTPA, p-SCN-Bn-CHX'A''-DTPA, p-SCN-Bn-TCMC, macropa-NCS, crown, p-SCN-Ph-Et-Py4Pa, 3,2-HOPO, and TCMC.

[0114] Examples of bifunctional chelating agents are 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), and related analogs of the aforementioned. Such chelating agents are suitable for coordinating metal ions, such as α- and β-emitting radionuclides.

[0115] In some embodiments, the chelator of the immunoconjugate or radioimmunoconjugate of the present disclosure is a bifunctional chelator, such as DOTA, DO3A-NHS, DOTAGA-NHS, DOTAGA-anhydride DOTAGA-TFP, p-SCN-Bn-DOTA, p-SCN-Bn-DTPA, p-SCN-Bn-CHX-A"-DTPA, p-SCN-Bn-TCMC, Macropa-NCS (Thiele NA, et al. Angew. Chem. Int. Ed. 56:1 (2017)), Crown (Yang H, et al. Chem. Eur. J. 26:11435 (2020)), P-SCN-Ph-Et-Py4Pa (Li L, et al. Bioconjugate Chem. ASAP (2020)), 3,2-HOPO (Wickstroem K, et al. al. Int. J. Rad. One. Biol. Phys. 105:410 (2019)) (for a review of these and other bifunctional chelators, see, e.g., Price EW and Orvig C Chem. Soc. Rev., 2014, 43:260 (2014) and Brechbiel MW QJ Nucl. Med. Mol. Imaging 52:166 (2008)).

[0116] In some embodiments, the chelator of the immunoconjugate or radioimmunoconjugate of the present disclosure is a bifunctional chelator, such as DOTA, DO3A-NHS, DOTAGA-NHS, DOTAGA-anhydride DOTAGA-TFP, p-SCN-Bn-DOTA, p-SCN-Bn-DTPA, p-SCN-Bn-CHX-A"-DTPA, p-SCN-Bn-TCMC, Macropa-NCS (Thiele NA, et al. Angew. Chem. Int. Ed. 56:1 (2017)), Crown (Yang H, et al. Chem. Eur. J. 26:11435 (2020)), P-SCN-Ph-Et-Py4Pa (Li L, et al. Bioconjugate Chem. ASAP (2020)), 3,2-HOPO (Wickstroem K ... al. Int. J. Rad. One. Biol. Phys. 105:410(2019)) (for a review of these and other bifunctional chelators, see, e.g., Price EW and Orvig C Chem. Soc. Rev., 2014, 43:260(2014) and Brechbiel MW QJ Nucl. Med. Mol. Imaging 52:166(2008)).

[0117] For 225-Ac immunoconjugates, there are a variety of acyclic and cyclic ligands known in the art as suitable chelators (see, e.g., Davis I, et al., Nucl Med Biol 26:581 (1999); Chappell L, et al., Bioconjug Chem 11:510 (2000); Chappell, L, et al., Nucl Med Biol 30:581 (2003); McDevitt M, et al., Appl Radiat Isot 57:841 (2002); Gouin S, et al., Org Biomol Chem 3:453 (2005); Thiele N, et al., Angew Chem Int Ed Engl 56:14712 (2017)).

[0118] In certain embodiments, the chelator is a chelator suitable for chelation of α-emitters. Some chelators suitable for α-emitters are described in Yang et al., "Harnessing α-Emitting Radionuclides for Therapy: Radiolabeling Method Review." J Nucl Med. 2022 Jan;63(1):5-13.

[0119] In certain embodiments, chelators suitable for alpha emitter chelation include DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DO3A 1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane, DOTAGA α-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAGA anhydride (2,2′,2′-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, Py4Pa 6,6',6'',6''''-(((pyridine-2,6-diylbis(methylene))bis(azatriyl))tetrakis(methylene))tetrapicolinic acid, Py4Pa-NCS is 6,6'-((((4-isothiocyanatopyridine-2,6-diyl)bis(methylene))bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid, Crown 2,2',2'',2'''-(1,10-dioxa-4,7,13,16-tetraazacyclooctadecane-4,7,13,16-tetrayl)tetraacetic acid, Macropa 6,6'-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))dipicolinic acid, Macropa-NCS 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)-4-isothiocyanatopicolinic acid, HEHA 1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid, CHX Octapa 6,6'-[(1R,2R)-1,2-cyclohexanediylbis[[(carboxymethyl)imino]methylene]]bis[2-pyridinecarboxylic acid], Bispa 3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylic acid, 7-[(6-carboxy-2-pyridinyl)methyl]-9-hydroxy-3-methyl-2,4-di-2-pyridinyl-, 1,5-dimethyl ester, Nonane-1,5-dicarboxylic acid, 6,6'-(((oxybis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid, and combinations thereof.

[0120] In certain embodiments, the chelator is a chelator suitable for β- or γ-emitter chelation. In certain embodiments, chelators suitable for β- or γ-emitter chelation include DOTMA (1R,4R,7R,10R)-a,a',a",a"'-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid, DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid), DOTP 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid), DOTMP 1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylenephosphonic acid, DOTA-4AMP 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonic acid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), NOTA 1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTP 1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid), TETPA 1,4,8,11-Tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid, TETA 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid, PEPA 1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N'",N""-pentaacetic acid, H4Octapa N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid, H2Dedpa 1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane, H6phospa N,N'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane, TTHA Triethylenetetramine-N,N,N',N'',N''',N"'-Hexaacetic acid, DO2P tetraazacyclododecanedimethanephosphonic acid, HP-DO3A hydroxypropyltetraazacyclododecanetriacetic acid, EDTA ethylenediaminetetraacetic acid, DTPA diethylenetriaminepentaacetic acid, DTPA-BMA diethylenetriaminepentaacetic acid-bismethylamide, HOPO octadentate hydroxypyridinone, 3,2,3-LI(HOPO)N,N'-(butane-1,4-diyl)bis(1-hydroxy-N-(3-(1-hydroxy-6-oxo-1,6-dihydropyridine-2-carboxamido)propyl)-6-oxo-1,6-dihydropyridine-2-carboxamido), 3,2-HOPO N,N'-(((2-(4-aminobenzyl)-3-((2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)(2-(3-hydroxy-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)propyl)azanediyl)bis(ethane-2,1-diyl))bis(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamide), Neunpa 6,6'-(((azanediylbis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid, Neunpa-NCS = 6,6'-(((((4-isothiocyanatophenethyl)azanediyl)bis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid, Octox 6,6'-((ethane-1,2-diylbis((carboxymethyl)(azanediyl))bis(methylene))dipicolinic acid, Octox 2,2'-(ethane-1,2-diylbis(((8-hydroxyquinolin-2-yl)methyl)(azanediyl))diacetic acid, PyPa 6,6'-(((pyridine-2,6-diylbis(methylene))bis((carboxymethyl)(azadiyl))bis(methylene))dipicolinic acid, porphyrin 21,22,23,24-tetraazapentacyclo[16.2.1.13,6.18,11.113,16]tetracosa-1,3,5,7,9,11(23),12,14,16,18(21),19-undecene, deferoxamine 30-amino-3,14,25-trihydroxy-3,9,14,20,25-pentaazatriacontane-2,10,13,21,24-pentaone, DFO, * N1-[5-(acetylhydroxyamino)pentyl]-N26-(5-aminopentyl)-N26,5,16-trihydroxy-4,12,15,23-tetraoxo-5,11,16,22-tetraazahexacosanediamide, and combinations thereof.

[0121] Alternatively, or in addition, an isothiocyanate linker, such as p-SCN-Bn-DOTA, may be used that includes a lysine residue in the immunoconjugates of the present disclosure.

[0122] Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), and the linker reagents: N-succinimidyl 4-(2-pyridylthio)pentanoate forming linker moiety 4-mercaptopentanoic acid ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate forming linker moiety 4-((2,5-dioxopyrrolidin-1-yl)methyl)cyclohexanecarboxylic acid ("SMCC" also referred to herein as "MCC"), 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfanyl)butanoate forming linker moiety Linker moieties include 4-mercaptobutanoic acid ("SPDB"), N-succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB"), and those resulting from conjugation with ethyleneoxy-CHCHO- as one or more repeating units ("EO," "PEO," or "PEG"). Additional linker components are known in the art, some of which are described herein. A variety of linker components are known in the art, some of which are described below.

[0123] In certain embodiments, the linker is SCN. In certain embodiments, the chelator is a linker-chelator selected from the list consisting of TFP-Ad-PEG5-DOTAGA, p-SCN-Bn-DOTA, p-SCN-Ph-Et-Py4Pa, and TFP-Ad-PEG5-Ac-Py4Pa. In certain embodiments, the chelator is TFP-Ad-PEG5-DOTAGA. In certain embodiments, the chelator is p-SCN-Bn-DOTA. In certain embodiments, the chelator is p-SCN-Ph-Et-Py4Pa. In certain embodiments, the chelator is TFP-Ad-PEG5-Ac-Py4Pa. Such linkers are shown in Figure 18.

[0124] The linker may be a "cleavable linker" that facilitates drug release in cells. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52:127-31 (1992); US Patent No. 5,208,020) may be used.

[0125] In certain embodiments, the linker has the following formula (Formula I):

[0126] [ka] As shown in the figure.

[0127] wherein A is , a is an integer from 0 to 1, W is an amino acid unit, w is an integer from 0 to 12, Y is a spacer unit, y is 0, 1 or 2, and Ab, D, and p are as defined above for Formula I. Exemplary embodiments of such linkers are described in US20050238649.

[0128] In some embodiments, a linker component may comprise a "stretcher unit" that links the immunoconjugate to another linker component or a drug moiety. Exemplary stretcher units are:

[0129] [ka] (where the wavy line indicates the site of covalent attachment to the immunoconjugate).

[0130] In some embodiments, linkers can be conjugated to antibodies via cysteine ​​bridging functionalities such as ThioBridge® or DBM (dibromomaleimide). These linkers can act to restabilize intrachain disulfides after reduction and conjugation (Bird M, et al., Antibody-Drug Conjugates pp. 113-129 (2019), and Behrens CR, et al. Mol. Pharmaceutics 12:3986 (2015)). Exemplary rebridging stretcher elements are listed below.

[0131] [ka] (where the wavy line indicates the site of covalent attachment to the immunoconjugate).

[0132] In some embodiments, the linker component may comprise an amino acid unit. In one such embodiment, the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating release of the drug from the immunoconjugate upon exposure to intracellular proteases, such as lysosomal enzymes (see, e.g., Doronina et al. (2003) Nat. Biotechnol. 21:778-4). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys), or N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). The amino acid units can include naturally occurring amino acid residues, as well as minor amino acids and unnatural amino acid analogs, such as citrulline. The amino acid units can be designed and optimized for selectivity for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0133] In some embodiments, the linker component may include a "spacer" unit that links the immunoconjugate to the drug moiety directly or via a stretcher unit and / or an amino acid unit. The spacer unit may be "self-immolative" or "non-self-immolative." A "non-self-immolative" spacer unit is one in which some or all of the spacer unit remains attached to the drug moiety upon enzymatic (e.g., proteolytic) cleavage of the ADC. Examples of non-self-immolative spacer units include, but are not limited to, a glycine spacer unit and a glycine-glycine spacer unit. Other combinations of peptide spacers susceptible to sequence-specific enzymatic cleavage are also contemplated. For example, enzymatic cleavage of an ADC containing a glycine-glycine spacer unit by a tumor cell-associated protease would result in release of the glycine-glycine-drug moiety from the remainder of the ADC. In one such embodiment, the glycine-glycine-drug moiety is then subjected to a separate hydrolysis step in the tumor cell, thus cleaving the glycine-glycine spacer unit from the drug moiety.

[0134] The "self-immolative" spacer unit allows for release of the drug moiety without a separate hydrolysis step. In certain embodiments, the spacer unit of the linker comprises a p-aminobenzyl unit. In one such embodiment, p-aminobenzyl alcohol is attached to the amino acid unit via an amide bond, and a carbamate, methylcarbamate, or carbonate is generated between the benzyl alcohol and the cytotoxic agent (see, e.g., Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-103). In one embodiment, the spacer unit is p-aminobenzyloxycarbonyl (PAB). In certain embodiments, the phenylene moiety of the p-aminobenzyl unit is substituted with Qm, where Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano, and m is an integer ranging from 0 to 4. Examples of self-immolative spacer units further include, but are not limited to, aromatic compounds electronically similar to p-aminobenzyl alcohol (see, e.g., US2005 / 0256030 A1), such as 2-aminoimidazole-5-methanol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237), and ortho- or para-aminobenzyl acetals. Spacers that undergo cyclization upon amide bond hydrolysis can be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 1995, 2, 223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., J. Amer. Chem. Soc., 1972, 94:5815), and 2-aminophenylpropionic acid amides (Amsberry et al., J. Org. Chem., 1990, 55:5867). Elimination of amine-containing drugs substituted at the alpha position of glycine (Kingsbury et al., J. Med. Chem., 1984, 27:1447) is also an example of a self-immolative spacer useful in ADCs.

[0135] In one embodiment, the spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) unit, shown below, which can be used to incorporate and release multiple drugs:

[0136] [ka] wherein Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano; m is an integer ranging from 0 to 4; n is 0 or 1; and p is ranging from 1 to about 20.

[0137] In some embodiments, the immunoconjugate comprises a linker, such as a dendritic-type linker, for covalently attaching more than one drug moiety to an antibody, e.g., via a branched polyfunctional linker moiety (Sun et al (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-5; Sun et al (2003) Bioorganic & Medicinal Chemistry 11:1761-8). Dendritic linkers can increase the drug-to-antibody molar ratio, i.e., loading, which is related to the potency of the ADC. Thus, cysteine-engineered antibodies have only one reactive cysteine ​​thiol group, and multiple drug moieties can be attached via the dendritic linker.

[0138] Examples of linker moieties and combinations thereof are shown below, which are also suitable for use in the above formula.

[0139] [ka]

[0140] Further non-limiting examples of linkers include those described in WO2015095953:

[0141] Linker components, including stretcher, spacer, and amino acid units, can be synthesized by methods known in the art, such as those described in US20050238649.

[0142] In some embodiments, the chelator comprises a linker and is selected from one of the compounds described in U.S. Application No. 63 / 373,189, filed August 22, 2022, or a U.S. non-provisional or international application claiming priority thereto, which are incorporated herein by reference with respect to such compounds. In some embodiments, the chelator comprises a linker and is selected from compounds 1-1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-26, 1-27, 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, and 1-34 described in U.S. Application No. 63 / 373,189, filed August 22, 2022, which is incorporated herein by reference for such compounds.

[0143] In some aspects, the chelator comprises a linker and is selected from one of the compounds described in U.S. Application No. 63 / 373,183, filed August 22, 2022, or a U.S. non-provisional or international application claiming priority thereto, which are incorporated by reference herein with respect to such compounds. In some embodiments, the chelator comprises a linker and is selected from compounds 2-1, 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, and 2-13, described in U.S. Application No. 63 / 373,183, filed August 22, 2022, which are incorporated by reference herein with respect to such compounds.

[0144] In some embodiments, the chelator comprises a linker and is selected from one of the compounds described in U.S. Application No. 63 / 373,190, filed August 22, 2022, or U.S. non-provisional applications claiming priority thereto, which are incorporated by reference herein for such compounds. In some embodiments, the chelator comprises a linker and is selected from compounds 3-1, 3-2, 3-3, 3-4, 3-5, 3-13, 3-16, described in U.S. Application No. 63 / 373,190, filed August 22, 2022, which is incorporated by reference herein in its entirety for such compounds.

[0145] Radioimmunoconjugates In one embodiment, the present disclosure provides an immunoconjugate. In one embodiment, the immunoconjugate, when so labeled, linked, or loaded with an α-emitter, is capable of delivering an α-emitter in vivo. In one embodiment, the immunoconjugate, when so labeled, linked, or loaded, is also capable of delivering other radioisotopes (β-emitters and / or γ-emitters) and / or other atoms in vivo. In one embodiment, the immunoconjugate, when so labeled, linked, or loaded, is capable of delivering an imaging metal (e.g., 111-In, 89-Zr, 64-Cu, 68-Ga, or 134-Ce) in vivo.

[0146] The immunoconjugates of the present disclosure may be loaded with a radioisotope for therapeutic or diagnostic effects. In certain embodiments, the chelating agent may further comprise a radioisotope. In certain embodiments, the radioisotope is an alpha emitter. In certain embodiments, the radioisotope is an alpha emitter selected from the list consisting of 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi. In certain embodiments, the radioisotope is 225-Ac. In certain embodiments, the radioisotope is a beta emitter. In certain embodiments, the radioisotope is a beta emitter selected from 177-Lu, 90-Y, 67-Cu, and 153-Sm.

[0147] Also described herein are methods for producing radioimmunoconjugates, including loading or conjugating the immunoconjugates of the present disclosure to a radioisotope. In certain embodiments, the radioisotope is an α-emitter. In certain embodiments, the radioisotope is an α-emitter selected from the list consisting of 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi. In certain embodiments, the radioisotope is 225-Ac. In certain embodiments, the radioisotope is a β-emitter. In certain embodiments, the radioisotope is a β-emitter selected from 177-Lu, 90-Y, 67-Cu, and 153-Sm.

[0148] In one aspect, the present disclosure provides a radioimmunoconjugate comprising an immunoconjugate of the present disclosure and an α-emitting isotope. In one embodiment, the α-emitting isotope of the radioimmunoconjugate is selected from the group including 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi. In one embodiment, the α-emitting isotope of the radioimmunoconjugate is selected from the group consisting of 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi. In one embodiment, the α-emitting isotope of the radioimmunoconjugate is 225-Ac. In one embodiment, the α-emitting isotope of the radioimmunoconjugate is 223-Ra. In one embodiment, the α-emitting isotope of the radioimmunoconjugate is 224-Ra. In one embodiment, the alpha-emitting isotope of the radioimmunoconjugate is 227-Th. In one embodiment, the alpha-emitting isotope of the radioimmunoconjugate is 212-Pb. In one embodiment, the alpha-emitting isotope of the radioimmunoconjugate is 212-Bi. In one embodiment, the alpha-emitting isotope of the radioimmunoconjugate is 213-Bi.

[0149] In some embodiments, the immunoconjugates of the present disclosure are combined with a radioisotope to provide a radioactive immunoconjugate of the present disclosure. In some embodiments, the radioisotope is 225-Ac, 86-Y, 90-Y, 177-Lu, 186-Re, 188-Re, 89-Sr, 153-Sm, 213-Bi, 213-Po, 212-Bi, 223-Ra, 224-Ra, 227-Th, 149-Tb, 68-Ga, 64-Cu, 67-Cu, 89-Zr, 137-Cs, 212-Pb, or 103-Pd. In some embodiments, the radioisotope is an alpha emitter, such as, for example, 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi. In some embodiments, the radioisotope is a beta particle emitter, such as, for example, 177-Lu, 90-Y, 67-Cu, 153-Sm, etc. In some embodiments, the radioisotope is both an alpha particle emitter and a beta and / or gamma particle emitter. In some embodiments, the radioisotope is both a beta particle emitter and a gamma particle and / or photon emitter. In some embodiments, the radioimmunoconjugate is labeled, linked, or loaded with, and thus comprises, both an alpha-emitter and a beta-emitter. In some embodiments, the radioisotope is selected for use in radioimaging from, for example, 68-Ga, 64-Cu, 89-Zr, 111-In, 134-Ce.

[0150] The immunoconjugates and radioimmunoconjugates of the present disclosure may contain, in addition to radioisotopes, other cargoes or payloads, including, for example, various cytotoxic agents, such as a small molecule chemotherapeutic agent, a cytotoxic antibiotic, an alkylating agent, antimetabolite, a topoisomerase inhibitor, and / or a tubulin inhibitor. For example, the immunoconjugates of the present disclosure may be used to deliver a non-radioisotope cytotoxin to a target cell. Non-limiting examples of cytotoxic agents include aziridine, cisplatin, tetrazine, procarbazine, hexamethylmelamine, vinca alkaloids, taxanes, camptothecin, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, aclarubicin, anthracyclines, actinomycin, bleomycin, plicamycin, mitomycin, daunorubicin, epirubicin, idarubicin, dolastatins, maytansine, docetaxel, adriamycin, calicheamicin, auristatins, pyrrolobenzodiazepines, carboplatin, 5-fluorouracil (5-FU), capecitabine, mitomycin C, paclitaxel, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), rifampicin, cisplatin, methotrexate, and gemcitabine.

[0151] In some embodiments, the radioimmunoconjugates of the present disclosure comprise a radioisotope selected from the group including 225-Ac, 86-Y, 90-Y, 177-Lu, 186-Re, 188-Re, 89-Sr, 153-Sm, 213-Bi, 213-Po, 211-At, 212-Bi, 223-Ra, 224-Ra, 227-Th, 149-Tb, 68-Ga, 64-Cu, 67-Cu, 89-Zr, 137-Cs, 212-Pb, and 103-Pd.

[0152] In some embodiments, the radioimmunoconjugates of the present disclosure comprise a radioisotope selected from the group consisting of 225-Ac, 86-Y, 90-Y, 177-Lu, 186-Re, 188-Re, 89-Sr, 153-Sm, 213-Bi, 213-Po, 211-At, 212-Bi, 223-Ra, 224-Ra, 227-Th, 149-Tb, 68-Ga, 64-Cu, 67-Cu, 89-Zr, 137-Cs, 212-Pb, and 103-Pd.

[0153] In some embodiments, the radioisotope is an alpha-particle-emitting radioisotope, including 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, or 213-Bi.

[0154] In some embodiments, the radioisotope is an alpha particle-emitting radioisotope selected from the group consisting of 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi.

[0155] In some embodiments, immunoconjugates of the present disclosure comprise antibody constructs comprising humanized immunoglobulin domains (used herein as antigen-binding regions).

[0156] Humanized forms of non-human (e.g., camelid, mouse, or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibodies) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as camelid, mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some examples, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being those of a human immunoglobulin consensus sequence. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321:522-5 (1986); Riechmann et al., Nature, 332:323-9 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-6 (1992)).

[0157] Methods for humanizing non-human antibodies are well known in the art. Generally, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are typically obtained from an "import" variable domain. Humanization can be performed essentially according to the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0158] According to another method, antigen binding can be restored during antibody humanization through the selection of restored hypervariable regions (see, e.g., U.S. Application No. 11 / 061,841, filed February 18, 2005). The method involves incorporating non-human hypervariable regions onto an acceptor framework and further introducing one or more amino acid substitutions in one or more hypervariable regions without modifying the acceptor framework sequence. Alternatively, the introduction of one or more amino acid substitutions can be accompanied by modifications in the acceptor framework sequence.

[0159] Any cysteine ​​residue not involved in maintaining the proper conformation of the immunoconjugates of the present disclosure may also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bond(s) may be added to the immunoconjugates of the present disclosure to improve their stability (particularly when the antibody is an Fv fragment or V H(If it is an antibody fragment such as H fragment).

[0160] In some embodiments, it may be desirable to create cysteine-engineered immunoconjugates in which one or more residues of the immunoconjugate are substituted with cysteine ​​residues. In some embodiments, the substituted residues occur at accessible sites of the immunoconjugate. By substituting these residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the immunoconjugate and can be used to conjugate the immunoconjugate with other moieties, such as drug moieties or linker-drug moieties. In some embodiments, any one or more of the following residues can be substituted with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies can be generated, for example, as described in US Pat. No. 7,521,541.

[0161] In some embodiments, the immunoconjugates provided herein are altered to increase or decrease the extent to which the immunoconjugate is glycosylated and / or to change the glycosylation pattern. "Altering the native glycosylation pattern," for purposes of this specification, is intended to mean the deletion of one or more carbohydrate moieties found in a parent immunoconjugate of the present disclosure (either by removal of the underlying glycosylation site or by loss of glycosylation by chemical and / or enzymatic means), and / or the addition of one or more glycosylation sites not present in a native sequence immunoconjugate of the present disclosure. In addition, this phrase includes qualitative changes in the glycosylation of a native protein, involving a change in the nature and proportion of the various carbohydrate moieties present.

[0162] If the immunoconjugate contains an Fc region, the carbohydrate attached thereto can be varied. Natural antibodies produced by mammalian cells typically contain a branched, biantennary oligosaccharide, usually N-linked to Asn297 in the CH2 domain of the Fc region (see, e.g., Wright et al., TIBTECH 15:26-32 (1997)). The oligosaccharide can contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in the immunoconjugates of the present disclosure can be made to generate immunoconjugate variants with specific improved properties.

[0163] In one embodiment, a radioimmunoconjugate is described herein that includes an antigen-binding region and an immunoglobulin heavy chain constant region, wherein the immunoglobulin heavy chain constant region includes an immunoglobulin hinge region, CH2 and CH3 domains of an immunoglobulin, and the antigen-binding region includes a VHH, wherein the VHH includes a heavy chain complementarity-determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 407 to 409 and a heavy chain complementarity-determining region 2 (HCDR3) set forth in any one of SEQ ID NOs: 410 to 412. and a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs:413 to 415 or SEQ ID NO:431, wherein the hinge region comprises a C220S substitution according to EU numbering; the immunoglobulin heavy chain constant region comprises L234A, L235E, G237A, A330S, P331S, and H435Q substitutions according to EU numbering; and the radioimmunoconjugate comprises a chelating agent. In certain embodiments, the chelating agent comprises DOTA. In certain embodiments, the chelating agent is p-SCN-Bn-DOTA. In certain embodiments, the chelating agent is conjugated to an α-, β-, or γ-radioactive isotope. In certain embodiments, the α-radioactive isotope is 225-Ac. In certain embodiments, the β-radioactive isotope is 177-Lu. In a particular embodiment, the gamma-emitting isotope is 111-In.

[0164] In one embodiment, described herein is a radioimmunoconjugate comprising an antigen binding region and an immunoglobulin heavy chain constant region, wherein the immunoglobulin heavy chain constant region comprises an immunoglobulin hinge region, CH2 and CH3 domains of an immunoglobulin, wherein the antigen binding region comprises a VHH, wherein the VHH comprises heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 307 to 309, heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 310 to 312, and heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 313 to 315, wherein the hinge region comprises a C220S substitution according to EU numbering, and wherein the immunoglobulin heavy chain constant region comprises L234A, L235E, G237A, A330S, P331S, and H435Q substitutions according to EU numbering, and wherein the radioimmunoconjugate comprises a chelator. In certain embodiments, the chelating agent comprises DOTA. In certain embodiments, the chelating agent is p-SCN-Bn-DOTA. In certain embodiments, the chelating agent is conjugated with an alpha-radioisotope, a beta-radioisotope, or a gamma-radioisotope. In certain embodiments, the alpha-radioisotope is 225-Ac. In certain embodiments, the beta-radioisotope is 177-Lu. In certain embodiments, the gamma-radioisotope is 111-In.

[0165] In one embodiment, a radioimmunoconjugate is described herein, comprising an antigen-binding region and an immunoglobulin heavy chain constant region, wherein the immunoglobulin heavy chain constant region comprises an immunoglobulin hinge region, CH2 and CH3 domains of an immunoglobulin, wherein the antigen-binding region comprises a VHH, wherein the VHH comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 403, wherein the hinge region comprises a C220S substitution according to EU numbering, and wherein the immunoglobulin heavy chain constant region comprises L234A, L235E, G237A, A330S, P331S, and H435Q substitutions according to EU numbering, and wherein the radioimmunoconjugate comprises a chelating agent. In certain embodiments, the chelating agent comprises DOTA. In certain embodiments, the chelating agent is p-SCN-Bn-DOTA. In certain embodiments, the chelating agent is conjugated with an alpha-radioisotope, a beta-radioisotope, or a gamma-radioisotope. In certain embodiments, the alpha-radioisotope is 225-Ac. In certain embodiments, the beta-radioisotope is 177-Lu. In certain embodiments, the gamma-radioisotope is 111-In.

[0166] In one embodiment, a radioimmunoconjugate is described herein, comprising an antigen-binding region and an immunoglobulin heavy chain constant region, wherein the immunoglobulin heavy chain constant region comprises an immunoglobulin hinge region, CH2 and CH3 domains of the immunoglobulin; the antigen-binding region comprises a VHH, wherein the VHH comprises the amino acid sequence set forth in SEQ ID NO: 403, wherein the hinge region comprises a C220S substitution according to EU numbering; and the immunoglobulin heavy chain constant region comprises L234A, L235E, G237A, A330S, P331S and H435Q substitutions according to EU numbering; and the radioimmunoconjugate comprises a chelating agent. In certain embodiments, the chelating agent comprises DOTA. In certain embodiments, the chelating agent is p-SCN-Bn-DOTA. In certain embodiments, the chelating agent is conjugated to an alpha-, beta-, or gamma-radioisotope. In certain embodiments, the alpha-radioisotope is 225-Ac. In certain embodiments, the beta-emitting isotope is 177-Lu. In certain embodiments, the gamma-emitting isotope is 111-In.

[0167] In one embodiment, a radioimmunoconjugate is described herein, comprising an antigen-binding region and an immunoglobulin heavy chain constant region, wherein the immunoglobulin heavy chain constant region comprises an immunoglobulin hinge region, CH2 and CH3 domains of an immunoglobulin, wherein the antigen-binding region comprises a VHH, wherein the VHH comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 317, wherein the hinge region comprises a C220S substitution according to EU numbering, and wherein the immunoglobulin heavy chain constant region comprises L234A, L235E, G237A, A330S, P331S, and H435Q substitutions according to EU numbering, and wherein the radioimmunoconjugate comprises a chelating agent. In certain embodiments, the chelating agent comprises DOTA. In certain embodiments, the chelating agent is p-SCN-Bn-DOTA. In certain embodiments, the chelating agent is conjugated with an alpha-radioisotope, a beta-radioisotope, or a gamma-radioisotope. In certain embodiments, the alpha-radioisotope is 225-Ac. In certain embodiments, the beta-radioisotope is 177-Lu. In certain embodiments, the gamma-radioisotope is 111-In.

[0168] In one embodiment, a radioimmunoconjugate is described herein, comprising an antigen-binding region and an immunoglobulin heavy chain constant region, wherein the immunoglobulin heavy chain constant region comprises an immunoglobulin hinge region, CH2 and CH3 domains of an immunoglobulin, wherein the antigen-binding region comprises a VHH, wherein the VHH comprises the amino acid sequence set forth in SEQ ID NO: 317, wherein the hinge region comprises a C220S substitution according to EU numbering, and the immunoglobulin heavy chain constant region comprises L234A, L235E, G237A, A330S, P331S and H435Q substitutions according to EU numbering, and the radioimmunoconjugate comprises a chelating agent. In certain embodiments, the chelating agent comprises DOTA. In certain embodiments, the chelating agent is p-SCN-Bn-DOTA. In certain embodiments, the chelating agent is conjugated to an alpha-, beta-, or gamma-radioisotope. In certain embodiments, the alpha-radioisotope is 225-Ac. In certain embodiments, the beta-emitting isotope is 177-Lu. In certain embodiments, the gamma-emitting isotope is 111-In.

[0169] In one embodiment, a radioimmunoconjugate is described herein, comprising an antigen-binding region and an immunoglobulin heavy chain constant region, wherein the immunoglobulin heavy chain constant region comprises an immunoglobulin hinge region, CH2 and CH3 domains of an immunoglobulin, wherein the antigen-binding region comprises a VHH, wherein the VHH comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 318, wherein the hinge region comprises a C220S substitution according to EU numbering, and wherein the immunoglobulin heavy chain constant region comprises L234A, L235E, G237A, A330S, P331S, and H435Q substitutions according to EU numbering, and wherein the radioimmunoconjugate comprises a chelating agent. In certain embodiments, the chelating agent comprises DOTA. In certain embodiments, the chelating agent is p-SCN-Bn-DOTA. In certain embodiments, the chelating agent is conjugated with an alpha-radioisotope, a beta-radioisotope, or a gamma-radioisotope. In certain embodiments, the alpha-radioisotope is 225-Ac. In certain embodiments, the beta-radioisotope is 177-Lu. In certain embodiments, the gamma-radioisotope is 111-In.

[0170] In one embodiment, a radioimmunoconjugate is described herein, comprising an antigen-binding region and an immunoglobulin heavy chain constant region, wherein the immunoglobulin heavy chain constant region comprises an immunoglobulin hinge region, CH2 and CH3 domains of the immunoglobulin; the antigen-binding region comprises a VHH, wherein the VHH comprises the amino acid sequence set forth in SEQ ID NO: 318, wherein the hinge region comprises a C220S substitution according to EU numbering; and the immunoglobulin heavy chain constant region comprises L234A, L235E, G237A, A330S, P331S and H435Q substitutions according to EU numbering; and the radioimmunoconjugate comprises a chelating agent. In certain embodiments, the chelating agent comprises DOTA. In certain embodiments, the chelating agent is p-SCN-Bn-DOTA. In certain embodiments, the chelating agent is conjugated to an alpha-, beta-, or gamma-radioisotope. In certain embodiments, the alpha-radioisotope is 225-Ac. In certain embodiments, the β-emitting isotope is 177-Lu. In certain embodiments, the γ-emitting isotope is 111-In.

[0171] In one embodiment, a radioimmunoconjugate is described herein, comprising an antigen-binding region and an immunoglobulin heavy chain constant region, wherein the immunoglobulin heavy chain constant region comprises an immunoglobulin hinge region, CH2 and CH3 domains of an immunoglobulin; the antigen-binding region comprises a VHH, wherein the VHH comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 319; the hinge region comprises a C220S substitution according to EU numbering; and the immunoglobulin heavy chain constant region comprises L234A, L235E, G237A, A330S, P331S, and H435Q substitutions according to EU numbering; and the radioimmunoconjugate comprises a chelating agent. In certain embodiments, the chelating agent comprises DOTA. In certain embodiments, the chelating agent is p-SCN-Bn-DOTA. In certain embodiments, the chelating agent is conjugated with an alpha-radioisotope, a beta-radioisotope, or a gamma-radioisotope. In certain embodiments, the alpha-radioisotope is 225-Ac. In certain embodiments, the beta-radioisotope is 177-Lu. In certain embodiments, the gamma-radioisotope is 111-In.

[0172] In one embodiment, a radioimmunoconjugate is described herein, comprising an antigen-binding region and an immunoglobulin heavy chain constant region, wherein the immunoglobulin heavy chain constant region comprises an immunoglobulin hinge region, CH2 and CH3 domains of the immunoglobulin; the antigen-binding region comprises a VHH, wherein the VHH comprises the amino acid sequence set forth in SEQ ID NO: 319, wherein the hinge region comprises a C220S substitution according to EU numbering; and the immunoglobulin heavy chain constant region comprises L234A, L235E, G237A, A330S, P331S and H435Q substitutions according to EU numbering; and the radioimmunoconjugate comprises a chelating agent. In certain embodiments, the chelating agent comprises DOTA. In certain embodiments, the chelating agent is p-SCN-Bn-DOTA. In certain embodiments, the chelating agent is conjugated to an alpha-, beta-, or gamma-radioisotope. In certain embodiments, the alpha-radioisotope is 225-Ac. In certain embodiments, the β-emitting isotope is 177-Lu. In certain embodiments, the γ-emitting isotope is 111-In.

[0173] Immunoconjugate Derivatives and Other Modifications Covalent modification of the immunoconjugates of the present disclosure is included within the scope of the present disclosure.One type of covalent modification involves reacting the target amino acid residue of the immunoconjugates of the present disclosure with an organic derivatizing agent that can react with selected side chains or N- or C-terminal residues of the immunoconjugates.Derivatization with bifunctional agents is useful, for example, for crosslinking the immunoconjugates of the present disclosure to a water-insoluble support matrix or surface for use in methods for purifying the immunoconjugates of the present disclosure, and vice versa. Commonly used cross-linking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, e.g., with 4-azidosalicylic acid, 3,3′-dithiobis(succinimidyl propionate), homobifunctional imidoesters including disuccinimidyl esters, bifunctional maleimides such as bis-N-maleimido-1,8-octane, and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.

[0174] Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, respectively, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0175] In some embodiments, the immunoconjugates provided herein can be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Moieties suitable for derivatization of immunoconjugates include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymers can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the immunoconjugate can vary, and when multiple polymers are attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the immunoconjugate to be improved, whether the immunoconjugate derivative will be used therapeutically under defined conditions, etc.

[0176] PEG-derivatized immunoconjugates of the present disclosure may contain one or more -CH2CHO- containing linkers, which can be used to alter the biodistribution and pharmacokinetics of the immunoconjugate. PEG can be prepared in polymeric form or as individual oligomers. Bifunctionalized versions of these polymers can link the immunoconjugate with chelators and / or provide additional size and / or solubility to the overall molecule. In some embodiments, PEG-derivatized immunoconjugates exhibit reduced immunogenicity compared to their underivatized parent molecules.

[0177] Methods for Making Immunoconjugates of the Present Disclosure The present disclosure provides compositions comprising any of the above embodiments or one or more of the immunoconjugates described herein. In another aspect, the present disclosure provides isolated nucleic acids encoding the radioisotope delivery platforms described herein. Also provided herein are nucleic acids encoding protein components of the immunoconjugates of the present disclosure, expression vectors comprising the nucleic acids, and host cells comprising the expression vectors.

[0178] In another aspect, the present disclosure provides a host cell comprising a nucleic acid and / or vector provided herein. In some embodiments, the host cell of the present disclosure is isolated or purified. In some embodiments, the host cell of the present disclosure is in a cell culture medium. The nucleic acids, expression vectors, and host cells of the present disclosure can be used to generate compositions comprising one or more of the immunoconjugates of the present disclosure. In some embodiments, the host cell is a eukaryote. In some embodiments, the host cell is a mammal. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the host cell is a prokaryote. In some embodiments, the host cell is Escherichia coli.

[0179] Exemplary techniques for producing the immunoconjugates and radioimmunoconjugates of the present disclosure for use according to the methods of the present disclosure are described below. In some embodiments, the present disclosure provides a process for making the immunoconjugates of the present disclosure, the method comprising culturing a host cell provided herein under conditions suitable for an expression vector encoding a radioisotope delivery platform, and recovering or purifying the radioisotope delivery platform. In some embodiments, the method further comprises radiolabeling the radioisotope delivery platform with a suitable isotope, such as, for example, an α- or β-particle emitter.

[0180] Production of immunoconjugates, host cells and expression vectors of the present disclosure The following description primarily relates to producing the antibody constructs of the present disclosure by culturing cells transformed or transfected with nucleic acids encoding the immunoconjugates containing vectors of the present disclosure. Of course, it is contemplated that alternative methods, well known in the art, may be used to prepare the antibody constructs of the present disclosure. For example, the appropriate amino acid sequence, or portions thereof, may be generated by direct peptide synthesis using solid-phase techniques (e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J., Am. Chem. Soc., 85:2149-54 (1963)). In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using the manufacturer's instructions. The various portions of the immunoconjugates of the present disclosure can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired immunoconjugate of the present disclosure.

[0181] Antibody constructs can be produced using recombinant methods and compositions such as those described in U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an antibody described herein is provided. Such a nucleic acid can encode an amino acid sequence comprising an amino acid sequence comprising the VH and / or VL amino acid sequence (e.g., VL) of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In some embodiments, the host cell comprises (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody (e.g., has been transformed with the vector). In some other embodiments, the host cell comprises (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., YO, NSO, Sp20 cell). In one embodiment, a method of making an immunoconjugate of the present disclosure is provided, the method comprising culturing a host cell comprising nucleic acid encoding an antibody provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0182] For recombinant production of the immunoconjugates of the present disclosure, nucleic acids encoding antibody constructs, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and / or light chains of the antibody). Nucleic acid molecules encoding the amino acid sequences (including sequence variants) of the immunoconjugates of the present disclosure can be prepared by a variety of methods known to those skilled in the art. These methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared mutant or non-mutant versions of the antibody construct.

[0183] Engineering host cells for immunoconjugate production For production of the immunoconjugates of the present disclosure, host cells are transfected or transformed with the expression or cloning vectors described herein and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. Culture conditions, such as medium, temperature, and pH, can be selected by those skilled in the art without undue experimentation. Generally, principles, protocols, and practical techniques for maximizing cell culture productivity can be found in Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

[0184] Suitable host cells for cloning or expressing immunoconjugate-encoding nucleic acids and vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199, US 5,840,523, and Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describe the expression of antibody fragments in E. coli. After expression, the immunoconjugate can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0185] Selection and use of replicable vectors For recombinant production of the radioisotope delivery platform of the present disclosure, the nucleic acid encoding it (e.g., cDNA or genomic DNA) is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or expression. DNA encoding the immunoconjugate is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of an antibody). Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, suitable host cells are of either prokaryotic or eukaryotic (generally mammalian) origin.

[0186] Vectors can be, for example, in the form of plasmids, cosmids, virus particles, or phage.Appropriate nucleic acid sequences can be inserted into vectors by various procedures.Generally, DNA is inserted into an appropriate restriction endonuclease site using techniques known in the art.Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.Construction of suitable vectors containing one or more of these components uses standard ligation techniques known to those skilled in the art.

[0187] The immunoconjugates of the present invention can be recombinantly produced not only directly but also as fusion polypeptides with heterologous polypeptides, which can be signal sequences or other polypeptides with specific cleavage sites at the N-terminus of the mature protein or polypeptide. Generally, the signal sequence can be a component of the vector, or it can be part of the immunoconjugate encoded by DNA inserted into the vector. The signal sequence can be, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion, the signal sequence can be, for example, the yeast invertase leader, the alpha-factor leader (including the Saccharomyces and Kluyveromyces alpha-factor leaders, the latter of which is described in U.S. Pat. No. 5,010,182), or the acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179, filed April 4, 1990), or the signals described in WO 90 / 13646, filed November 15, 1990. In mammalian cell expression, mammalian signal sequences can be used to direct protein secretion, for example, signal sequences from secreted polypeptides of homologous or related species, as well as viral secretory leaders.

[0188] Purification of immunoglobulin-derived structures of the present disclosure The immunoconjugates of the present disclosure can be recovered from culture medium or host cell lysates. If membrane-bound, they can be released from the membrane using an appropriate detergent solution (e.g., Triton-X 100) or by enzymatic cleavage. Cells used to express the immunoconjugates of the present disclosure can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.

[0189] It may be desirable to purify the immunoconjugates of the present disclosure from recombinant cell proteins or polypeptides. The following procedures are examples of suitable purification procedures, including fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica or a cation exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75, a protein A Sepharose column to remove contaminants such as IgG, and a metal chelate column to bind epitope-tagged forms of the immunoconjugates of the present disclosure. Various methods of protein purification can be used, and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification steps selected will depend, for example, on the nature of the production process used and the particular immunoconjugate of the present disclosure being produced.

[0190] When using recombinant technology, immunoconjugates can be produced intracellularly in the periplasmic space or directly secreted into the culture medium. If the immunoconjugate is produced intracellularly, the first step is to remove particulate debris, either host cells or lysed fragments, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-7 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. If the immunoconjugate is secreted into the culture medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0191] Immunoconjugate compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the immunoconjugate. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the immunoconjugate contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on anion or cation exchange resins (such as heparin SEPHAROSE™ chromatography (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available, depending on the immunoconjugate to be recovered.

[0192] After any preliminary purification steps, the mixture containing the immunoconjugate of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography, using an elution buffer with a pH between about 2.5 and 4.5, and generally a low salt concentration (e.g., about 0 to 0.25 M salt).

[0193] Immunoconjugates using chelators and / or linkers Methods for attaching radioisotopes to immunoconjugates or antibody constructs (i.e., "labeling" antibodies with radioisotopes) are well known to those skilled in the art. Some of these methods are described, for example, in WO2017 / 155937.

[0194] For example, bifunctional chelators such as DOTA, DTPA, and related analogs are suitable for coordinating metal ions such as α- and β-radionuclides. For example, these chelating molecules can be linked to targeting molecules by forming a new amide bond between an amine (e.g., a functional group of a lysine residue) on the antibody construct and a carboxylate on DOTA / DTPA. In the case of peptide synthesis, characterization and purification of the linker addition can be part of the overall synthesis of an antibody platform or immunoconjugate for radioisotope conjugates.

[0195] In some embodiments, the method of producing the immunoconjugate comprises a click chemistry step as described by Poty, S et al., Chem Commun. (Camb) 54:2599 (2018).

[0196] In some embodiments, peptides may be biosynthesized or synthesized by chemical amino acid synthesis using appropriate amino acid precursors that contain, for example, fluorine-19, instead of hydrogen. In some embodiments, a radiolabel may be incorporated into the peptide. In some embodiments, a radiolabel may be linked to the peptide. To incorporate iodine-123, the IODOGEN method (Fraker et al. (1978) Biochem Biophys Res Commun. 80:49-57) may be used. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.

[0197] Characterization of the Immunoconjugates of the Invention Immunoconjugates of the present invention can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art. Immunoconjugates and antibody constructs of the present disclosure can be characterized for their physical / chemical properties and / or biological activity by various assays known in the art. Immunoconjugates of the present disclosure can be characterized by a range of assays, including, but not limited to, polypeptide sequencing, amino acid analysis, non-denaturing size-exclusion high-pressure liquid chromatography (HPLC), mass spectrometry, ion-exchange chromatography, and papain digestion.

[0198] antigen binding The immunoconjugates of the present invention can be tested for their antigen-binding activity by methods known in the art, such as ELISA, Western blot, etc. The binding affinity of an antibody can be determined, for example, by Scatchard analysis as described in Munson et al., Anal Biochem. 107:220 (1980). Furthermore, the antigen-binding ability of the immunoconjugates of the present disclosure can be quantified using methods known in the art, such as quantitative ELISA, quantitative Western blot, surface plasmon resonance assay, and / or Scatchard analysis.

[0199] In one embodiment, the KD of the immunoconjugate is measured using a radiolabeled antigen ELISA performed on the immunoconjugate. According to another embodiment, the KD is measured by using surface-plasmon resonance assays using a BIACORE®-2000 or BIACORE®-3000 instrument (BIAcore, Inc., Piscataway, NJ), e.g., by using an immobilized antigen CM5 chip at 25°C and 10 response units.

[0200] In another aspect, binding competition assays can be used to identify immunoconjugates that compete for binding to the same antigen or epitope thereof. In some embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) of an immunoconjugate of the present disclosure (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)).

[0201] The epitope and / or contact residues within the antigen to which the immunoconjugate of the present disclosure binds can be identified or mapped using methods known to those of skill in the art. Detailed exemplary methods for mapping the epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology (3rd ed., Humana Press, Totowa, NJ).

[0202] Pharmaceutical Compositions and Formulations of the Present Disclosure As will be recognized by those skilled in the art, certain teachings herein below apply to the immunoconjugates and radioimmunoconjugates of the present disclosure, regardless of specific textual reference to a certain type of invention, and such applications are encompassed in their entirety by this disclosure.

[0203] In another aspect, the present disclosure provides compositions comprising the immunoconjugates or radioimmunoconjugates of the present invention. The present disclosure further provides pharmaceutical compositions and formulations comprising at least one immunoconjugate of the present invention and at least one pharmaceutically acceptable excipient or carrier. In some embodiments, the pharmaceutical formulation comprises (1) an immunoconjugate or radioimmunoconjugate of the present disclosure and (2) a pharmaceutically acceptable carrier.

[0204] The immunoconjugate or radioimmunoconjugate may be formulated in any form suitable for delivery to the target cells / tissues. Pharmaceutical formulations of the immunoconjugates of the present invention are prepared by mixing such immunoconjugates having the desired purity with one or more pharmaceutically acceptable carriers, diluents, and / or excipients in the form of a lyophilized formulation or aqueous solution (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers, diluents, and excipients are generally non-toxic to recipients at the dosages and concentrations employed and include, for example, sterile water, buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. azoles), low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0205] Pharmaceutical formulations to be used for in vivo administration are generally sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0206] Examples of lyophilized antibody formulations are described in US 6,267, 958. Aqueous antibody formulations include those described in US 6,171,586 and WO 2006 / 044908, the latter formulations containing a histidine-acetate buffer.

[0207] Pharmaceutically acceptable carriers herein further include an interstitial drug dispersing agent, such as a soluble neutral-active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

[0208] The formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0209] The active ingredient can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacylate) microcapsules, respectively, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[0210] In some embodiments, the immunoconjugate may be formulated as an immunoliposome. A "liposome" is a vesicle composed of various types of lipids, phospholipids, and / or surfactants that is useful for delivering drugs to mammals. The components of a liposome are usually arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing immunoconjugates are prepared by methods known in the art, such as those described in Epstein et al., Proc Natl Acad Sci USA 82:3688 (1985); Hwang et al., Proc Natl Acad Sci USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO 1997 / 38731, published October 23, 1997. Particularly useful liposomes can be produced by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters with defined pore sizes to obtain liposomes with desired diameters. Chemotherapeutic agents are optionally contained within the liposomes (see Gabizon et al., J. National Cancer Inst. 81:1484 (1989)). Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

[0211] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0212] Methods of using immunoconjugates and radioimmunoconjugates and compositions thereof In one aspect, the disclosure provides a method of treating a disease, disorder, or condition in a patient in need thereof, the method comprising administering to a subject in need thereof a pharmaceutically effective amount of an immunoconjugate or radioimmunoconjugate or composition of the invention. In some further embodiments, the method is for inhibiting the growth and / or death of cancer cells or tumors. In another aspect, the disclosure provides use of an immunoconjugate described herein for the preparation and / or manufacture of a medicament for treating a disease, disorder, or condition, such as cancer, in a subject.

[0213] The pharmaceutical composition of the present invention can be administered in a manner appropriate for the disease to be treated (or prevented). The amount and frequency of administration are determined by factors such as the patient's disease and the type and severity of the patient's disease, but the appropriate dosage can be determined by clinical trials.

[0214] In one embodiment, the immunoconjugate or radioimmunoconjugate or composition of the present disclosure can be used in a method for binding a target antigen in an individual suffering from a disorder associated with increased target antigen expression and / or activity, the method comprising administering the immunoconjugate or radioimmunoconjugate or composition to the individual such that the target antigen in the individual is bound. In one embodiment, the target antigen is a human target antigen, and the individual is a human individual. The immunoconjugate or radioimmunoconjugate or composition of the present disclosure can be administered to a human for therapeutic purposes. Furthermore, the immunoconjugate or radioimmunoconjugate or composition of the present disclosure can be administered to a non-human mammal (e.g., a primate, pig, rat, or mouse) expressing a target antigen with which the immunoconjugate or radioimmunoconjugate cross-reacts for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models can be useful for evaluating the therapeutic efficacy (e.g., testing dosages and time courses of administration) of the immunoconjugate or radioimmunoconjugate or composition of the present disclosure.

[0215] The immunoconjugates or radioimmunoconjugates or compositions of the present disclosure (and any additional therapeutic agents or adjuvants) can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, as well as intralesional administration if local treatment is desired. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, antibodies are suitably administered by pulse infusion, particularly with declining doses of antibody. Dosing can be by any suitable route, e.g., injections, such as intravenous or subcutaneous injections, depending in part on whether administration is brief or chronic.

[0216] The immunoconjugates or radioimmunoconjugates or compositions of the present disclosure are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to physicians. The immunoconjugates of the present disclosure are administered to human patients according to known methods, for example, intravenous administration as a bolus or by continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. In some embodiments, intravenous or subcutaneous administration of the immunoconjugates or radioimmunoconjugates or compositions of the present disclosure is preferred.

[0217] For disease prevention or treatment, the dosage and mode of administration will be selected by a physician according to known criteria. The appropriate dosage of the immunoconjugate or radioimmunoconjugate or composition of the present disclosure depends on the type of disease being treated, as defined above, the severity and course of the disease, whether the immunoconjugate or radioimmunoconjugate or composition of the present disclosure is administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to the immunoconjugate or radioimmunoconjugate or composition, and the discretion of the attending physician. The immunoconjugate or radioimmunoconjugate or composition of the present disclosure is appropriately administered to the patient at one time or over a series of treatments. Preferably, the immunoconjugate or radioimmunoconjugate or composition is administered by intravenous infusion or subcutaneous injection. Depending on the type and severity of the disease, about 1 μg / kg to about 50 mg / kg body weight (e.g., about 0.1 to 15 mg / kg / dose) of the immunoconjugate or radioimmunoconjugate or composition may be an initial candidate dosage for administration to a patient, whether by one or more separate administrations or by continuous infusion, for example. A dosing regimen may involve administering an initial loading dose of about 4 mg / kg, followed by a weekly maintenance dose of about 2 mg / kg of the immunoconjugate or radioimmunoconjugate or composition of the present disclosure. However, other dosing regimens may also be useful. A typical daily dose may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors described above. For repeated administration over several days or longer, depending on the disease, treatment is continued until a desired suppression of disease symptoms occurs. The progress of this therapy can be easily monitored by conventional methods and assays and based on criteria known to the physician or other skilled artisan.

[0218] Dosage and administration schedule can be selected and adjusted based on the level of disease or tolerance in the subject, and this can be monitored during the course of treatment.The conjugate of the present invention can be administered once a day, once a week, multiple times a week, less than once a day, multiple times a month, less than once a day, multiple times a month, less than once a week, once a month, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, or intermittently to alleviate or relieve disease symptoms.Administration can continue at any of the intervals disclosed until the symptoms of the tumor or cancer being treated are alleviated.Administration can continue after symptom alleviation or relief is achieved, and such alleviation or relief is prolonged by such continued administration.

[0219] In some embodiments, an effective amount of the immunoconjugate or radioimmunoconjugate or composition may be provided as a single dose.

[0220] The immunoconjugates and radioimmunoconjugates of the present invention may be used in combination with conventional and / or novel methods of treatment or therapy, or separately as monotherapy.

[0221] The immunoconjugates and radioimmunoconjugates of the present invention may (i) inhibit the growth or proliferation of cells to which they bind, (ii) induce the death of cells to which they bind, (iii) inhibit delamination of cells to which they bind, (iv) inhibit metastasis of cells to which they bind, or (v) inhibit the angiogenesis of tumors containing cells to which they bind. In this context, "inhibiting cell growth or proliferation" means reducing cell growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death.

[0222] By way of example, an immunoconjugate that inhibits tumor cell proliferation is one that results in measurable growth inhibition of tumor cells (e.g., cancer cells). In one embodiment, an immunoconjugate or radioimmunoconjugate of the present disclosure is capable of inhibiting the growth of cancer cells that present the antigen bound by the immunoconjugate or radioimmunoconjugate. Preferred growth-inhibitory immunoconjugates or radioimmunoconjugates inhibit the growth of antigen-expressing tumor cells by more than 20%, preferably about 20% to about 50%, and even more preferably more than 50% (e.g., about 50% to about 100%), relative to a suitable control, which is typically tumor cells that have not been treated with the immunoconjugate or radioimmunoconjugate being tested.

[0223] In some embodiments, the majority of the immunoconjugate or radioimmunoconjugate or composition administered to a subject typically consists of unlabeled immunoconjugate, with a minority being labeled radioimmunoconjugate. The ratio of labeled to unlabeled immunoconjugate can be adjusted using known methods. Thus, according to certain aspects of the invention, the immunoconjugate / radioimmunoconjugate contains up to 100 mg of total protein, e.g., less than 60 mg, or 5 mg to 45 mg, or 0.1 μg / kg to 1 mg / kg of patient weight, e.g., 1 μg / kg to 1 mg / kg of patient weight, or 10 μg / kg to 1 mg / kg of patient weight, or 100 μg / kg to 1 mg / kg of patient weight, or 0.1 μg / kg The protein may be provided in a total protein amount of from 0.1 μg / kg to 100 μg / kg patient weight, or from 0.1 μg / kg to 50 μg / kg patient weight, or from 0.1 μg / kg to 10 μg / kg patient weight, or from 0.1 μg / kg to 40 μg / kg patient weight, or from 1 μg / kg to 40 μg / kg patient weight, or from 0.1 mg / kg to 1.0 mg / kg patient weight, for example from 0.2 mg / kg patient weight to 0.6 mg / kg patient weight.

[0224] In certain embodiments, the immunoconjugate / radioimmunoconjugate may be administered at about 0.5 mg / kg to about 30 mg / kg.In certain embodiments, the immunoconjugate / radioimmunoconjugate is administered at a concentration of about 0.5 mg / kg to about 1 mg / kg, about 0.5 mg / kg to about 2 mg / kg, about 0.5 mg / kg to about 5 mg / kg, about 0.5 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 3 mg / kg, about 0.5 mg / kg to about 4 mg / kg, about 0.5 mg / kg to about 5 mg / kg, about 0.5 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 20 mg / kg, about 0.5 mg / kg to about 30 mg / kg, about 1 mg / kg to about 2 mg / kg, about 1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 3 mg / kg, about 1 mg / kg to about 4 mg / kg, about 1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 20 mg / kg, about 1 mg / kg to about 30 mg / kg, about 2 m g / kg~about 5mg / kg, about 2mg / kg~about 10mg / kg, about 2mg / kg~about 3mg / kg, about 2mg / kg~about 4mg / kg, about 2mg / kg~about 5mg / kg, about 2mg / kg~about 10mg / kg, about 2mg / kg~about 20mg / kg, about 2mg / kg~about 30 mg / kg, approximately 5 mg / kg to approximately 10 mg / kg, approximately 5 mg / kg to approximately 3 mg / kg, approximately 5 mg / kg to approximately 4 mg / kg, approximately 5 mg / kg to approximately 5 mg / kg, approximately 5 mg / kg to approximately 10 mg / kg, approximately 5 mg / kg to approximately 20 mg / kg, approximately 5 mg / kg to approximately 30 mg / kg, approximately 10mg / kg~about 3mg / kg, about 10mg / kg~about 4mg / kg, about 10mg / kg~about 5mg / kg, about 10mg / kg~about 10mg / kg, about 10mg / kg~about 20mg / kg, about 10mg / kg~about 30mg / kg, about 3mg / kg~about 4mg / kg, about 3m It may be administered at a dose of about 10 mg / kg to about 5 mg / kg, about 3 mg / kg to about 10 mg / kg, about 3 mg / kg to about 20 mg / kg, about 3 mg / kg to about 30 mg / kg, about 4 mg / kg to about 5 mg / kg, about 4 mg / kg to about 10 mg / kg, about 4 mg / kg to about 20 mg / kg, about 4 mg / kg to about 30 mg / kg, about 5 mg / kg to about 10 mg / kg, about 5 mg / kg to about 20 mg / kg, about 5 mg / kg to about 30 mg / kg, about 10 mg / kg to about 20 mg / kg, about 10 mg / kg to about 30 mg / kg, or about 20 mg / kg to about 30 mg / kg.In certain embodiments, the immunoconjugate / radioimmunoconjugate may be administered at about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, or about 30 mg / kg. In certain embodiments, the immunoconjugate / radioimmunoconjugate may be administered at at least about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, or about 20 mg / kg. In certain embodiments, the immunoconjugate / radioimmunoconjugate may be administered at up to about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, or about 30 mg / kg.

[0225] In some embodiments, the method comprises administering an effective amount of a radioimmunoconjugate comprising 225-Ac that is between 0.01 and 0.1 mCi, or between 0.1 mCi and 1.0 mCi, or between 1.0 mCi and 2.0 mCi, or between 2.0 mCi and 4.0 mCi.

[0226] In some embodiments, the method comprises administering an effective amount of an immunoconjugate comprising 225-Ac at 0.1 μCi / kg to 2.0 μCi / kg of the subject's body weight, or 0.1 μCi / kg to 1.0 μCi / kg of the subject's body weight, or 1.0 μCi / kg to 3.0 μCi / kg of the subject's body weight, or 3.0 μCi / kg to 10.0 μCi / kg of the subject's body weight, or 10.0 μCi / kg to 20.0 μCi / kg of the subject's body weight, or 10.0 μCi / kg to 30.0 μCi / kg of the subject's body weight.

[0227] In certain embodiments, the effective amount of 225-Ac is about 0.1 microcuries to about 20 microcuries. In certain embodiments, the effective amount of 225-Ac is about 0.1 microcuries to about 0.2 microcuries, about 0.1 microcuries to about 0.5 microcuries, about 0.1 microcuries to about 1 microcurie, about 0.1 microcuries to about 2 microcuries, about 0.1 microcuries to about 3 microcuries, about 0.1 microcuries to about 4 microcuries, about 0.1 microcuries to about 5 microcuries, about 0.1 microcuries to about 10 microcuries, about 0.1 microcuries to about 20 microcuries, about 0.2 microcuries to about 0.5 microcuries, about 0.2 microcuries to about 1 microcurie, About 0.2 microcuries to about 2 microcuries, about 0.2 microcuries to about 3 microcuries, about 0.2 microcuries to about 4 microcuries, about 0.2 microcuries to about 5 microcuries, about 0.2 microcuries to about 10 microcuries, about 0.2 microcuries to about 20 microcuries, about 0.5 microcuries to about 1 microcurie, about 0.5 microcuries to about 2 microcuries, about 0.5 microcuries to about 3 microcuries, about 0.5 microcuries to about 4 microcuries, about 0.5 microcuries to about 5 microcuries, about 0.5 microcuries to about 10 microcuries, about 0.5 microcuries to about 20 microcuries, about 1 microcurie to about 2 microcuries, about 1 microcurie to about 3 microcuries, about 1 microcurie to about 4 microcuries, about 1 microcurie to about 5 microcuries, about 1 microcurie to about 10 microcuries, about 1 microcurie to about 20 microcuries, about 2 microcuries to about 3 microcuries, about 2 microcuries to about 4 microcuries, about 2 microcuries to about 5 microcuries, about 2 microcuries to about 10 microcuries, about 2 microcuries Curies to about 20 microcuries, about 3 microcuries to about 4 microcuries, about 3 microcuries to about 5 microcuries, about 3 microcuries to about 10 microcuries, about 3 microcuries to about 20 microcuries, about 4 microcuries to about 5 microcuries, about 4 microcuries to about 10 microcuries, about 4 microcuries to about 20 microcuries, about 5 microcuries to about 10 microcuries, about 5 microcuries to about 20 microcuries, or about 10 microcuries to about 20 microcuries. In certain embodiments, the effective amount of 225-Ac is about 0.1 microcuries, about 0.2 microcuries, about 0.5 microcuries, about 1 microcuries, about 2 microcuries, about 3 microcuries, about 4 microcuries, about 5 microcuries, about 10 microcuries, or about 20 microcuries. In certain embodiments, the effective amount of 225-Ac is at least about 0.1 microcuries, about 0.2 microcuries, about 0.5 microcuries, about 1 microcuries, about 2 microcuries, about 3 microcuries, about 4 microcuries, about 5 microcuries, or about 10 microcuries. In certain embodiments, the effective amount of 225-Ac is at most about 0.2 microcuries, about 0.5 microcuries, about 1 microcuries, about 2 microcuries, about 3 microcuries, about 4 microcuries, about 5 microcuries, about 10 microcuries, or about 20 microcuries. According to aspects in which the radioisotope of the radioimmunoconjugate is 111-In, the effective amount is, for example, 15.0 mCi (i.e., the amount of 111-In administered to the subject delivers a total body radiation dose of less than 15.0 mCi).

[0228] According to embodiments in which the radioactive isotope of the radioimmunoconjugate is 111-In, an effective amount is less than 15.0 mCi, less than 14.0 mCi, less than 13.0 mCi, less than 12.0 mCi, less than 11.0 mCi, less than 10.0 mCi, less than 9.0 mCi, less than 8.0 mCi, less than 7.0 mCi, less than 6.0 mCi, less than 5.0 mCi, less than 4.0 mCi, less than 3.5 mCi, less than 3.0 mCi, less than 2.5 mCi, less than 2.0 mCi, less than 1.5 mCi, less than 1.0 mCi, less than 0.5 mCi, less than 0.4 mCi, less than 0.3 mCi, less than 0.2 mCi, or less than 0.1 mCi.

[0229] According to an embodiment in which the radioactive isotope of the radioimmunoconjugate is 111-In, the effective amount is 0.1 mCi to 1.0 mCi, 0.1 mCi to 2.0 mCi, 1.0 mCi to 2.0 mCi, 1.0 mCi to 3.0 mCi, 1.0 mCi to 4.0 mCi, 1.0 mCi to 5.0 mCi, 1.0 mCi to 10.0 mCi, 1.0 mCi to 15.0 mCi, 1.0 mCi to 20.0 mCi, 2.0mCi~3.0mCi, 3.0mCi~4.0mCi, 4.0mCi~5.0mCi, 5.0mCi~10.0mCi, 5.0mCi~15.0mCi, 5.0mCi~20.0m Ci, 6.0mCi~14.0mCi, 7.0mCi~13.0mCi, 8.0mCi~12.0mCi, 9.0mCi~11.0mCi, or 10.0mCi~15.0mCi.

[0230] According to embodiments in which the radioactive isotope of the radioimmunoconjugate is 111-In, an effective amount is 15.0 mCi, 14.0 mCi, 13.0 mCi, 12.0 mCi, 11.0 mCi, 9.0 mCi, 8.0 mCi, 7.0 mCi, 6.0 mCi, 5.0 mCi, 4.0 mCi, 3.5 mCi, 3.0 mCi, 2.5 mCi, 2.0 mCi, 1.5 mCi, 1.0 mCi, 0.5 mCi, 0.4 mCi, 0.3 mCi, 0.2 mCi, or 0.1 mCi.

[0231] According to embodiments in which the radioactive isotope of the radioimmunoconjugate is 225-Ac, an effective amount is, for example, less than 30.0 μCi / kg (i.e., when the amount of 225-Ac administered to a subject delivers a radiation dose of less than 30.0 μCi per kilogram of the subject's body weight).

[0232] According to embodiments in which the radioactive isotope of the radioimmunoconjugate is 225-Ac, effective amounts are 30 μCi / kg, 25 μCi / kg, 20 μCi / kg, 17.5 μCi / kg, 15.0 μCi / kg, 12.5 μCi / kg, 10.0 μCi / kg, 9 μCi / kg, 8 μCi / kg, 7 μCi / kg, 6 μCi / kg, 5 μCi / kg, 4.5 μCi / kg, 4.0 μCi / kg g, 3.5μCi / kg, 3.0μCi / kg, 2.5μCi / kg, 2.0μCi / kg, 1.5μCi / kg, 1.0μCi / kg, 0.9μCi / kg, 0.8μCi / kg, 0.7 less than μCi / kg, 0.6 μCi / kg, 0.5 μCi / kg, 0.4 μCi / kg, 0.3 μCi / kg, 0.2 μCi / kg, 0.1 μCi / kg, or 0.05 μCi / kg.

[0233] According to embodiments in which the radioactive isotope of the radioimmunoconjugate is 225-Ac, the effective amount is 0.05 μCi / kg to 0.1 μCi / kg, 0.1 μCi / kg to 0.2 μCi / kg, 0.2 μCi / kg to 0.3 μCi / kg, 0.3 μCi / kg to 0.4 μCi / kg, 0.4 μCi / kg to 0.5 μCi / kg, 0.5 μCi / kg to 0.6 μCi / kg, 0.6 μCi / kg to 0.7 μCi / kg, 0.7 μCi / kg to 0.8 μCi / kg, 0. 8μCi / kg~0.9μCi / kg, 0.9μCi / kg~1.0μCi / kg, 1.0μCi / kg~1.5μCi / kg, 1.5μCi / kg~2.0μCi / kg, 2.0μCi / kg~2.5μCi / kg, 2.5μ Ci / kg~3.0μCi / kg, 3.0μCi / kg~3.5μCi / kg, 3.5μCi / kg~4.0μCi / kg, 4.0μCi / kg~4.5μCi / kg, or 4.5μCi / kg~5.0μCi / kg.

[0234] According to an embodiment in which the radioactive isotope of the radioimmunoconjugate is 225-Ac, an effective amount is 0.05 μCi / kg, 0.1 μCi / kg, 0.2 μCi / kg, 0.3 μCi / kg, 0.4 μCi / kg, 0.5 μCi / kg, 0.6 μCi / kg, 0.7 μCi / kg, 0.8 μCi / kg, 0.9 μCi / kg, 1.0 μCi / kg, 1.5 μCi / kg, 2.0 μCi / kg, 2.5 μCi / kg, 3.0 μCi / kg, 3.5 μCi / kg, 4.0 μCi / kg, 4.5 μCi / kg, 5.0 μCi / kg, 6.0 μCi / kg, 6.5 μCi / kg, 7.0 μCi / kg, 7.5 μCi / kg, 8.0 μCi / kg, 8.5 μCi / kg, 9.0 μCi / kg, 9.5 μCi / kg, 10.0 μCi / kg, 10.5 μCi / kg, 11.0 μCi / kg, 11.5 μCi / kg, 12.0 μCi / kg, 12.5 μCi / kg, 13.0 μCi / kg, 14.0 μCi / kg, 15.0 μCi / kg, 16.0 μCi / kg, 17.0 μCi / kg, 18.0 μCi / kg, 19.0 μCi / kg, 20.0 μCi / kg, 21.0 μCi / kg, 22.0 μCi / kg, 23.0 μCi / kg, 24.0 μCi / kg, 25.0 μCi / kg i / kg, 3.0μCi / kg, 3.5μCi / kg, 4.0μCi / kg, or 4.5μCi / kg, 5.0μCi / kg, 6.0μCi / kg, 7.0μCi / kg, 8.0μCi / kg, 9.0μCi / kg, 10.0μCi / kg, 12.5μCi / kg, 15.0μCi / kg, 17.5μCi / kg, 20.0μCi / kg, 25μCi / kg, or 30μCi / kg.

[0235] In certain embodiments where the radioisotope of the radioimmunoconjugate is 177-Lu, the effective amount is between 0.1 uCi and 100 mCi per square meter of body surface area.

[0236] In certain embodiments, where the radioisotope of the radioimmunoconjugate is 177-Lu, an effective amount is between 1 mCi and 100 mCi per square meter of body surface area. In certain embodiments, an effective amount is between about 1 mCi per square meter and about 100 mCi per square meter. In certain embodiments, an effective amount is between about 1 mCi per square meter and about 5 mCi per square meter, between about 1 mCi per square meter and about 10 mCi per square meter, between about 1 mCi per square meter and about 15 mCi per square meter, between about 1 mCi per square meter and about 20 mCi per square meter, between about 1 mCi per square meter and about 25 mCi per square meter, between about 1 mCi per square meter and about 75 mCi per square meter, between about 1 mCi per square meter and about 100 mCi per square meter. Approximately 100, approximately 5 per square meter to approximately 10 per square meter, approximately 5 per square meter to approximately 15 per square meter, approximately 5 per square meter to approximately 20 per square meter, approximately 5 per square meter to approximately 25 per square meter, approximately 5 per square meter to approximately 75 per square meter, approximately 5 per square meter to approximately 100 per square meter, approximately 10 per square meter to approximately 15 per square meter, approximately Approximately 10 per square meter to approximately 20 per square meter, approximately 10 per square meter to approximately 25 per square meter, approximately 10 per square meter to approximately 75 per square meter, approximately 10 per square meter to approximately 100 per square meter, approximately 15 per square meter to approximately 20 per square meter, approximately 15 per square meter to approximately 25 per square meter, approximately 15 per square meter to approximately 75 per square meter, approximately about 15 per square meter to about 100 per square meter, about 20 per square meter to about 25 per square meter, about 20 per square meter to about 75 per square meter, about 20 per square meter to about 100 per square meter, about 25 per square meter to about 75 per square meter, about 25 per square meter to about 100 per square meter, or about 75 per square meter to about 100 per square meter.In certain embodiments, the effective amount is about per square meter, about per 5 square meters, about per 10 square meters, about per 15 square meters, about per 20 square meters, about per 25 square meters, about per 75 square meters, or about per 100 square meters. In certain embodiments, the effective amount is at least about 1 per square meter, about 5 per square meter, about 10 per square meter, about 15 per square meter, about 20 per square meter, about 25 per square meter, or about 75 per square meter. In certain embodiments, the effective amount is at most about 5 per square meter, about 10 per square meter, about 15 per square meter, about 20 per square meter, about 25 per square meter, about 75 per square meter, or about 100 per square meter.

[0237] According to certain aspects of the invention, a preparation of a radioimmunoconjugate of the present disclosure or a composition thereof (e.g., a pharmaceutical composition) can include a radiolabeled fraction (radioimmunoconjugate) and an unlabeled fraction (immunoconjugate), and the ratio of labeled:unlabeled can be about 1:1000 to 1:1.

[0238] Additionally, pharmaceutical compositions can be provided as single-dose compositions tailored to a particular patient, i.e., as patient-specific therapeutic compositions, where the amounts of labeled and unlabeled immunoconjugates (for clarity, labeled immunoconjugates are the same as radioactive immunoconjugates herein) in the composition can depend, at least, on the patient's weight, height, body surface area, age, sex, and / or disease or health condition. Thus, the total volume of the patient-specific therapeutic composition can be provided in a vial configured to be administered entirely to a patient in a single treatment session, such that little or no composition remains in the vial after administration.

[0239] Currently, depending on the stage of the cancer, cancer treatment includes one or a combination of the following therapies: surgery to remove cancerous tissue, radiation therapy, and chemotherapy. Treatment using the radioimmunoconjugates (interchangeably, "radiolabeled immunoconjugates") of the present disclosure may be particularly desirable in elderly patients who do not tolerate the toxicity and side effects of chemotherapy well, and in metastatic disease where radiation therapy has limited usefulness. In some embodiments, treatment using the radiolabeled immunoconjugates of the present disclosure is useful for alleviating target antigen-expressing cancers at the time of initial diagnosis of disease or during recurrence.

[0240] In some embodiments, determining whether a cancer is suitable for treatment with the methods disclosed herein involves detecting the presence of a target antigen in a subject or a sample derived from the subject. Various detection assays are available for determining target antigen expression in cancer. In one aspect, target antigen overexpression is analyzed by immunohistochemistry (IHC). Paraffin-embedded tissue sections from tumor biopsies are subjected to IHC assays and subjected to target antigen staining intensity criteria. Alternatively, or additionally, FISH assays such as INFORM® (sold by Ventana, AZ, USA) or PATHVISION® (Vysis, IL, USA) can be performed on formalin-fixed, paraffin-embedded tumor tissue to determine the degree (if any) of target antigen overexpression in the tumor.

[0241] Overexpression or amplification of a target antigen can be assessed using an in vivo detection assay, for example, by administering a molecule (such as an antibody construct or immunoconjugate of the present disclosure) that binds to the molecule to be detected and is tagged with a detectable label (e.g., a radioisotope or fluorescent label) and externally scanning the patient for label localization.

[0242] The immunoconjugates or radioimmunoconjugates of the present disclosure can be used, for example, in vitro, ex vivo, and in vivo methods. In one aspect, the present disclosure provides a method for inhibiting cell growth or proliferation either in vivo or in vitro, the method comprising exposing cells to the immunoconjugates or radioimmunoconjugates of the present disclosure under conditions that allow the immunoconjugates or radioimmunoconjugates to bind to the target antigen. The immunoconjugates or radioimmunoconjugates of the present disclosure can also (i) inhibit the growth or proliferation of cells to which they bind, (ii) induce the death of cells to which they bind, (iii) inhibit the delamination of cells to which they bind, (iv) inhibit the metastasis of cells to which they bind, or (v) inhibit the angiogenesis of tumors containing cells to which they bind.

[0243] In one aspect, the present disclosure provides a method for killing antigen-expressing cells, the method comprising contacting cells with an immunoconjugate or radioimmunoconjugate of the invention (or a composition thereof). This method can be used, for example, to kill, deplete, or eliminate target antigen-expressing cells from a population of mixed cells. This method can be used, for example, to kill, deplete, or eliminate target antigen-expressing cells from a population of mixed cells as a step in the purification of other cells. This method can be performed in vitro or in vivo, including ex vivo, on primary patient cell or tissue compositions to prepare such compositions for transplantation.

[0244] In one aspect, the immunoconjugates or radioimmunoconjugates of the present disclosure are used to treat or prevent cell proliferative disorders. In certain embodiments, the cell proliferative disorder includes solid tumor cancer. Solid tumor cancers are cancers involving an abnormal mass of tissue, e.g., carcinomas and sarcomas. In certain other embodiments, the cell proliferative disorder includes tumor cancer or hematological cancer, e.g., leukemia and lymphoma, used interchangeably, where such cancers are present in bodily fluids. In certain embodiments, the cell proliferative disorder is associated with increased expression and / or activity of a target antigen. For example, in certain embodiments, the cell proliferative disorder is associated with increased expression of a target antigen on the surface of a cell. In certain embodiments, the cell proliferative disorder is a tumor or cancer. In certain embodiments, the cell proliferative disorder includes solid tumor cancer. Solid tumor cancers include cancers involving an abnormal mass of tissue, e.g., carcinomas and sarcomas. In certain other embodiments, the cell proliferative disorder includes tumor cancer or hematological cancer, e.g., leukemia and lymphoma, used interchangeably, where such cancers are present in bodily fluids.

[0245] In one aspect, the present disclosure provides a method for treating a cell proliferative disorder, the method comprising administering to an individual an effective amount of an immunoconjugate or radioimmunoconjugate of the present disclosure.

[0246] In addition to direct cell killing of target cells expressing the cell surface antigen specifically bound by the immunoconjugates or radioimmunoconjugates of the present disclosure, the immunoconjugates or radioimmunoconjugates of the present disclosure can optionally be used to deliver additional cargo to the vicinity of or inside the target cells. Delivery of additional exogenous substances can be used, for example, for cytotoxicity, cytostatic, interrogation, and / or diagnostic functions. Non-cytotoxic, or optionally toxic, variants of the immunoconjugates or radioimmunoconjugates of the present disclosure can be used to deliver and / or label cargo inside cells expressing the target antigen. Non-limiting examples of cargo include cytotoxic agents, detection-enhancing agents, and small molecule chemotherapeutic agents.

[0247] As described herein, in some embodiments, the antibody constructs, immunoconjugates, radioimmunoconjugates, and targeted imaging conjugates of the present disclosure have various non-therapeutic applications. In some embodiments, the compositions of the present disclosure can be used to identify patient populations predicted to benefit from a particular therapeutic approach or treatment modality, such as treatment with an immunoconjugate or radioimmunoconjugate of the present disclosure. In some embodiments, the compositions of the present disclosure can be useful for staging target antigen-expressing cancers (e.g., by radioimaging) or as a prognostic indicator of disease progression. In some embodiments, the compositions are also useful for in vitro detection and quantification of target epitopes, for example, in ELISA or Western blots, and for purifying or immunoprecipitation of target antigens from cell or tissue samples.

[0248] In some embodiments, the immunoconjugate or radioimmunoconjugate of the present disclosure is used in a method for detecting the presence or level of an antigen, for example, in vitro in a biological sample or in vivo using imaging technology.Detection of immunoconjugates and radioimmunoconjugates can be achieved through different techniques known to those skilled in the art and described herein, such as IHC and PET imaging.When the immunoconjugate or radiolabeled immunoconjugate of the present disclosure is used for detection, it can contain a radioactive atom, such as 99m-Tc or 111-In, for scintigraphy studies.

[0249] Another embodiment of the present invention provides a method for diagnosing the presence of a tumor in a subject, the method comprising: (a) contacting a test sample comprising tissue cells obtained from a mammal with an immunoconjugate that binds to a target antigen, and (b) detecting the formation of a conjugate between the immunoconjugate and the target antigen in the test sample, wherein the formation of the conjugate indicates the presence of a tumor in the mammal. Optionally, the immunoconjugate is detectably labeled, attached to a solid support, or the like, and / or the test sample of tissue cells is obtained from an individual suspected of having a cancerous tumor.

[0250] In some embodiments, the immunoconjugates of the present invention, including compositions including those described above and / or provided herein, are useful, for example, for detecting the presence of a target antigen in vivo or in a biological sample. The immunoconjugates of the present disclosure can be used in a variety of different assays, including, but not limited to, ELISA, bead-based immunoassays, and mass spectrometry.

[0251] Kits and Articles of the Invention Another aspect of the present invention is an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of diseases and disorders characterized by target antigen-expressing cells (e.g., cancer cells). The article of manufacture of the present disclosure includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition effective for the treatment, prevention, and / or diagnosis of cancer disease and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an immunoconjugate of the present disclosure. The label or package insert indicates that the composition is used to treat cancer. The label or package insert further includes instructions for administering the immunoconjugate composition to a cancer patient. In addition, the article of manufacture may further include a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0252] In another aspect, the present disclosure provides kits comprising any of the immunoconjugates described herein and additional reagents or pharmaceutical devices. In some further embodiments, the kits comprise a composition (e.g., a pharmaceutical composition or a diagnostic composition) provided herein. Another aspect of the present invention is a kit useful for various purposes, such as target antigen-expressing cell killing, target antigen-expressing cell detection, target antigen quantification, purification, or immunoprecipitation from cells.

[0253] In some embodiments, the kit of the present disclosure is an immunoassay kit for specifically detecting an antigen in a biological sample, comprising (a) an immunoconjugate and / or composition thereof described herein, and (b) instructions for detecting the immunoconjugate. The target antigen detection assay of the present invention can be provided in the form of a kit. In some embodiments, such a kit comprises an immunoconjugate of the present disclosure or a composition comprising the foregoing, such as those described herein. The kit may further comprise a solid support for a capture reagent, which may be provided as a separate component or on which the capture reagent may already be immobilized. For target antigen isolation and purification, the kit may contain an immunoconjugate of the present disclosure bound to beads (e.g., Sepharose beads). The present disclosure provides kits containing antibodies for in vitro detection and / or quantification of target antigens, for example, in ELISA or Western blot. In some embodiments, the capture reagent (e.g., an immunoconjugate of the present disclosure) is coated or attached to a solid material (e.g., a bead, microtiter plate, or comb). The detectable antibody can be a labeled antibody that is detected directly, or it can be an unlabeled antibody that is detected by a labeled antibody against the unlabeled antibody, e.g., an antibody raised in a different species. If the label is an enzyme, the kit will usually include substrates and cofactors required by the enzyme, a dye precursor that provides the detectable chromophore if the label is a fluorophore, and an avidin, e.g., avidin, streptavidin, or streptavidin conjugated with MUG to HRP or β-galactosidase if the label is biotin.

[0254] Similar to the products of the present disclosure, kits of the present disclosure include a container and a label or package insert on or associated with the container. The container holds a composition comprising at least one immunoconjugate of the present disclosure. Additional containers containing, for example, diluents and buffers, control immunoconjugates, or antibodies may be included. The label or package insert may provide a description of the composition and instructions for its intended in vitro or detection use. The kit also typically includes additives such as stabilizers, washing and incubation buffers, etc. for performing the assay method. The components of the kit are provided in predetermined ratios, with the relative amounts of the various reagents appropriately varied to provide concentrations in solution of the reagents that substantially maximize the sensitivity of the assay. In particular, the reagents are provided as dry powders, usually lyophilized, including excipients, which, upon dissolution, provide a reagent solution having the appropriate concentration for combination with the sample to be tested.

[0255] The present invention is further illustrated by the following non-limiting examples of immunoconjugates comprising the aforementioned structures and functions, particularly platforms having VHH polypeptides with molecular weights of 60-110 kDa and serum half-lives of less than 96 hours, which in some embodiments exhibit enhanced stability at temperatures required for certain radiolabeling processes compared to other antibody fragment platforms, and in some embodiments exhibit reduced loss of targeting ability due to radiolysis compared to other possible delivery platforms.

[0256] Specific Definitions In this description, certain specific details are set forth to provide a thorough understanding of various embodiments. However, those skilled in the art will understand that the provided embodiments may be practiced without these details. Unless the context otherwise requires, throughout the following specification and claims, the word "comprise" and variations thereof, such as "comprises" and "comprising," should be construed in the inclusive sense, i.e., "including, but not limited to." As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally used in its sense to include "and / or" unless the content clearly dictates otherwise. Additionally, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0257] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are fully explained in such publications as "Molecular Cloning: A Laboratory Manual," second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F.M. Usubel et al., eds., 1987, and periodic updates); "PCR: The Polymerase Chain Reaction" (Mullis et al., ed., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001). Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.For purposes of the present invention, a number of terms are defined below.

[0258] As used in this specification and the appended claims, the terms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0259] Throughout this specification, the term "including" is used to mean "including but not limited to," "including," and "including but not limited to" are used interchangeably.

[0260] The term "about" as used herein refers to the normal error range of each value, which is readily known to those skilled in the art. Reference to "about" a value or parameter herein includes (and describes) an embodiment that is directed to the value or parameter itself. When used before a numerical indication, such as a numerical temperature, time, amount, or concentration, including a range, the term "about" indicates an approximation that may vary by ±10%.

[0261] The term "amino acid residue" or "amino acid" includes reference to an amino acid incorporated into a protein, polypeptide, and / or peptide. The term "polypeptide" includes any polymer of amino acids or amino acid residues. The term "polypeptide sequence" refers to a series of amino acids or amino acid residues that physically comprise a polypeptide and can be of any length. A "protein" is a macromolecule containing one or more polypeptides or polypeptide "chains." A "peptide" is a small polypeptide ranging in size from 2 to 20 amino acid residues. The term "amino acid sequence" refers to a series of amino acids or amino acid residues that physically comprise a peptide or polypeptide, depending on its length. Unless otherwise indicated, polypeptide and protein sequences disclosed herein are written from left to right, representing their order from the amino terminus to the carboxy terminus.

[0262] The terms "amino acid," "amino acid residue," "amino acid sequence," or polypeptide sequence include naturally occurring amino acids (including L- and D-isostereoisomers) and, unless otherwise specified, also include known analogs of natural amino acids that can function similarly to common natural amino acids, such as selenocysteine, pyrrolysine, N-formylmethionine, gamma-carboxyglutamate, hydroxyproline hypusine, pyroglutamic acid, and selenomethionine (see, e.g., Ho J et al., ACS Synth Biol 5:163-71 (2016); Wang Y, Tsao M, Chembiochem 17:2234-9 (2016)). The amino acids referred to herein are described by the abbreviations in Table A below.

[0263] As used herein, the term "radioisotope" refers to any radioisotope, e.g., 86-Y, 90-Y, 177-Lu, 186-Re, 188-Re, 89-Sr, 153-Sm, 225-Ac, 213-Bi, 213-Po, 212-Bi, 223-Ra, 224-Ra, 227-Th, 149-Tb, 68-Ga, 64-Cu, 67- Including, but not limited to, alpha-emitting isotopes (interchangeably, alpha-emitting isotopes), beta-emitting isotopes (interchangeably, beta-emitting isotopes), and / or gamma-emitting isotopes (interchangeably, gamma-emitting isotopes), such as any one of Cu, 89-Zr, 137-Cs, 212-Pb, 103-Pd, 111-In, 89-Zn, 123-I, and 99m-Tc.

[0264] As used herein, the term "radioimmunoconjugate" refers to a molecular conjugate comprising (1) an immunoconjugate according to the present disclosure and (2) a radioisotope. In a preferred embodiment, the radioisotope is an α-emitting isotope. In another embodiment, the radioisotope is a β-emitting isotope. In another embodiment, the radioisotope is a γ-emitting isotope. In another embodiment, the present disclosure provides a radioimmunoconjugate comprising an α-emitting isotope and a β-emitting isotope. The term "radioconjugate" is used interchangeably herein with "radioimmunoconjugate." In one embodiment, the radioisotope is bound to a chelator of the radioimmunoconjugate. In one embodiment, the radioisotope is directly linked to the immunoconjugate.

[0265] As used herein, the term "immunoconjugate" refers to a molecular conjugate comprising at least one antigen-binding region (e.g., a variable region or a complementarity-determining region) derived from an antibody, further conjugated to at least one non-antibody-derived molecule, such as a chelator or cytotoxic agent. The non-antibody-derived molecule may be conjugated, for example, to one or more lysine or cysteine ​​residues of the antigen-binding region or to a constant region attached (by peptide bond or otherwise) to the antigen-binding region. In some embodiments, the immunoconjugate further comprises a chelator (interchangeably, "chelator"). In one embodiment, the immunoconjugate comprises an antibody construct of the present disclosure linked directly or indirectly to a cytotoxic agent or a radioisotope.

[0266] The immunoconjugates and radioimmunoconjugates described herein contain antigen-binding regions. These antigen-binding regions can be derived from "antibodies." The term "antibody" is used in the broadest sense herein and includes monoclonal antibodies, as well as intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, such as single-chain variable fragments (sFv or scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific (e.g., bispecific) antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv, etc. Unless otherwise specified, the term "antibody" should be understood to include functional antibody fragments thereof. The term also includes intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. The antibody may comprise a human IgG1 constant region. The antibody may comprise a human IgG4 constant region.

[0267] The terms "complementarity determining region" and "CDR," which are synonymous with "hypervariable region" or "HVR," are known in the art to refer to non-contiguous sequences of amino acids in an antibody variable region that confer antigen specificity and / or binding affinity. Generally, each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) has three CDRs, and each light chain variable region (CDR-L1, CDR-L2, CDR-L3) has three CDRs. The terms "framework regions" and "FR" are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4) has four FRs, and each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4) has four FRs.The exact amino acid sequence boundaries of a given CDR or FR can be determined using the methods of Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 Jan;27(1):55-77 ("IMGT" numbering scheme); Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool", J Mol Biol, 2001 Jun 8;309(3):657-70, ("Aho" numbering scheme); and Whitelegg NR and Rees AR, "WAM: an improved algorithm for modeling antibodies on the WEB", Protein Eng. 2000 Dec;13(12):819-24 ("AbM" numbering scheme).In certain embodiments, the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0268] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering of both the Kabat scheme and the Chothia scheme is based on the most common antibody region sequence length, and insertions are represented by an insertion letter, for example, "30a", and deletions appear in some antibodies. The two schemes place specific insertions and deletions ("indels") at different positions, resulting in differential numbering. The contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.

[0269] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three CDRs (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., pages 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen require the use of a complementary VL or VH domain, respectively. HTo screen a library of domains, a VH or VL domain from an antibody that binds to the antigen can be used to isolate (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0270] The antigen-binding region of the immunoconjugates described herein may be humanized. "Humanized" with respect to an immunoconjugate refers to an antigen-binding region in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized immunoconjugate may optionally include at least a portion of an antibody constant region derived from a human antibody.

[0271] Among the immunoconjugates provided are human immunoconjugates. "Human immunoconjugates" are immunoconjugates having an antigen-binding region with an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or a non-human source utilizing a human antibody repertoire, including a human antibody library, or other human antibody-encoding sequence. The term excludes humanized forms of non-human antibodies that contain non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human.

[0272] As used herein, the phrase "antigen binding arm" refers to a single polypeptide chain comprising an "antigen binding region," a hinge region, and a variant constant region. Other elements (e.g., chelators, imaging metals) can be attached to the antigen binding arms directly or via one or more linkers in the compositions of the present disclosure. The immunoconjugates of the present disclosure comprise two antigen binding arms covalently linked together. In one embodiment, the antigen binding arms are linked via a hinge region. In one embodiment, the antigen binding arms are linked via an immunoglobulin heavy chain constant region. In one embodiment, the antigen binding arms are linked via a variant constant region. In one embodiment, the antigen binding arms are linked via a disulfide bond (e.g., via cysteine ​​residues in the hinge region).

[0273] As used herein, the phrase "antigen binding region" refers to the region of an immunoconjugate responsible for specific binding to an antigen; such region refers to one or more antigen-binding domains, including complementarity-determining regions, variable regions, and framework regions, which may be derived from, modeled on, or mimic an antibody or fragment thereof, as known to those skilled in the art. In one embodiment, the "antigen binding region" of an antigen-binding arm comprises one or two antigen-binding domains. In a preferred embodiment, the "antigen-binding region" of an antigen-binding arm consists of a single antigen-binding domain, which is preferably a VHH polypeptide. In a preferred embodiment, the antigen-binding regions of both antigen-binding arms of an immunoconjugate independently consist of a single antigen-binding domain, which is preferably a VHH polypeptide, which may be the same or different.

[0274] As used herein, the term "VHH polypeptide" encompasses natural and synthetic compositions and includes any VHH polypeptide known in the art. H Polypeptides constituting the H fragment, i.e., the variable domain fragment of only a single domain heavy chain, or V H"VHH" refers to a polypeptide structurally and functionally similar to an H fragment, such as described further below, having the ability to specifically bind to an antigen, both of which are well known in the art. In a preferred embodiment, the VHH polypeptide comprises a heavy chain variable region comprising three heavy chain CDRs; in one embodiment, the VHH polypeptide is derived from a camelid; in another embodiment, the VHH polypeptide is derived from a library; the VHH polypeptide binds to the antigen with specificity and high affinity. In a preferred embodiment, the VHH polypeptide is a single heavy chain variable domain comprising the following sequences: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. VHH polypeptides can be obtained, for example, as antigen-binding fragments of heavy chain-only antibodies generated in vivo (e.g., in camelids). VHH polypeptides can also be obtained from synthetic libraries, such as phage display libraries. For example, McMahon et al.,Nature Structural & Molecular Biology|VOL 25|MARCH 2018|289-296 Yeast surface display platform for rapid discovery of conformationally selective nanobodies;Moutel et al.,eLife 2016;5:e16228 NaLi-H1:A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies.De Genst E,Saerens D,Muyldermans S,Conrath K.Antibody repertoire development in camelids.Dev Comp Immunol.2006;30(1-2):187-98.doi:10.1016 / j.dci.2005.06.010.PMID:16051357.Vincke C,Gutierrez C,Wernery U,Devoogdt N, Hassanzadeh-Ghassabeh G, Muyldermans S.Generation of single domain antibody fragments derived from camelids and generation of manifold constructs. Methods Mol Biol. 2012;907:145-76. doi:10.1007 / 978-1-61779-974-7_8. PMID:22907350. Arbabi Ghahroudi M, Desmyter A, Wyns L, Hamers R, Muyldermans S. Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett. 1997 Sep 15;414(3):521-6. doi:10.1016 / s0014-5793(97)01062-4. PMID:9323027.

[0275] For VHH humanization, see, e.g., Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath K. General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. J Biol Chem. 2009 Jan 30;284(5):3273-84. doi:10.1074 / jbc.M806889200. Epub 2008 Nov 14. PMID:19010777.

[0276] For VHH stability, see e.g. Kunz P,Flock T,Soler N,Zaiss M,Vincke C,Sterckx Y,Kastelic D,Muyldermans S,Hoheisel JD.Exploiting sequence and stability information for directing nanobody stability engineering.Biochim Biophys Acta Gen Subj.2017 Sep;1861(9):2196-2205.doi:10.1016 / j.bbagen.2017.06.014.Epub 2017 Jun 20.PMID:28642127;PMCID:PMC5548252;Kunz P, Zinner K, Mucke N, Bartoschik T, Muyldermans S, Hoheisel JD.The structural basis of nanobody unfolding reversibility and thermoresistance.Sci Rep.2018 May 21;8(1):7934. doi:10.1038 / s41598-018-26338-z. PMID:29784954; PMCID:PMC5962586.

[0277] As used herein, a "linker" is also referred to as a "linker sequence," a "spacer," a "tethering sequence," or grammatical equivalents. As referred to herein, a "linker" connects two separate molecules that themselves have target binding, catalytic activity, or are naturally expressed and assembled as separate polypeptides, or comprise separate domains of the same polypeptide. For example, two different binding moieties, or heavy / light chain pairs, or an antigen-binding region and an immunoglobulin heavy chain constant region. Several strategies can be used to covalently link molecules. The linkers described herein can be utilized to link the light chain variable region and heavy chain variable region in an scFv molecule, or can be used to tether an scFv or other antigen-binding fragment to the N- or C-terminus of an antibody heavy chain. These include, but are not limited to, polypeptide bonds between the N- and C-termini of the protein or protein domain, bonds via disulfide bonds, and bonds via chemical cross-linking reagents. In one aspect of this embodiment, the linker is a peptide bond generated by recombinant techniques or peptide synthesis.

[0278] An antibody that "binds" an antigen or epitope of interest is one that binds to the antigen or epitope with sufficient affinity that it is measurably different from nonspecific interactions. Specific binding can be measured, for example, by determining binding of a molecule relative to binding of a control molecule (e.g., an isotype control), which is generally a molecule of similar structure that lacks binding activity.

[0279] "Specific binding" refers to an antibody or immunoconjugate that can bind to an antigen with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting that antigen. In one embodiment, the extent of binding of the antibody to unrelated proteins is less than about 10% of the binding of the antibody to its antigen, as measured, for example, by radioimmunoassay. As used herein, an "antigen-specific" antibody or immunoconjugate is one that specifically binds to an antigen with sufficient specificity and affinity to be useful in methods of targeting, targeting diagnostics, or detecting the antigen in a biological sample from a subject. In some embodiments, an immunoconjugate or antibody construct or targeted imaging conjugate or radioimmunoconjugate that binds to its target antigen has an affinity of ≦1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 -8 Less than M, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 Dissociation constant (K D In some embodiments, the immunoconjugates or antibody constructs or targeted imaging conjugates or radioimmunoconjugates of the invention bind to multiple antigens, e.g., epitopes that are conserved among homologs from different species such that the amino acid identity of the epitope is not identical in different species.

[0280] As used herein, the term "variant constant region" refers to a polypeptide comprising a portion of an immunoglobulin heavy chain constant region modified, preferably at one to several amino acid positions, from a native immunoglobulin amino acid sequence. Unless otherwise specified herein, the numbering of amino acid residues in an Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Modifications to Fc regions for various purposes are well known in the art. See, e.g., Kevin O. Saunders, Frontiers in Immunology, June 2019 | Volume 10 | Article 1296, titled "Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life."

[0281] Percent (%) sequence identity to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways using available computer software. Appropriate parameters for aligning sequences can be determined, including the algorithms required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, % amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code, along with user documentation, has been submitted to the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0282] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B, to a given amino acid sequence A, to a given amino acid sequence B, or to a given amino acid sequence B (which may alternatively be referred to as a given amino acid sequence A having or containing a particular % amino acid sequence identity to a given amino acid sequence B, to a given amino acid sequence B, or to a given amino acid sequence B) is hereinafter calculated as the 100-fold fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of A to B. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0283] The term "cytotoxic agent," as used herein, refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes, chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents), growth inhibitory agents, enzymes such as nucleases and fragments thereof, antibiotics, toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof, and the various cytotoxic agents described herein.

[0284] The term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope). Unless otherwise indicated, "binding affinity," as used herein, refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen or epitope). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by common methods known in the art, including those described herein. Specific exemplary embodiments for measuring binding affinity are described herein.

[0285] The term "antagonist" is used in the broadest sense and includes any molecule that partially or completely blocks, inhibits, or neutralizes the biological activity of an antigen. Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, or derivatives thereof.

[0286] A "blocking" or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds, or of a protein conjugate containing the antigen. Preferred blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen or of a protein conjugate containing the antigen.

[0287] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0288] The terms "cancer" and "cancerous," as used herein, refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), skin cancer, melanoma, lung cancer, including small cell lung cancer, non-small cell lung cancer ("NSCLC"), small cell neuroendocrine lung cancer, large cell neuroendocrine lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer or stomach cancer, including gastrointestinal cancer, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), glioblastoma, cervical cancer, ovarian cancer (e.g., high-grade serous ovarian cancer), and others. cancer), liver cancer (e.g., hepatocellular carcinoma (HCC)), bladder cancer (e.g., urothelial bladder cancer), testicular (germ cell tumor) cancer, hepatoma, breast cancer, brain cancer (e.g., astrocytoma), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer (e.g., renal cell carcinoma, nephroblastoma, or Wilms' tumor), prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.Further examples of cancer include retinoblastoma, sarcoma, arrhythmiablastoma, hepatocellular carcinoma, hematologic malignancies including non-Hodgkin's lymphoma (NHL), multiple myeloma and acute hematologic malignancies, endometrial or uterine cancer, endometriosis, fibrosarcoma, choriocarcinoma, salivary gland cancer, vulvar cancer, thyroid cancer, esophageal cancer, liver cancer, anal cancer, penile cancer, nasopharyngeal cancer, laryngeal cancer, melanoma, skin cancer, schwannoma, oligodendroglioma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcoma, urinary tract cancer, anaplastic astrocytoma, basal cell carcinoma (basal cell epithelioma), bile duct cancer, small cell bladder cancer, metastatic breast cancer, metastatic colorectal cancer, epithelial ovarian cancer, fallopian tube cancer, gastric adenocarcinoma, and glioblastoma multiforme. These include, but are not limited to, glioblastoma multiforme (GBM), glioma, gliosarcoma, head and neck squamous cell carcinoma (HNSCC), recurrent head and neck squamous cell carcinoma, malignant pleural mesothelioma, Hodgkin's lymphoma, metastatic renal cell carcinoma, metastatic renal clear cell carcinoma, squamous non-small cell lung cancer, squamous cell carcinoma of the lung, recurrent or refractory small cell lung cancer, treatment-resistant melanoma, metastatic melanoma, Merkel cell carcinoma, neuroendocrine carcinoma, large cell neuroendocrine carcinoma, neuroendocrine tumors (NETS), ovarian cancer, papillary carcinoma, peritoneal carcinoma, neuroendocrine prostate cancer, hormone-refractory prostate cancer, castration-resistant prostate cancer, soft tissue sarcoma, and squamous cell carcinoma.

[0289] The term "metastatic cancer" refers to a cancerous condition in which cancer cells in a tissue of origin are transmitted by blood or lymphatic vessels from the original site to one or more sites elsewhere in the body and form one or more secondary tumors in one or more organs other than the tissue of origin. A notable example is metastatic breast cancer.

[0290] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0291] The terms "associated," "associating," "linked," or "linking," in relation to the claimed invention, refer to the state in which two or more components of a molecule are joined, attached, linked, or bound to form a single molecule (or a single-molecular conjugate), or refer to the act of joining two molecules together to form a single molecule (or a single-molecular conjugate) by creating an association, linkage, attachment, and / or any other bond between the two molecules. For example, the term "linked" can refer to two or more components joined by one or more atomic interactions such that a single molecule is formed, and the individual atomic interactions can be covalent or non-covalent. Non-limiting examples of covalent bonds between two components include peptide bonds and cysteine-cysteine ​​disulfide bonds. Non-limiting examples of non-covalent bonds between two molecular components include ionic bonds.

[0292] A "bispecific" antibody refers to an antibody that has binding specificities for at least two different epitopes, whether the epitopes are in the same molecule and / or partially overlap. In some embodiments, a bispecific immunoconjugate of the invention binds to two different epitopes of a single antigen as described herein.

[0293] As used herein, the terms "expressed," "expressing," or "expresses," and grammatical variants thereof, refer to the translation of a polynucleotide or nucleic acid into a protein. The expressed protein may remain intracellular, become a component of the cell surface membrane, or be secreted into the extracellular space.

[0294] For purposes of the present invention, the phrase "derived from," when referring to a polypeptide or polypeptide region, means that the polypeptide or polypeptide region comprises a highly similar amino acid sequence originally found in the "parental" protein, which may include additions, deletions, truncations, rearrangements of specific amino acid residues, or other changes, relative to the original polypeptide or polypeptide region, so long as the specific function (e.g., antigen binding affinity) and structure of the "parental" molecule are substantially preserved. One of skill in the art can easily identify a polypeptide or polypeptide region (e.g., a VHH polypeptide, CDRs, HVRs, VHH polypeptide ... H , and / or V L ) can be derived from the parent molecule (e.g., antibody sequence).

[0295] As used herein, cells that express at least one extracellular target biomolecule or antigen on their cell surface are "target positive cells" or "target+ cells," which are cells physically bound to a particular extracellular target biomolecule. Descriptions of additional target biomolecules are provided below. The terms "target biomolecule," "target antigen molecule," "target antigen," "antigen of interest," and grammatical variants and equivalents are used interchangeably herein and include molecular determinants of antibody binding, as recognized by those skilled in the art given the context of use. Such antigens can be bound by the immunoconjugates described herein via the antigen-binding region or antigen-binding arm of the immunoconjugate.

[0296] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation that is in a form that effectively effects the biological activity of the active ingredient contained therein and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0297] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0298] An "isolated" antibody or immunoconjugate or radioimmunoconjugate is one that is separated from components of its natural environment or artificial production. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). Routine methods for assessing antibody purity in a composition are known to those of skill in the art; see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007). In particular, undesired components (contaminants) that are purified away are those that would interfere with the desired use, e.g., therapeutic use, of the antibody and may include, inter alia, bacterial agents, enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0299] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its native environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present in an extrachromosomal location or at a chromosomal location that is different from its native chromosomal location.

[0300] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny, regardless of the number of transfers. The progeny may not be completely identical in nucleic acid content as the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0301] As used herein, the term "administer," with respect to an immunoconjugate or composition thereof (e.g., a radioimmunoconjugate, a pharmaceutical composition, or a diagnostic composition), means delivering the immunoconjugate or composition thereof to the body of a subject by any known method suitable for delivering an immunoconjugate or composition thereof. Specific modes of administration include, but are not limited to, intravenous, transdermal, subcutaneous, intraperitoneal, and intrathecal administration.

[0302] An "effective amount" of a drug, eg, a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0303] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated and may be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or palliation of the disease state, and remission or improved prognosis. In some embodiments, the radioimmunoconjugates of the present disclosure are used to delay the onset of disease or slow the progression of the disease.

[0304] A "therapeutically effective amount" is at least the minimum concentration required to bring about a measurable improvement or prevention of a particular disorder. The therapeutically effective amount herein may vary according to factors such as the patient's disease state, age, sex, and weight, as well as the ability of the disclosed compositions to elicit a desired response in an individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the disclosed compositions are outweighed by the therapeutically beneficial effects.

[0305] The terms "predictive" and "prognostic" are interchangeable as used herein. In one sense, a predictive or prognostic method is one that allows one practicing the disclosed predictive / prognostic method to select patients (usually, but not necessarily, prior to treatment) who are deemed likely to respond to treatment with an immunoconjugate of the invention or an aforementioned composition (e.g., pharmaceutical composition).

[0306] The term "detecting" is used in the broadest sense to include both qualitative and quantitative measurements of target antigen molecules. In one aspect, the detection methods described herein are used to identify the mere presence of an antigen of interest in a biological sample. In another aspect, the methods are used to test whether an antigen of interest in a sample is present at a detectable level. In yet another aspect, the methods can be used to quantify the amount of an antigen of interest in a sample and further compare antigen levels from different samples. In another aspect, the methods can be used in vivo to determine the location of target cells, for example, using the targeted imaging complex of the present disclosure.

[0307] The term "biological sample" refers to any biological material that may contain an antigen of interest. The sample may be a biological fluid such as whole blood or a whole blood component, including red blood cells, white blood cells, platelets, serum and plasma, ascites, vitreous humor, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, saliva, sputum, tears, sweat, mucus, cerebrospinal fluid, and other components of the body that may contain the antigen of interest. In various embodiments, the sample is a biological sample from any animal. In some embodiments, the sample is from a mammal. In some embodiments, the sample is from a human subject. In some embodiments, the biological sample is serum from a clinical patient. In some embodiments, the biological sample is biopsy material. In some embodiments, the biological sample is biopsy material from a clinical patient. In some embodiments, the biological sample is serum from a clinical patient. In some embodiments, the biological sample is primary cell culture material. In some embodiments, the biological sample is primary cell culture material from a clinical patient. In some embodiments, the biological sample is derived from a clinical patient or a patient treated with a composition of the disclosure, e.g., a radioimmunoconjugate, or a patient treated with a different therapeutic agent, e.g., an antibody-drug conjugate targeting an antigen of interest or beta-irradiation or a small molecule therapeutic agent.

[0308] The term "package insert" is used to refer to instructions customarily included in commercial packaging of therapeutic products that contain information regarding directions, usage, dosage, administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic products.

[0309] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that are integrated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors." [Example]

[0310] The following illustrative examples are representative of embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

[0311] The following examples describe radioisotope delivery platforms with sizes ranging from 60 to 110 kDa that have shorter half-lives (e.g., 4 days or less) than conventional IgG but longer half-lives (e.g., more than 10 hours) than smaller monomeric antibody fragment formats. Furthermore, certain radioisotope delivery platforms provided herein exhibit high stability in vitro or in vivo, low immunogenicity, and an appropriate therapeutic window. These radioisotope delivery platforms are preferred for targeting radioisotopes in vivo to treat diseases. These radioisotope delivery platforms are particularly useful for the safe and effective targeted delivery of alpha emitters in subjects by exhibiting half-lives of up to 4 days and / or reduced adverse effects compared to antibodies with molecular weights less than 60 kDa.

[0312] In certain phrases below, "Fc portion" is used in reference to the variant constant domain and "hinge" is used in reference to the "hinge region," as will be understood by those skilled in the art.

[0313] Example 1. Antibody production VHH-Fc plasmids were generated by cloning VHH sequences containing the hinge and Fc regions (human IgG1 CH2-CH3) into a mammalian expression vector. In some cases, mutations were introduced into the Fc region. To produce recombinant VHH-Fc and its variants, the plasmids were transfected into HEK293.SUS cells (ATUM, or similar). After 3–5 days of secretion, the antibody-containing supernatant was centrifuged and sterile filtered to remove the cells. The antibodies were purified using a Mab Select SuRe PCC column (GE, Cat#: 11003495) and buffer exchanged into PBS, pH 7.0. Protein was quantified using A280 or BCA. Antibody purity was tested by SDS-PAGE, capillary electrophoresis, HPLC-SEC, and LC-MS using standard protocols. Regarding VHH polypeptides, see, for example, McMahon et al., Nature Structural & Molecular Biology | VOL 25 | MARCH 2018 | 289-296 Yeast surface display platform for rapid discovery of conformationally selective nanobodies; Moutel et al., eLife 2016;5:e16228 NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. De Genst E, Saerens D, Muyldermans S, Conrath K. Antibody repertoire development in camelids. Dev Comp Immunol. 2006;30(1-2):187-98. doi:10.1016 / j.dci.2005.06.010. PMID:16051357.Vincke C,Gutierrez C,Wernery U,Devoogdt N,Hassanzadeh-Ghassabeh G,Muyldermans S.Generation of single domain antibody fragments derived from camelids and generation of manifold constructs.Methods Mol Biol.2012;907:145-76.doi:10.1007 / 978-1-61779-974-7_8.PMID:22907350.Arbabi Ghahroudi M,Desmyter A,Wyns L,Hamers R,Muyldermans S.Selection and identification of single domain antibody fragments from camel heavy-chain antibodies.FEBS Lett.1997 Sep 15;414(3):521-6.doi:10.1016 / s0014-5793(97)01062-4.PMID:9323027.

[0314] For VHH humanization, see, e.g., Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath K. General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. J Biol Chem. 2009 Jan 30;284(5):3273-84. doi:10.1074 / jbc.M806889200. Epub 2008 Nov 14. PMID:19010777.

[0315] For VHH stability, see e.g. Kunz P,Flock T,Soler N,Zaiss M,Vincke C,Sterckx Y,Kastelic D,Muyldermans S,Hoheisel JD.Exploiting sequence and stability information for directing nanobody stability engineering.Biochim Biophys Acta Gen Subj.2017 Sep;1861(9):2196-2205.doi:10.1016 / j.bbagen.2017.06.014.Epub 2017 Jun 20.PMID:28642127;PMCID:PMC5548252;Kunz P, Zinner K, Mucke N, Bartoschik T, Muyldermans S, Hoheisel JD.The structural basis of nanobody unfolding reversibility and thermoresistance.Sci Rep.2018 May 21;8(1):7934. doi:10.1038 / s41598-018-26338-z. PMID:29784954; PMCID:PMC5962586.

[0316] Several VHH-Fc prototypes and variants were engineered using VHH sequences such as the anti-HER2 clone 2RS15d VHH (see, e.g., WO2016 / 016021) (SEQ ID NO: 20) and the anti-DLL3 clone hz10D9v7.251 VHH sequence (see, e.g., WO2020 / 07967) (SEQ ID NO: 30), and unless otherwise stated herein, the data collected and presented was obtained using the VHH antigen-binding regions of these clones.

[0317] [Table 1]

[0318] Example 2. Antibody Binding Properties: Target Protein and Target Cell AssaysVHH-Fc was assessed by ELISA for binding to target soluble proteins—human, mouse, and cynomolgus monkey orthologs, as appropriate—according to standard protocols. Antigens were either commercially available or produced by cloning known antigen sequences (Uniprot) into mammalian expression vectors with HIS, FLAG, or equivalent tags for purification and detection purposes. A commercially available control anti-target IgG was included. Plates (96-well maxisorp, Corning 3368) were coated with 50–100 μL of each target protein of interest at a concentration optimized for coating. Purified VHH-Fc and hIgG1 isotype control (Sigma, Cat# II5154) were prepared at starting concentrations of 200–400 nM and titrated 1:4. The primary antibody was incubated for 1 hour at room temperature (RT) and washed, after which 0.2 μg / ml of secondary HRP-labeled antibody was added and incubated for 1 hour at RT (goat anti-human IgG-Fc-HRP Jackson, Cat#109-035-098). The reaction was detected using 50 μL / well of TMB (Neogen, Cat#308177). Color development was stopped with 1 M HCl (50 μL). Optical density (OD) was measured at 450 nm using a Spectromax plate reader, and the data were processed using SoftMaxPro. The data show that the anti-target VHH-Fc binds to human, mouse, and cynomolgus monkey target proteins. The recombinant DLL3 proteins used were human DLL3.FLAG (Adipogen #AG-40B-0151, amino acids 27-466), human DLL3.HIS (Abcam #ab255797, amino acids 27-492), mouse DLL3.HIS (IPA Custom, amino acids 25-477), or cynomolgus monkey DLL3.HIS (Acrobiosystems #, amino acids 27-490). The control antibody for DLL3 binding was rovalpituzumab (Creative Biolabs #TAB-216CL). The recombinant HER2 proteins used were Her2.HIS (Sinobiologics, #10004-H08H) and mouse HER2.HIS (Sinobiologics #50714-M08H).The control antibody for HER2 binding was trastuzumab (DIN: 02240692, ROCHE). Figures 1A and 1B show anti-Her2 and anti-DLL3 VHH-Fcs that specifically bind to soluble target antigen in ELISA. Additional VHH-Fcs containing Fc region mutations that reduce effector function and / or FcRn binding were tested but did not significantly affect binding to the target antigen.

[0319] VHH-Fc was screened for binding to various target-positive cancer cell lines by flow cytometry. All cell lines were obtained from ATCC unless otherwise noted and were cultured according to the manufacturer's instructions and recommended media. HER2-positive cell lines used were SKBR3 (ATCC #HTB-30), BT474 (ATCC #HTB-20), and HEK293-6E (NRC) cells. DLL3-positive cell lines tested included SHP-77 (ATCC CRI-2195), NCI-H82 (ATCC HTB-175), NCI-H69 (ATCC HTB-119), and HEK-DLL3 (Creative Biogene #CSC-RO0531). HER2-negative cell lines tested included SHP-77. DLL3-negative cell lines tested included HCT-116 (CCL-247), BT-474, and SKBR3. Primary antibodies, diluted in the same manner as in ELISA, were added to the cells and incubated on ice for 1 hour. The cells were washed twice with 1% FBS in PBS, centrifuged at 450xG for 4 minutes, and incubated on ice for 30 minutes with 2 μg / mL AlexaFluor 647-conjugated anti-human IgG (Jackson, Cat. #109-605-098) or 1:1000 DAPI (Biolegend, Cat. #422801) with AlexaFluor 647-conjugated anti-mouse IgG (Jackson, Cat. #115-605-164). After two additional washes, the cells were resuspended and analyzed by flow cytometry on the iQue screening platform (Intellicyt). Data were processed in Forecyt according to standard protocols. Figures 2A, 2B, and 2C show binding to target-positive cell lines, demonstrating that binding was specific to target-positive cells (i.e., through comparison with binding to negative control cells). Additional experiments showed that Fc mutations to reduce effector function and / or FcRn binding did not affect binding to cancer cells compared to wild-type Fc.

[0320] Example 3. Internalization assay VHH-Fc was tested for internalization by target-expressing cells using a secondary antibody conjugated to a pH-sensitive dye. A goat anti-huIgG-Fc secondary antibody was amine-conjugated to a pH-sensitive pHAb dye (Promega Cat# G9845) according to the manufacturer's instructions. pHAb dyes have low or no fluorescence at pH >7 but become fluorescent in acidic environments upon antibody internalization. Target-positive and target-negative cells were seeded at 1.0 × 106 / mL in a 96-well V-bottom plate. VHH-Fc and hIgG1 isotype controls were diluted to 75 nM in medium. Cells were centrifuged, the supernatant removed, and the cells were resuspended in the prepared primary antibody and incubated on ice for 1 hour. Excess primary antibody was washed from the cells, followed by incubation with pHAb-labeled secondary antibody on ice for 30 minutes. Excess secondary antibodies were then washed away, and the cells were resuspended in medium. One set of samples was placed in a 37°C incubator for internalization, while the other set was placed on ice (0°C) as a binding-only control. Cells were sampled at different time points ranging from 0 to 24 hours. Cells were stained with DAPI and analyzed by flow cytometry in the 572 / 28 channel using the iQue screening platform. VHH-Fc exhibited higher fluorescence on target-positive cells than negative controls (isotype, buffer). Figures 3A and 3B show that H101 and D102 were internalized by SHP-77 and HEK-DLL3 cells.

[0321] Example 4. Antibody Thermal Stability Determination The denaturation temperature (Tm) of VHH-Fc was determined by differential scanning fluorimetry (DSF) using the Protein Thermo Shift Dye Kit™ (ThermoFisher, Cat#: 4461146). Briefly, a total of 1 μg of antibody was used for each reaction. Antibody melting curves were generated using an Applied Biosystems QuantStudio 7 Flex Real-Time PCR System with the recommended settings described in the kit manual. The Tm of the antibodies in Table 1 was then determined using ThermoFisher Protein Thermal Shift software (v.1.3). The Tm1 of VHH-Fc was determined by DSF. Both H101 and D102 showed good thermal stability of 67.5 ± 0.1 °C. Furthermore, VHH-Fcs containing mutations in the Fc region to reduce effector function and / or FcRn binding were tested for thermostability, resulting in slightly lower thermostability (1–2°C), but still within an acceptable range.

[0322] Example 5. Determination of receptor density To test the efficacy of immunoconjugate binding relative to target density, target receptor density was measured on target-positive cell lines. Target density was measured using an ABC (antibody binding capacity) assay. Cancer cells expressing the target of interest, as well as negative control cell lines, were harvested with cell dissociation buffer and seeded at approximately 5 × 10 cells per well in a 96-well V-bottom plate (Sarstedt 82.1583.001). Cells were tested for receptor expression using QuantiBRITE PE beads (BD Cat# 340495) and PE-conjugated anti-hu IgG (Biolegend clone HP6017) according to the manufacturer's instructions. Briefly, VHH-Fc and isotype control antibodies were prepared at appropriate saturating concentrations based on previous experiments. Antibody sample dilutions were incubated with the cell line panel on ice for 1 hour. Cells were washed twice with 1% FBS in 1x PBS (FACS buffer) and centrifuged at 400×G for 4 minutes. The cells were then incubated with 4 μg / mL mouse PE-conjugated anti-hu and DAPI (1:1000) on ice for 30 minutes. The cells were washed twice with FACS buffer, centrifuged at 400×G for 4 minutes, and resuspended in FACS buffer. The fluorescence intensity on the PE channel was measured using the iQue Screener platform, and the data was processed using ForeCyt software. The amount of PE signal generated from different primary antibodies was then fitted to a standard curve based on known PE molecule / Quantibrite bead samples to determine the number of antibody binding sites per cell. The relative antibody binding sites correlate with the number of antigens or receptors on the cell surface. Table 2 shows the receptor density numbers of anti-DLL3 and anti-HER2 VHH-Fc binding to a panel of cancer cell lines, which were in a range similar to those reported in the literature.

[0323] [Table 2]

[0324] Example 6. Affinity of antibodies to target proteins Antibody affinity was assessed using Octet Red 96e (ForteBio). The association rate constant (k), dissociation rate constant (K), and affinity constant (K) were measured by biolayer interferometry using an anti-hIgG Fc (AHC) capture biosensor (ForteBio cat# 18-5063). Each cycle was performed at an orbital shake speed of 1,000 rpm. The antigen was titrated 1:2 from the appropriate starting concentration in kinetics buffer (ForteBio, cat# 18-1105). The AHC biosensor set was immersed in kinetics buffer for a 60-second baseline step. Anti-target VHH-Fc (5 μg / mL in kinetics buffer) was loaded onto the biosensor for 240 seconds, followed by a second 30-second baseline step. To compensate for the natural dissociation of the captured IgG, the IgG capture sensor was immersed in a buffer for single reference subtraction. Each biosensor was then immersed in the corresponding concentration of target protein (human, mouse, or cynomolgus monkey monomeric protein) for 600 seconds, followed by dissociation for 1800 seconds in kinetics buffer or under optimized conditions. A new set of AHC biosensors was used for every VHH-Fc. Data were analyzed using a global fit 1:1 model for the binding and dissociation steps (Octet software version v11.0). Table 3 shows the binding affinity data.

[0325] [Table 3]

[0326] Example 7. FcRn and Fc effector mutant affinity determination The FcRn affinity of VHH-Fc can generally be used to predict the half-life of antibody serum clearance. (See, for example, Datta-Mannan A et al. "FcRn affinity-pharmacokinetic relationship of 5 human IgG4 antibodies engineered for improved in vitro FcRn binding properties in cynomolgus monkeys," Drug Metab Dispos. 2012 Aug;40(8):1545-55.) Briefly, 10 nM biotinylated hFcRn (Sino Biological, Cat#:CT071-H27H-B) was captured on an SA biosensor using an Octet RED96e (Fortebio). Binding was measured by immersing the hFcRN-coated biosensor in a sample solution in sodium phosphate buffer (100 mM NaHPO, 150 mM NaCl / 0.05% Tween-20, pH 6.0) containing serial concentrations of test antibody. Dissociation was measured by immersing the biosensor in sodium phosphate buffer without antibody. KD values ​​were determined using Octet Data Analysis HT 11.0 software. A 2:1 (heterologous ligand) binding model was used for the analysis. Table 4 shows the effect of specific mutations in the Fc on FcRN affinity for wild-type VHH-Fc and for the mutants. The changes in FcRn affinity were consistent between targets. Constructs with only Fc effector mutations do not affect FcRn affinity. The addition of Fc effector mutations to FcRn mutant constructs does not affect FcRn affinity. Table 4A shows the affinity of VHH-Fc and Fc variants for FcRn.

[0327] [Table 4]

[0328] VHH-Fc was also tested for affinity to FcγR by biolayer interferometry using the Octet Red96e platform. Each cycle was performed at an orbital shaking speed of 1,000 rpm. Streptavidin (SA) biosensors (Sartorius 18-5019) were rehydrated for 10 minutes using kinetics buffer (PBS + 0.1% BSA + 0.02% Tween-20). Biotinylated FcγR (Acro Biosystems) was then loaded onto the SA biosensor at concentrations ranging from 1 to 5 μg / mL in PBS for 40 to 100 seconds. VHH-Fc was serially diluted 1:2 in sample buffer (PBS + 0.02% Tween-20) to starting concentrations ranging from 5000 nM to 37.5 nM. The loaded biosensor was then allowed to bind to VHH-Fc for 60 to 120 seconds. VHH-Fc dissociation was measured in sample buffer for 30 to 900 seconds. Bound VHH-Fc was then removed using three cycles of 5 seconds of regeneration buffer (150 mM NaCl, 300 mM sodium citrate) and 5 seconds of sample buffer. Data were analyzed using either a global fit 1:1 Langmuir binding model (FcγRI) or steady-state analysis (Octet software version HT v11.1).

[0329] The analysis shows reduced binding to FcγR (represented by a higher KD) for constructs with mutations incorporated as shown in Table 4B.

[0330] [Table 5]

[0331] Example 8. Self-binding studies using AC-SINS The self-association tendency of VHH-Fc was determined by affinity capture self-interaction nanoparticle spectroscopy (AC-SINS) using gold nanoparticles (Au-NPs) (Ted Pella, Cat#: 15705). (PMID: 24492294, 30395473) Briefly, Au-NPs were coated with goat IgG and goat anti-human Fc IgG (1:4 molar ratio). The conjugated Au-NPs were mixed with 5 μg of each VHH-Fc in quadruplicates in a 96-well plate. Wavelength scans were measured using a Synergy Neo2 plate reader. The maximum absorbance difference (Δλmax) was calculated by subtracting the λmax of each reaction from the λmax of PBS buffer. Data were analyzed using the Linest function in Excel using second-order polynomial fitting. A control antibody with a known high ACSINS score (above the literature established cutoff of 11 for IgG) was included in the assay. Figure 4 shows the ACSINS scores for the test articles and controls.

[0332] Example 9. Polyreactivity studies The polyreactivity of VHH-Fc against negatively charged biomolecules was determined by ELISA (e.g., Avery et al., "Establishing in vitro and in vivo correlations to screen monoclonal antibodies for physicochemical properties related to the physicochemical properties" MAbs. 2018 Feb / Mar;10(2):244-255). Briefly, ELISA plates were coated overnight with 5 μg / mL human insulin (SigmaAlrich, Cat#: I9278) and 10 μg / mL double-stranded DNA (SigmaAlrich, Cat#: D1626-250MG). The plates were blocked with ELISA buffer (PBS, 1 mM EDTA, 0.05% Tween-20, pH 7.4). 10 μg / mL of test VHH-Fc was loaded onto the plates in quadruplicate and incubated for 2 hours. HRP-conjugated goat anti-human Fc (0.01 μg / ml) was then added, and the plate was incubated for 1 hour. Signals were developed with TMB, and A450 absorbance was measured on a Synergy Neo2 plate reader. Signals were normalized to the signal of uncoated wells for each antibody tested. Table 5 shows the polyreactivity scores compared to the control antibody.

[0333] [Table 6]

[0334] Example 10. Fc variants effectively decrease VHH-Fc half-life. In certain instances, shortening the drug half-life of α-emitters is important for safety and to avoid unwanted toxicity associated with treatment.However, antibodies usually have a half-life of 14 days or more.Therefore, the half-life of VHH-Fc variants was tested to observe and measure any reduction in half-life.

[0335] Twenty-eight 8-week-old male B6.Cg-Fcgrt mice tm1Dcr Tg(FCGRT)32Dcr / DcrJ (Tg32 hom, JAX stock #014565) mice were distributed into seven groups with four mice per group, as outlined in the table. Tg32 mice contain a humanized FcRn and are generally considered surrogate for human antibody pharmacokinetics when compared with non-human primates. (See, e.g., Avery LB et al. "Utility of a human FcRn transgenic mouse model in drug discovery for early assessment and prediction of human pharmacokinetics of monoclonal antibodies," MAbs. 2016 Aug-Sep;8(6):1064-78.) On day 0, body weights were measured, and all mice were administered the test article IV at 3 mg / kg and 5 ml / kg. 25 μL blood samples were collected from each mouse at timed intervals. Blood samples were collected in 1 μL of K3EDTA, processed to plasma, diluted 1 / 10 with 50% glycerol in PBS, transferred to specialized 96-well storage plates, and stored at −20° C. All plasma samples were evaluated by hIgG ELISA, selected for its high sensitivity to all seven test articles.

[0336] [Table 7]

[0337] As can be seen in Table 6, the introduction of mutations within FcRn generally shortened the half-life of anti-HER2 VHH-Fc. Interestingly, in contrast to published results in the field, not all Fc variants when included in the tested immunoconjugates showed a decrease in half-life consistent with previously published results found in the literature (see, e.g., Burvenich IJ et al., "Cross-species analysis of Fc engineered anti-Lewis-Y human IgG1 variants in human neonatal receptor transgenic mice reveal impertmental of S254 and Y436 in binding human neonatal Fc receptor," MAbs. 2016 May-Jun;8(4):775-86).

[0338] [Table 8]

[0339] As can be seen in Table 7, the introduction of mutations within FcRn was generally able to shorten the half-life of anti-DLL3 VHH-Fc. Similar to the HER2-binding immunoconjugates, and contrary to published results, not all Fc variants showed a decrease in half-life consistent with previously published results found in the literature.

[0340] Example 11. Complete mass analysis of VHH-Fc The conjugates were deglycosylated and then analyzed with in-house Endo-S enzyme (final concentration 10 μg / mL) for 1 hour at 37°C. For intact mass analysis, 8 μL of sample was injected into a Waters Acquity UPLC-Q-TOF equipped with a UPLC BEH200SEC 1.7 μM 4.6 × 150 mm column. The samples were eluted with a mobile phase of water / ACN (70 / 30, v / v) containing 0.1% TFA and 0.1% FA (formic acid) for 11 minutes at a flow rate of 0.25 mL / min.

[0341] Example 12. Procurement of bifunctional chelating agents Several chelators pre-functionalized for antibody conjugation are known to those skilled in the art. p-SCN-Bn-DOTA (1) is available from Macrocyclics (Plano, TX). Other linker variants of DOTA can be generated from the advanced intermediate DOTAGA-tetra(t-Bu ester) (2) (Macrocyclics, Plano, TX) according to the following general procedure.

[0342] Other reagents used in these procedures are available from Millipore Sigma, CombiBlocks, Chem-Impex, and Broadpharm. All solvents were obtained from VWR and used as received without anhydrous handling conditions unless otherwise indicated. Mass spectra were acquired using an Agilent HPLC-MS or Waters HPLC-MS with a C18 reverse-phase column and an acetonitrile / water (+0.1% formic acid) gradient. Flash chromatography was performed using a Biotage IsoleraOne instrument equipped with an appropriately sized normal-phase silica gel cartridge, with fractions collected at 254 nm. The final compound was purified by Agilent preparative-scale HPLC using an acetonitrile / water (+0.1% TFA) gradient. NMR spectra were acquired on a Bruker 400 MHz NMR instrument and processed with MestReNova v.14. Detailed NMR data were compiled using the multiplet analysis function in manual mode.

[0343] Figure 5 shows the synthesis of PEG5-DOTA, including compounds numbered (2) to (5) below. Compound 3 was prepared by HATU coupling followed by TFA deprotection and is available without chromatographic purification.

[0344] Synthesis of Compound (3) 4-({2-[2-(2-aminoethoxy)ethoxy]ethyl}carbamoyl)-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid Tetrakis(trifluoroacetic acid): Compound 2 (100 mg, 0.143 mmol) was dissolved in DMF (2 mL) and HATU (65.1 mg, 0.171 mmol) was added, followed by DIPEA (0.099 mL, 73.8 mg, 0.57 mmol). After 3 minutes, a solution of Boc-NH-PEG5-amine (65.1 mg, 0.17 mmol) was added to the reaction. After stirring for 10 minutes, HPLC indicated the reaction was complete. After 1 hour, the reaction was quenched with approximately 5 mL of NaHCO3 (sat.), then 5 mL of water was added, and the mixture was extracted four times with 30 mL of Et2O. The combined organics were washed with saturated brine, dried over sodium sulfate, filtered, and concentrated in vacuo to give the crude protected intermediate in good purity. m / z found = 1063.6 (M+H).

[0345] The above intermediate was directly dissolved in DCM (5 mL) and TFA (5 mL) was added. The reaction was stirred for 24 h until HPLC showed complete removal of the Boc and tBu esters. The reaction solution was concentrated in vacuo and coevaporated twice with 25 mL of DCM. The residue was precipitated from DCM containing EtO, and the remaining solid was then thoroughly triturated with sonication (15–30 min) to afford the title compound (128 mg, 86% two-step) as an off-white powder in good purity. 1 H NMR (400 MHz, deuterium oxide) δ 4.15-3.68(m,7H),3.62(d,J=4.7 Hz,2H),3.59-3.49(m,20H),3.47(t,J=5.5 Hz,2H),3.35-2.78(m,16H),2.52-2.37(m,2H),1.97-1.79(m,2H).m / z found=739.5(M+H).

[0346] Synthesis of compound (4) bis(2,3,5,6-tetrafluorophenyl)hexanedioate: Adipic acid (1.00 g, 6.84 mmol) and EDC (3.28 g, 17.1 mmol) were dissolved in 20 mL of DCM and cooled to 0° C. in an ice bath, followed by the addition of a solution of 2,3,5,6-tetrafluorophenol in 20 mL of DCM. Conversion to the product was monitored by TLC (R f =0.5; 75% DCM / Hexanes). The reaction mixture was concentrated in vacuo and purified by flash chromatography (0-100% DCM / Hexanes) to afford the title compound (2.48 g, 82%) as a crystalline white powder. 1 H NMR (400 MHz, chloroform-d) δ 7.03 (tt, J = 9.9, 7.0 Hz, 2H), 3.00-2.63 (m, 4H), 1.95 (t, J = 3.3 Hz, 4H). This compound has poor signals by LCMS.

[0347] Compound (5) -{[2-(2-{2-[6-oxo-6-(2,3,5,6 tetrafluorophenoxy)hexanamido]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid: To a solution of compound 3 (22.1 mg, 0.017 mmol) in DMF (1.5 mL) was added bis(2,3,5,6-tetrafluorophenyl)hexanedioate (4) (45.2 mg, 0.102 mmol) and triethylamine (0.0086 mL, 6.2 mg, 0.061 mmol). Complete conversion to the product was confirmed by HPLC. After stirring for 2 h, the reaction was diluted with DMSO (1.5 mL) and purified by direct injection onto preparative HPLC (Agilent, Hanover, CT) with a gradient of 15–50% MeCN / water 0.1% TFA to afford the title compound (10.6 mg, 50%) as a white powder (2× TFA salt). 1H NMR (400 MHz, deuterium oxide) δ 7.20(tt,J=10.4,7.2 Hz,1H),3.97-3.65(m,5H),3.58-3.51(m,20H),3.49(q,J=5.1 Hz,2H),3.43-3.32(m,6H),3.26(t,J=5.3 Hz,2H),3.20-2.82(m,12H),2.69(t,J=6.8 Hz,2H),2.52-2.34(m,2H),2.19(t,J=6.8 Hz,2H),1.99-1.82(m,2H),1.75-1.46 (m,4H).m / z found=1015.3(M+H).

[0348] FIG. 6 shows the synthesis of PEG5-Py4Pa, including compounds numbered (6) to (10) below.

[0349] Synthesis of compound (6) tert-butyl 6-[({[4-(benzyloxy)-6-{[bis({6-[(tert-butoxy)carbonyl]pyridin-2-yl}methyl)amino]methyl}pyridin-2-yl]methyl}({6-[(tert-butoxy)carbonyl]pyridin-2-yl}methyl)amino)methyl]pyridine-2-carboxylate. To a stirred solution of 1-[6-(aminomethyl)-4-(benzyloxy)pyridin-2-yl]methanamine (0.65 g, 2.67 mmol) (available from N. Delsuc, et al. Angew Chem. Int. Ed. 2007, 46, 214-217) in acetonitrile (50 mL), DIPEA (1.40 mL, 1.04 mg, 8.01 mmol) and tert-butyl 6-(bromomethyl)pyridine-2-carboxylate (4.36 g, 16.0 mmol) (available from P. Coomba, et al. Inorg. Chem. 2016, 55, 12531-12543) were added, and the solution was heated to reflux. After 16 hours, the reaction was cooled, and the solvent was removed in vacuo. The crude material was dissolved in 200 mL of DCM and washed with 2 × 75 mL of NaHCO (sat.) and 2 × 75 mL of saturated brine. The DCM layer was then dried over sodium sulfate, filtered, and concentrated in vacuo to give a crude brown oil (950 mg), which could be used in the next step without further purification. The intermediate from above was dissolved in EtOH, ammonium formate (297 mg, 4.71 mmol) was added, and the flask was purged with N. 10% Pd / C (250 mg, 0.23 mmol) was added, followed by another N purge, and then 30% Pd / C (50 mg, 0.14 mmol). After another N purge, the reaction was heated to 50 °C and stirred for 6 h, at which point the reaction was complete by LCMS. The reaction mixture was filtered through Celite, washed with 3 × 50 mL of MeOH, and then concentrated in vacuo to give a pale yellow oil. The crude material was purified by flash chromatography using a Biotage Sfar amino D cartridge and a gradient of 40-100% EtOAc / hexanes followed by 0-20% MeOH / DCM to afford the title compound as a yellow solid (278 mg, 11%). 1H NMR (400 MHz, methanol-d4) δ 7.88(dd,J= 7.7,1.3 Hz,4H),7.82 (t,J= 7.7 Hz,4H),7.73(dd,J=7.7,1.2 Hz,4H),6.41(s,2H),4.00(s,8H),3.94(s,4H),1.61(s,36H).m / z found=918.4(M+H).

[0350] Synthesis of compound (7) tert-butyl N-[17-(2-bromoacetamido)-3,6,9,12,15-pentaoxaheptadecan-1-yl]carbamate: A solution of tert-butyl N-(17-amino-3,6,9,12,15-pentaoxaheptadecan-1-yl)carbamate (200 mg, 0.53 mmol) and DIPEA (0.146 mL, 109 mg, 0.84 mmol) in 5 mL of DCM was cooled to 0 °C. A solution of 2-bromoacetyl bromide (0.069 mL, 159 mg, 0.79 mmol) in 5 mL of DCM cooled to 0 °C was added dropwise over 2 minutes. The reaction was allowed to warm to room temperature, and after 90 minutes, HPLC showed full conversion to the product. The reaction was concentrated and partitioned between EtO and water, NaHCO (sat.) was added, and the mixture was then extracted 3 x 25 mL with EtO. The combined organics were washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The crude residue was co-evaporated once with acetonitrile to remove water. The title compound was recovered as a brownish oil (261 mg, 99%). 1 H NMR(400 MHz,chloroform-d)δ 3.90 (s,2H),3.75-3.64(m,18H),3.61(d,J= 4.5 Hz,2H),3.56(t,J=5.1 Hz,2H),3.52 (t,J=5.2 Hz,2H),3.37-3.30(m,2H),1.46(s,9H).m / z found=523.2(M+Na).

[0351] Synthesis of compound (8) tert-butyl 6-({[(6-{[bis({6-[(tert-butoxy)carbonyl]pyridin-2-yl}methyl)amino]methyl}-4-{[(17-{[(tert-butoxy)carbonyl]amino}-3,6,9,12,15-pentaoxaheptadecan-1-yl)carbamoyl]methoxy}pyridin-2-yl)methyl] ({6-[(tert-butoxy)carbonyl]pyridin-2-yl}methyl)amino}methyl)pyridine-2-carboxylate. Compound 6 (100 mg, 0.11 mmol) and compound 7 (81.9 mg, 0.163 mmol) were dissolved in acetonitrile (5 mL), then potassium carbonate (30.1 mg, 0.218 mmol) was added and the reaction was stirred at 60° C. After 24 h, no starting material remained by HPLC. The reaction was concentrated and purified by flash chromatography (Biotage amino D cartridge, gradient 0.2-15% MeOH / DCM) to give the title compound as a yellow film (106 mg, 73%). 1 H NMR(400 MHz, methanol-d4)δ 7.89(d,J= 7.8 Hz,4H),7.83(t,J=7.7 Hz,4H),7.66(d,J=7.6 Hz,4H),6.95(s,2H),4.66(s,2H),4.04(s,8H),3.92(s,4H),3.75-3.55(m, 20H),3.53-3.43(m,2H),3.30-3.13(m,2H),1.52(s,36H),1.43(s,9H).m / z found=670.0(M+2H / 2).

[0352] Synthesis of compound (9) 6-({[(4-{[(17-amino-3,6,9,12,15-pentaoxaheptadecan-1-yl)carbamoyl]methoxy}-6-({bis[(6-carboxypyridin-2-yl)methyl]amino}methyl)pyridin-2-yl)methyl][(6-carboxypyridin-2-yl)methyl]amino}methyl)pyridine-2-carboxylic acid: Compound 8 (125 mg, 0.093 mmol) was dissolved in DCM (5 mL) and TFA (5 mL) was added. After 18 h, HPLC showed no starting material or t-butyl intermediate remained. The reaction was concentrated in vacuo and co-evaporated once with DCM. The crude oil was triturated twice with EtO with sonication and collected by filtration to give 100 mg (64%, 5× as the TFA salt) of the title compound as a brown solid. 1 H NMR (400 MHz, methanol-d4) δ 8.04(d,J=7.7 Hz,4H),7.96(t,J=7.8 Hz,4H),7.66(t,J= 8.4 Hz,4H),7.45(s,2H),4.84(s,2H),4.74-4.49(m,12H),3.74(t,J=5.0 Hz,2H),3.71-3.63(m,14H),3.60(t,J=5.3 Hz,2H),3.48(t,J=5.6 Hz,2H),3.20-3.12(m,2H).m / z found=1014.3(M+H).

[0353] Synthesis of compound (10) 6-[({[6-({bis[(6-carboxypyridin-2-yl)methyl]amino}methyl)-4-[({17-[6-oxo-6-(2,3,5,6-tetrafluorophenoxy)hexanamido]-3,6,9,12,15-pentaoxaheptadecan-1-yl}carbamoyl)methoxy]pyridin-2-yl]methyl}[(6-carboxypyridin-2-yl)methyl]amino)methyl]pyridine-2-carboxylic acid. To a solution of compound 9 (80 mg, 0.079 mmol) in DMF (2.5 mL) was added bis(2,3,5,6-tetrafluorophenyl)hexanedioate (4) (140 mg, 0.32 mmol) and triethylamine (0.027 mL, 20 mg, 0.197 mmol). Complete conversion to the product was confirmed by HPLC. After stirring for 4 h, the reaction was diluted with DMSO (1.5 mL) and purified by direct injection onto preparative HPLC (Agilent, Hanover, CT) with a gradient of 25–60% MeCN / water + 0.1% TFA to afford the title compound (57.5 mg, 56%) as a white powder (3× TFA salt). 1 H NMR (400 MHz, deuterium oxide) δ 7.85(t,J=7.8 Hz,4H),7.78(dd,J=7.8,1.2 Hz,4H),7.50(dd,J= 7.8,1.2 Hz,4H),7.11(tt,J=10.4,7.2 Hz,1H),6.99(s,2H),4.59(s,2H),4.49(s,8H),4.45(s,4H),3.60-3.45(m,18H),3.46(t,J=5.3 Hz,2H),3.36(t,J=5.3 Hz,2H),3.22(t,J=5.3 Hz,2H),2.59(t,J=6.7 Hz,2H),2.14(t,J=6.7 Hz,2H),1.61-1.46(m,4H).m / z found=1290.3(M+H).

[0354] Synthesis of compound (11) 6-[({[6-({bis[(6-carboxypyridin-2-yl)methyl]amino}methyl)-4-{2-[4-(cyanosulfanyl)phenyl]ethoxy}pyridin-2-yl]methyl}[(6-carboxypyridin-2-yl)methyl]amino)methyl]pyridine-2-carboxylic acid; bis(trifluoroacetic acid): The title compound was prepared according to the conditions in L Li et al. Bioconjugate Chem. 2021, 32, 1348-1363. Spectral and LCMS data matched the reported values.

[0355] Example 13. Conjugates of VHH-Fc proteins using chelator-linkers Conjugation can be performed using many of the methods available for preparing IgG radioconjugates and IgG antibody-drug conjugates. For information on the range of applicable methodologies, see P.W. Howard, "Antibody-Drug Conjugates (ADCs)," Protein Therapeutics, First Edition, chapter 9, pp. 278-279 (2017).

[0356] For a typical lysine-based conjugate, VHH-Fc was buffer-exchanged into 0.1 M NaHCO3, pH 8.5–9.5, using either a Microsep Advance Centrifugal Device (Pall 10K MWCO, Cat#: MCP010C41) or a Zeba column (ThermoFisher, Cat#: 87768), followed by sterilization in a Costar Spin-X Centrifuge Tube, 0.22 μm (Corning, Cat#: 8160). Buffer-exchanged antibody was quantified by BCA assay. An appropriate molar excess (5–20 equivalents) of chelator-linker (50 mM in DMSO) was added to VHH-Fc (2 mg / mL final concentration), and the reaction was incubated at 25°C for 2 hours or overnight in a Thermomixer. After the reaction was complete, the sample was passed through a Zeba column (ThermoFisher, Cat#: 87770) to remove unused chelator-linker and buffer exchanged into PBS (pH 7.4) (LifeTechnologies, Cat#: 10010-023) according to the manufacturer's protocol. The VHH-Fc-chelator conjugate (VFCC) was stored at 4°C until analysis and purification.

[0357] Example 14. Purification of VHH-Fc-chelator conjugates (VFCC) by SEC To remove high-molecular-weight species (HMWS) and low-molecular-weight species (LMWS), VHH-Fc was purified by SEC using an AKTA Pure FPLC system equipped with a Cytiva HiLoad 16 / 600 Superdex 200 pg column. TBS buffer (50 mM Tris, 150 mM NaCl, OmniTrace Ultra water [VWR, Cat#: CAWX0003-2]) at pH 7.6 was used as the SEC buffer. Fractions containing intact VHH-Fc were pooled together and concentrated using a Microsep Advance Centrifugal Device (Pall 10k MWCO, Cat#: MCP010C41). The concentrated sample was transferred to an Ultrafree-MC GV centrifugal filter, 0.22 μm, 0.5 mL (Millipore, Cat#: UFC30GV0S) and centrifuged at 3,000 × g for 3 minutes.

[0358] Example 15. Protein quantification VHH-Fc protein content was quantified with cetuximab (LIST / E:094822, DIN 02271249, 2 mg / mL) using a standardized Pierce BCA protein assay kit (Thermo, Cat#:23225).

[0359] Example 16. Chelating Agent (CAR) Analysis for VHH-Fc Ratio The chelator loading ratio, herein referred to as CAR, can be analyzed by methods applicable to those skilled in the art of antibody conjugates. For a review of these methods in the context of ADCs, see A Wakankar et al., mAbs 3:161 (2011). The CAR of each conjugate was analyzed by DG-SEC-MS.

[0360] The conjugates were analyzed by deglycosylation and UPLC-Q-TOF procedures described in Example 11. In this case, the mass distribution is obtained after spectral deconvolution, which allows the calculation of the average CAR of the preparation.

[0361] The conjugates were analyzed by deglycosylation and UPLC-Q-TOF procedures described in Example 11. In this case, the distribution of masses is obtained after spectral deconvolution, which allows the calculation of the average CAR of the preparation.

[0362] Example 17. Binding of VHH-Fc conjugates to cells expressing target proteins In some cases, conjugation can negatively affect the binding of VHH-Fc to target proteins. Therefore, the binding of VHH-Fc conjugates was tested as described above. Table 8 shows the cell binding data of VHH-Fc chelator conjugates.

[0363] [Table 9]

[0364] As seen in Table 8, binding was observed for both long and short DOTA linkers. As shown in Table 8, binding was also observed over increasing chelator VHH-Fc ratios (CAR).

[0365] Example 18. Percent Intact Analysis The percentage of intact immunoconjugate was established by HPLC-SEC. 12 μL of conjugate was added to a glass vial insert in a standard HPLC vial. 10 μL of sample was injected into an Agilent HPLC-SEC equipped with a Wyatt Technology WTC-050S5 SN:0429 BN WBD129 column and eluted with 1×PBS (100%) for 40 min at a flow rate of 0.5 mL / min.

[0366] Example 19. Endotoxin Level Determination Endotoxin testing was performed using Wako's Limulus Amebocyte Lysate Pyrostar™ ES-F Single Test (Cat#: WPESK-0015) according to the manufacturing protocol. QC cutoffs were set based on the maximum expected injected dose for each animal in the study, following appropriate animal care and FDA guidelines.

[0367] Example 20. Radiolabeling with In-111 40 μg of each of the four test substances was diluted to 100 μL with 0.1 M ammonium acetate buffer in a 500 μL Eppendorf tube, and 18–25 μL (20–22 MBq) of [ 111 [In]InCl was added and mixed with a pipette. The reaction mixture was incubated in an incubator at 37°C for 1 hour. The tube was then transferred to a 4°C refrigerator.

[0368] Radionuclide incorporation was determined by spotting 0.5 μL of sample at the origin of a 1.5 × 10 cm iTLC strip. The strip was then placed in a 50 mL Falcon tube containing 2 mL of mobile phase (25 mM E...

Claims

1. A polypeptide comprising an antigen-binding region that binds to DLL3, the antigen-binding region comprising: a. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 107 to 109, 207 to 209, 307 to 309, 407 to 409, or 507 to 509; b. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 110 to 112, 210 to 212, 310 to 312, 410 to 412, or 510 to 512, and / or c. A heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 113 to 115, 213 to 215, 313 to 315, 413 to 415, 513 to 515, 131, 231, 431, or 531. A polypeptide comprising:

2. The polypeptide of claim 1, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 101-106, 201-206, 301-306, 401-306, and 501-506.

3. the antigen-binding region a. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 107 to 109; b. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 110 to 112; c. A heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 113 to 115, or SEQ ID NO:

131. The polypeptide of claim 1 or 2, comprising:

4. The polypeptide of any one of claims 1 to 3, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 101 to 106.

5. The polypeptide of any one of claims 1 to 3, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NOs: 101 to 106.

6. the antigen-binding region a. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 207 to 209; b. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 210 to 212; c. A heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 213 to 215, or SEQ ID NO:

231. The polypeptide of claim 1 or 2, comprising:

7. 10. The polypeptide of claim 1, 2, or 6, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs:201 to 206.

8. 10. The polypeptide of claim 1, 2, or 6, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs:201 to 206.

9. the antigen-binding region a. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 307 to 309; b. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 310 to 312; c. A heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 313 to 315. The polypeptide of claim 1 or 2, comprising:

10. 10. The polypeptide of any one of claims 1, 2, or 9, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs:301 to 306.

11. 10. The polypeptide of any one of claims 1, 2, or 9, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 301 to 306.

12. 10. The polypeptide of any one of claims 1, 2, or 9, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:

303.

13. 10. The polypeptide of any one of claims 1, 2, or 9, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

303.

14. 10. The polypeptide of any one of claims 1, 2, or 9, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:

304.

15. 10. The polypeptide of any one of claims 1, 2, or 9, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

304.

16. 10. The polypeptide of any one of claims 1, 2, or 9, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:

305.

17. 10. The polypeptide of any one of claims 1, 2, or 9, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

305.

18. the antigen-binding region a. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 407 to 409; b. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 410 to 412; c. A heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 413 to 415, or 431. The polypeptide of claim 1 or 2, comprising:

19. 20. The polypeptide of any one of claims 1, 2, or 18, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs:401-406.

20. 20. The polypeptide of any one of claims 1, 2, or 18, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:

403.

21. 20. The polypeptide of any one of claims 1, 2, or 18, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

403.

22. 20. The polypeptide of any one of claims 1, 2, or 18, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs:401-406.

23. the antigen-binding region a. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 507 to 509; b. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 510 to 512; c. A heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 513 to 515, or SEQ ID NO:

531. The polypeptide of claim 1 or 2, comprising:

24. 24. The polypeptide of any one of claims 1, 2, or 23, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs:501-506.

25. 24. The polypeptide of any one of claims 1, 2, or 23, wherein the antigen-binding region comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs:501 to 506.

26. The polypeptide of any one of claims 1 to 25, wherein the antigen-binding region is humanized.

27. The polypeptide of any one of claims 1 to 26, wherein the antigen-binding region does not comprise an immunoglobulin light chain.

28. The polypeptide of any one of claims 1 to 27, wherein the antigen-binding region comprises a VHH.

29. The polypeptide of any one of claims 1 to 28, wherein the polypeptide comprises an immunoglobulin heavy chain constant region.

30. 30. The polypeptide of claim 29, wherein the immunoglobulin heavy chain constant region comprises an immunoglobulin CH2 domain, an immunoglobulin CH3 domain, or an immunoglobulin CH2 and CH3 domain.

31. 31. The polypeptide of claim 29 or 30, wherein the immunoglobulin heavy chain constant region comprises the CH2 and CH3 domains of an immunoglobulin.

32. 32. The polypeptide of any one of claims 29 to 31, wherein the immunoglobulin heavy chain constant region is of the IgA, IgG1, IgG2, IgG3, or IgG4 isotype.

33. The polypeptide of any one of claims 29 to 31, wherein the immunoglobulin heavy chain constant region is of the IgG1 isotype.

34. The polypeptide of any one of claims 29 to 31, wherein the immunoglobulin heavy chain constant region is of the IgG4 isotype.

35. 35. The polypeptide of any one of claims 29-34, wherein the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region or alters binding of the polypeptide to the neonatal Fc receptor (FcRn).

36. 35. The polypeptide of any one of claims 29-34, wherein the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region and alters binding of the polypeptide to the neonatal Fc receptor (FcRn).

37. 35. The polypeptide of any one of claims 29 to 34, wherein the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region.

38. 35. The polypeptide of any one of claims 29-34, wherein the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that alters binding of the polypeptide to the neonatal Fc receptor (FcRn).

39. 39. The polypeptide of any one of claims 35 to 38, wherein the change to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region is a change that reduces complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), or a combination thereof.

40. The changes to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region are, according to EU numbering, (a) 297A, 297Q, 297G, or 297D; (b) 279F, 279K, or 279L; (c) 228P; (d) 235A, 235E, 235G, 235Q, 235R, or 235S; (e) 237A, 237E, 237K, 237N, or 237R; (f) 234A, 234V, or 234F; (g) 233P; (h) 328A; (i) 327Q or 327T; (j) 329A. , 329G, 329Y, or 329R, (k) 331S, (l) 236F or 236R, (m) 238A, 238E, 238G, 238H, 238I, 238V, 238W, or 238Y, (n) 248A, (o) 254D, 254E, 254G, 254H, 254I, 254N, 254P, 254Q, 254T, or 254V, (p) 255N, (q) 256H, 256K, 256R, or 256V, (r) 264S, (s) 265H, 265K, 265S, 265Y, or 265A, (t) 267G, 267H, 267I, or 267K, (u) 268K, (v) 269N or 269Q, (w) 270A, 270G, 270M, or 270N, (x) 271T, (y) 272N, (z) 292E, 292F, 292G, or 292I, (aa) 293S, (bb) 301W, (cc) 304E, (dd) 311E, 311G, or 311S, (ee) 316F, (ff) 328V, (gg) 330R, (hh) 339E or 339L, (ii) 343I or 343V, (jj) 373A, 373G, or 373S, (kk) 376E, 376W , or 376Y, (ll) 380D, (mm) 382D or 382P, (nn) 385P, (oo) 424H, 424M, or 424V, (pp) 434I, (qq) 438G, (rr) 439E, 439H, or 439Q, (ss) 440A, 440D, 440E, 440F, 440M, 440T, or 440V, (tt) K322A, (uu) L235E, (vv) L234A and L235A, (ww) L234A, L235A, and G237A, (xx) L234A, L235A, and P329G, (yy) L234F,L235E, and P331S, (zz) L234A, L235E, and G237A, (aaa) L234A, L235E, G237A, and P331S, (bbb) L234A, L235A, G237A, P238S, H268A, A330S, and P331S, (ccc) L234A, L235A, and P329A, (ddd) G236R and L328R, (eee) G237A, (fff) F241A, (ggg) V264A, (hhh (iii) D265A, (iii) D265A and N297A, (jjj) D265A and N297G, (kkk) D270A, (II) A330L, (mmm) P331A or P331S, or (nnn) E233P, (ooo) L234A, L235E, G237A, A330S, and P331S, or (ppp) any combination of (a) to (ppp).

41. 41. The polypeptide of any one of claims 35 to 40, wherein the changes to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region comprise, according to EU numbering, L234A, L235E, G237A, A330S, and P331S.

42. 42. The polypeptide of any one of claims 35-41, wherein the amino acid changes to one or more amino acid residues that alter binding of the polypeptide to the neonatal Fc receptor (FcRn) decrease the serum half-life of the polypeptide.

43. 43. The polypeptide of any one of claims 35 to 42, wherein the change to one or more amino acid residues that alters binding of the polypeptide to neonatal Fc receptor (FcRn) is a change to an amino acid residue selected from the list consisting of 251, 252, 253, 254, 255, 288, 309, 310, 312, 385, 386, 388, 400, 415, 433, 435, 436, 439, 447, according to EU numbering, and combinations thereof.

44. 44. The polypeptide of any one of claims 35 to 43, wherein the change to one or more amino acid residues that alters binding of the polypeptide to neonatal Fc receptor (FcRn) is a change to an amino acid residue selected from the list consisting of 253, 254, 310, 435, 436, according to EU numbering, and combinations thereof.

45. 45. The polypeptide of any one of claims 35-44, wherein the change to one or more amino acid residues that alters binding of the polypeptide to neonatal Fc receptor (FcRn) is a change to an amino acid residue selected from the list consisting of: I253A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof, according to EU numbering.

46. 46. ​​The polypeptide of any one of claims 35 to 45, wherein the change to one or more amino acid residues that alters binding of the polypeptide to neonatal Fc receptor (FcRn) is a change to an amino acid residue selected from the list consisting of: I253A, S254A, H310A, H435Q, Y436A, and combinations thereof, according to EU numbering.

47. 47. The polypeptide of any one of claims 35 to 46, wherein the change to one or more amino acid residues that alters binding of the polypeptide to neonatal Fc receptor (FcRn) is a change to an amino acid residue selected from the list consisting of: I253A, H310A, H435Q, and combinations thereof, according to EU numbering.

48. 48. The polypeptide of any one of claims 35 to 47, wherein the alteration to one or more amino acid residues that alters binding of the polypeptide to the neonatal Fc receptor (FcRn) comprises I253A according to EU numbering.

49. 48. The polypeptide of any one of claims 35 to 47, wherein the changes to one or more amino acid residues that alter binding of the polypeptide to the neonatal Fc receptor (FcRn) comprise H310A according to EU numbering.

50. 48. The polypeptide of any one of claims 35-47, wherein the changes to one or more amino acid residues that alter binding of the polypeptide to the neonatal Fc receptor (FcRn) comprise H435Q according to EU numbering.

51. 51. The polypeptide of any one of claims 1 to 50, further comprising a linker amino acid sequence or a human IgG hinge region.

52. 52. The polypeptide of claim 51, wherein the human IgG hinge region comprises the amino acid sequence set forth in SEQ ID NO:

41.

53. 53. The polypeptide of any one of claims 1 to 52, wherein the antigen-binding region is linked to the immunoglobulin heavy chain constant region by a human IgG hinge region.

54. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:116 to 120, 216 to 220, 316 to 320, 416 to 420, and 516 to 520.

55. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises an amino acid sequence identical to any one of SEQ ID NOs:116 to 120, 216 to 220, 316 to 320, 416 to 420, and 516 to 520.

56. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:116 to 120.

57. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises an amino acid sequence identical to any one of SEQ ID NOs: 116 to 120.

58. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:216 to 220.

59. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises an amino acid sequence identical to any one of SEQ ID NOs: 216 to 220.

60. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:316 to 320.

61. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:

317.

62. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

317.

63. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:

318.

64. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

318.

65. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:

319.

66. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

319.

67. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises an amino acid sequence identical to any one of SEQ ID NOs: 316 to 320.

68. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NOs:416 to 420.

69. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:

417.

70. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

417.

71. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises an amino acid sequence identical to any one of SEQ ID NOs: 416 to 420.

72. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 516-520.

73. 54. The polypeptide of any one of claims 1 to 53, wherein the polypeptide comprises an amino acid sequence identical to any one of SEQ ID NOs: 516 to 520.

74. The polypeptide has a K Ｄ 74. The polypeptide of any one of claims 1 to 73, having the following structure:

75. The polypeptide has a K Ｄ 74. The polypeptide of any one of claims 1 to 73, having the following structure:

76. The polypeptide has a K Ｄ 74. The polypeptide of any one of claims 1 to 73, having the following structure:

77. The polypeptide has a K Ｄ 74. The polypeptide of any one of claims 1 to 73, having the following structure:

78. 78. An immunoconjugate comprising a polypeptide according to any one of claims 1 to 77 and a chelating agent.

79. 79. The immunoconjugate of claim 78, wherein the molecular weight of the immunoconjugate is between 60 and 110 kDa.

80. 80. The immunoconjugate of claim 78 or 79, wherein the immunoconjugate has a serum half-life of less than 15 days.

81. 80. The immunoconjugate of claim 78 or 79, wherein the immunoconjugate has a serum half-life of less than 10 days.

82. 80. The immunoconjugate of claim 78 or 79, wherein the immunoconjugate has a serum half-life of less than 120 hours.

83. 80. The immunoconjugate of claim 78 or 79, wherein the immunoconjugate has a serum half-life of less than 72 hours.

84. The immunoconjugate of any one of claims 78 to 83, wherein the chelating agent is a radioisotope chelating agent.

85. The immunoconjugate of any one of claims 78 to 83, wherein the chelating agent is an alpha emitter chelating agent.

86. 84. The immunoconjugate of any one of claims 78 to 83, wherein the chelator is a beta-emitter chelator or a gamma-emitter chelator.

87. 87. The immunoconjugate of any one of claims 78-86, wherein the chelating agent is selected from the list consisting of DOTA, DO3A, DOTAGA, DOTAGA anhydride, Py4Pa, Py4Pa-NCS, Crown, Macropa, Macropa-NCS, HEHA, CHXoctapa, Bispa, Noneunpa, and combinations thereof.

88. The chelating agent is DOTMA, DOTPA, DO3AM-acetic acid, DOTP, DOTMP, DOTA-4AMP, CB-TE2A, NOTA, NOTP, TETPA, TETA, PEPA, H4Octapa, H2Dedpa, DO2P, EDTA, DTPA-BMA, 3,2,3-LI (HOPO), 3,2-HOPO, Neunpa, Neunpa-NCS, Octapa, PyPa, porphyrin, deferoxamine, DFO ＊ 87. The immunoconjugate of any one of claims 78 to 86, selected from the list consisting of:

89. 87. The immunoconjugate of any one of claims 78-86, wherein the chelating agent is DOTA.

90. 87. The immunoconjugate of any one of claims 78-86, wherein the chelating agent is DOTAGA.

91. 87. The immunoconjugate of any one of claims 78 to 86, wherein the chelating agent is Py4Pa.

92. 92. The immunoconjugate of any one of claims 78 to 91, wherein the chelator is directly attached to the antigen binding region and / or the immunoglobulin heavy chain constant region.

93. 92. The immunoconjugate of any one of claims 78 to 91, wherein the chelator is attached to the antigen binding region and / or the immunoglobulin heavy chain constant region by a linker.

94. The linker may be 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and linker reagents: N-succinimidyl 4-(2-pyridylthio)pentanoate forming linker moiety 4-mercaptopentanoic acid (SPP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB), polyethylene glycol (PEG), polyethylene glycol polymer (PEG ｎ ), and those obtained from conjugates with S-2-(4-isothiocyanatobenzyl) (SCN) 94. The immunoconjugate of claim 93, selected from:

95. 94. The immunoconjugate of claim 93, wherein the linker is selected from polyethylene glycol (PEG), polyethylene glycol polymer (PEG), and S-2-(4-isothiocyanatobenzyl) (SCN).

96. The linker is PEG ５ 94. The immunoconjugate of claim 93, wherein:

97. 94. The immunoconjugate of claim 93, wherein the linker is SCN.

98. 98. The immunoconjugate of any one of claims 1 to 97, wherein the chelator is a linker-chelator selected from the list consisting of TFP-Ad-PEG5-DOTAGA, p-SCN-Bn-DOTA, p-SCN-Ph-Et-Py4Pa, and TFP-Ad-PEG5-Ac-Py4Pa.

99. The immunoconjugate of any one of claims 78 to 98, wherein the chelating agent is TFP-Ad-PEG5-DOTAGA.

100. 99. The immunoconjugate of any one of claims 78 to 98, wherein the chelating agent is p-SCN-Bn-DOTA.

101. 99. The immunoconjugate of any one of claims 78 to 98, wherein the chelating agent is p-SCN-Ph-Et-Py4Pa.

102. The immunoconjugate of any one of claims 78 to 98, wherein the chelator is TFP-Ad-PEG5-Ac-Py4Pa.

103. 103. The immunoconjugate of any one of claims 78 to 102, wherein the chelator is conjugated to the antigen binding region and / or the immunoglobulin heavy chain constant region in a ratio of 1:1 to 8:

1.

104. 103. The immunoconjugate of any one of claims 78 to 102, wherein the chelator is conjugated to the antigen binding region and / or the immunoglobulin heavy chain constant region in a ratio of 1:1 to 6:

1.

105. 103. The immunoconjugate of any one of claims 78 to 102, wherein the chelator is conjugated to the antigen binding region and / or the immunoglobulin heavy chain constant region in a ratio of 2:1 to 6:

1.

106. The immunoconjugate of any one of claims 78 to 105, further comprising a radioisotope.

107. The immunoconjugate of claim 106, wherein the radioisotope is an alpha emitter.

108. 107. The immunoconjugate of claim 106, wherein said radioisotope is an alpha emitter selected from the list consisting of 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi.

109. The immunoconjugate of claim 106, wherein the radioisotope is 225-Ac.

110. The immunoconjugate of claim 106, wherein the radioisotope is a beta emitter.

111. 107. The immunoconjugate of claim 106, wherein the radioisotope is a beta-emitter selected from 177-Lu, 90-Y, 67-Cu, and 153-Sm.

112. The immunoconjugate of claim 106, wherein the radioisotope is a gamma-ray emitter.

113. 107. The immunoconjugate of claim 106, wherein said radioisotope is a gamma emitter selected from 111-In, 89-Zn, 123-I, 99m-Tc, and 68-Ga.

114. 112. The immunoconjugate of any one of claims 78 to 111, wherein the molecular weight of the immunoconjugate is between 60 and 100 kDa.

115. 112. The immunoconjugate of any one of claims 78 to 111, wherein the molecular weight of the immunoconjugate is between 60 and 90 kDa.

116. 112. The immunoconjugate of any one of claims 78 to 111, wherein the molecular weight of the immunoconjugate is between 65 and 90 kDa.

117. 112. The immunoconjugate of any one of claims 78 to 111, wherein the molecular weight of the immunoconjugate is between 70 and 90 kDa.

118. The immunoconjugate of any one of claims 78 to 117, wherein the immunoconjugate forms a dimer with another immunoconjugate.

119. 119. A polypeptide or immunoconjugate according to any one of claims 1 to 118, further comprising a pharmaceutically acceptable excipient or carrier.

120. 120. A polypeptide or immunoconjugate according to any one of claims 1 to 119 formulated for intravenous administration.

121. 119. A method of making the immunoconjugate of any one of claims 1 to 118, comprising the step of charging said immunoconjugate with a radioisotope.

122. 122. The method of claim 121, wherein the radioisotope is an alpha emitter.

123. 123. The method of claim 122, wherein said radioisotope is an alpha emitter selected from the list consisting of 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi.

124. 123. The method of claim 122, wherein the radioisotope is 225-Ac.

125. 122. The method of claim 121, wherein the radioisotope is a beta emitter.

126. 122. The method of claim 121, wherein the radioisotope is a beta emitter selected from 177-Lu, 90-Y, 67-Cu, and 153-Sm.

127. 122. The method of claim 121, wherein the radioisotope is 177-Lu.

128. The immunoconjugate of claim 121, wherein the radioisotope is a gamma-ray emitter.

129. 122. The immunoconjugate of claim 121, wherein the radioisotope is a gamma emitter selected from 111-In, 89-Zn, 123-I, 99m-Tc, and 68-Ga.

130. 121. A method of treating cancer or tumor in an individual, comprising administering to said individual a polypeptide or immunoconjugate of any one of claims 1 to 120, thereby treating the cancer or tumor.

131. 131. The method of claim 130, wherein the individual is a human individual.

132. 132. The method of claim 130 or 131, wherein the cancer or tumor is a solid cancer or tumor.

133. 132. The method of claim 130 or 131, wherein the cancer or tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer.

134. 133. The method of any one of claims 130 to 132, comprising administering to the individual between 0.5 μCi and 30.0 μCi per kilogram.

135. 133. The method of any one of claims 130 to 132, comprising administering to the individual between 10 mCi and 75 mCi per square metre of body surface area.

136. 136. The method of any one of claims 130-135, wherein the cancer or tumor expresses an antigen that is specifically bound by the immunoconjugate.

137. 121. A polypeptide or immunoconjugate according to any one of claims 1 to 120 for use in a method for treating cancer or a tumor in an individual.

138. 138. The use of claim 137, wherein the individual is a human individual.

139. 139. The use of claim 137 or 138, wherein the cancer or tumor is a solid cancer or tumor.

140. 140. The use of any one of claims 137 to 139, wherein the cancer or tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer.

141. 141. The use of any one of claims 137 to 140, wherein 0.5 μCi to 30.0 μCi per kilogram is administered to the individual.

142. 142. The use of any one of claims 137 to 141, comprising administering to the individual between 10 mCi and 75 mCi per square metre of body surface area.

143. 143. The use of any one of claims 137 to 142, wherein the cancer or tumor expresses an antigen that is specifically bound by the immunoconjugate.

144. 121. A method of killing cancer cells in an individual, comprising the step of administering to said individual a polypeptide or immunoconjugate of any one of claims 1 to 120, thereby killing the cancer cells.

145. 145. The method of claim 144, wherein the individual is a human individual.

146. 146. The method of claim 144 or 145, wherein the cancer cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells.

147. 147. The method of claims 144-146, wherein the cancer cells express an antigen that is specifically bound by the immunoconjugate.

148. 121. A polypeptide or immunoconjugate according to any one of claims 1 to 120 for use in a method for killing cancer cells in an individual.

149. 149. The use of claim 148, wherein the individual is a human individual.

150. 150. The use of claim 148 or 149, wherein the cancer cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells.

151. 151. The use of any one of claims 148 to 150, comprising administering to the individual between 0.5 μCi and 30.0 μCi per kilogram.

152. 152. The use of any one of claims 148 to 151, wherein cancer cells express an antigen that is specifically bound by the immunoconjugate.

153. 121. A method of delivering a radioisotope to cancer or tumor cells in an individual, comprising administering to said individual a polypeptide or immunoconjugate of any one of claims 1 to 120, thereby delivering said radioisotope to said cancer or tumor cells.

154. 154. The method of claim 153, wherein the individual is a human individual.

155. 155. The method of claim 153 or 154, wherein the cancer or tumor cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells.

156. 156. The method of any one of claims 153 to 155, wherein the cancer or tumor cells express an antigen that is specifically bound by the immunoconjugate.

157. 121. A polypeptide or immunoconjugate according to any one of claims 1 to 120 for use in delivering a radioisotope to cancer or tumour cells in an individual.

158. 158. The use of claim 157, wherein the individual is a human individual.

159. 159. The use of claim 157 or 158, wherein the cancer cells or tumor cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells.

160. 160. The use of any one of claims 157 to 159, wherein the cancer or tumor cells express an antigen that is specifically bound by the immunoconjugate.

161. 121. A method of imaging a tumor in an individual, comprising administering to said individual an immunoconjugate of any one of claims 1 to 120.

162. 162. The method of claim 161, wherein the individual is a human individual.

163. 163. The method of claim 161 or 162, wherein the tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer.

164. 164. The method of any one of claims 161 to 163, wherein the tumor expresses an antigen specifically bound by the immunoconjugate.

165. 121. The immunoconjugate of any one of claims 1 to 120 for use in a method for imaging a tumor in an individual.

166. 166. The use of claim 165, wherein the individual is a human individual.

167. 167. The use of claim 165 or 166, wherein the cancer or tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer.

168. 168. The use of any one of claims 165 to 167, wherein the tumor expresses an antigen specifically bound by the immunoconjugate.

169. A nucleic acid encoding a polypeptide according to any one of claims 1 to 77.

170. An expression vector comprising the nucleic acid of claim 169.

171. 171. A cell comprising the nucleic acid of claim 169 or the expression vector of claim 170.

172. The cell of claim 171, wherein the cell is a eukaryotic cell.

173. The cell of claim 172, wherein the eukaryotic cell is a CHO cell.

Images