Immunoconjugates for targeted radioisotope therapy

Abstract

Described herein is an immunoconjugate comprising a) an antigen binding region, b) an immunoglobulin heavy chain constant region, and c) a radioisotope chelator, wherein the molecular weight of the immunoconjugate is between 60 and 110 kDa. The immunoconjugate can be used to deliver alpha and beta emitters for the treatment of tumors or cancer.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 152,079, filed February 22, 2021, which is incorporated herein by reference in its entirety. [Background technology]

[0002] The superior specificity of antibodies (e.g., IgG) for their antigens makes them a highly targeted platform for therapeutics. However, the typical serum half-life of at least three weeks for IgG is disadvantageous for the delivery of radioisotopes, including alpha-emitting isotopes such as Ac-225 and beta-emitting isotopes such as Lu-177 and Y-90, particularly due to prolonged exposure and chronic off-target toxicity. The emergence of engineered smaller antibody formats (e.g., monomeric scFvs, heavy chain antibodies, or single-domain antibody fragments) offers the superior specificity of full-sized antibodies (e.g., IgG (approximately 150 kDa)) with smaller formats (e.g., 15 to 30 kDa) and much shorter serum half-lives (e.g., 30 minutes to 2 hours) (Bates A, Power, C, Antibodies (Base 1) 8:28 (2019)). Unfortunately, their short half-lives do not allow sufficient time for effective target binding due to poor retention and tumor uptake, and furthermore, plasma clearance of these small antibody formats by the renal system can result in isotope accumulation in kidney tissue and problematic off-target toxicity.

[0003] 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 killers and can overcome the acquired resistance observed in response to other therapies. Unfortunately, however, many challenges remain regarding achieving the desired dosimetry in target versus non-target tissues as a result of systemic administration and decay events at different locations in vivo. The key to the application of α-emitting radionuclides as targeted drugs is the ability to modulate the distribution of the daughter nuclides in vivo to limit toxicity. This is related to the timing of parent nuclide production, the time of therapeutic administration, the decay pathway and half-life of the daughter nuclides, circulation time, and the biodistribution and pharmacokinetics of the delivery vehicle. Unfortunately, particle release also typically produces recoil energies large enough to separate the daughter nuclide from the chelator, potentially separating the daughter nuclide from its targeting vehicle and resulting in subsequent redistribution of the "free" daughter nuclide, which can induce multiple harmful effects. See, e.g., Robertson A et al., Curr Radiopharm 11:156 (2018). Thus, nephrotoxicity caused by 225-Ac recoil daughter nuclides (e.g., 213-Bi) has been a major limitation to the therapeutic use of 225-Ac (see, e.g., Jaggi J et al., Cancer Res. 65:4888 (2005)).

[0004] A further confounding issue regarding the use of antibodies and antibody fragments bearing α-emitting radioisotopes in therapeutics is that the accompanying radioactive decay can damage, particularly, antibody components and target sequences, even before treatment. Radiolysis of the antibody fragment can occur before the α-emitter-labeled antibody fragment can be administered to a patient, thereby reducing the amount of targeting (see, e.g., Iarsen R, Brull and O, J. Am. Chem. Radiopharm. 36:1009-18 (1995)). At the higher specific activities required for therapeutic administration, immunoreactivity can rapidly decline along with radiochemical quality. Salako et al., J. Nucl. Med. 39(4):667-670 (1998). For example, the high ionization density emitted by α-emitters impaired the immunoreactivity of isotope-labeled Fab fragments through radiolysis at doses of 1,000 Gray (Gy) or more. Similarly, significant radiolysis of luminescent isotope-labeled antibodies was observed at doses exceeding 1200 Gy (Zalutsky M et al., J Nucl Med. 42(10):1508-15(2001)). Therefore, identifying a suitable targeted delivery vehicle for alpha-emitting isotopes is not straightforward.

[0005] Furthermore, targeted radioscope delivery platforms, including alpha- and beta-emitting isotopes, present additional challenges, requiring simultaneous optimization of, for example, immunogenicity, specificity, tissue penetration, stability, ease of manufacture, and acceptable therapeutic window when designing such platforms. Summary of the Invention

[0006] The present invention relates to immunoconjugates or radioimmunoconjugates, compositions comprising the same, and methods of using such immunoconjugates and compositions. The immunoconjugates and compositions of the invention have numerous uses, for example, for the delivery of radioisotopes to kill target cells (e.g., cancer cells that express the target antigen to which the radioimmunoconjugate is bound), for the detection and characterization of malignant cells (e.g., target antigen expression) within a subject, and for the diagnosis and treatment of various diseases and disorders, such as cancers, tumors, and other proliferative disorders involving antigen-expressing cells.

[0007] The present invention addresses several challenges inherent in targeted delivery of α-particle emitters in vivo through the selection and specific combination of specific delivery platform components. The α-particle-emitting radioisotope-delivery platform of the present invention provides 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 α-emitter while preserving the antibody fragment long enough to exert therapeutic activity in the body. For example, the α-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. Additionally and surprisingly, the α-particle-emitting radioisotope-delivery platform of the present invention exhibits excellent tumor binding and labeling properties for tumors with different antigen densities, which can be a limitation for some uses of some immunoconjugates.

[0008] In one aspect, an immunoconjugate is described herein, the immunoconjugate comprising a) an antigen-binding region, b) an immunoglobulin heavy chain constant region, and c) a chelator, wherein the molecular weight of the immunoconjugate is between 60 and 110 kDa. In some embodiments, the antigen-binding region comprises an scFv polypeptide or a VHH polypeptide. In some embodiments, the antigen-binding region comprises an scFv polypeptide. In some embodiments, the antigen-binding region comprises a VHH polypeptide. In some embodiments, the antigen-binding region is humanized. In some embodiments, the antigen-binding region specifically binds to HER2 or D113. In some embodiments, the antigen-binding region specifically binds to HER2. In some embodiments, the antigen-binding region comprises a) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, b) a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and c) a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and binds to HER2. In some embodiments, the antigen-binding region comprises a sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 20 and binds to HER2. In some embodiments, the antigen-binding region specifically binds to D113. In some embodiments, the antigen-binding region comprises a) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, b) a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and c) a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33 and binds to D113. In some embodiments, the antigen-binding region comprises a sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 30 and binds to D113. In some embodiments, the immunoglobulin heavy chain constant region comprises an immunoglobulin CH2 domain, an immunoglobulin CH3 domain, or an immunoglobulin CH2 and CH3 domains. In some embodiments, the immunoglobulin heavy chain constant region comprises the CH2 and CH3 domains of an immunoglobulin. In some embodiments, the immunoglobulin heavy chain constant region is a human immunoglobulin heavy chain constant region. In some embodiments, the immunoglobulin heavy chain constant region is an IgA, IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments,The immunoglobulin heavy chain constant region is of the IgG1 isotype. In some embodiments, the immunoglobulin heavy chain constant region is of the IgG4 isotype. In some 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 immunoconjugate to fetal Fc receptor (FcRn). In some 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 immunoconjugate to fetal Fc receptor (FcRn). In some 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 some embodiments, the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that alters binding of the immunoconjugate to fetal Fc receptor (FcRn). In some embodiments, the 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 cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or a combination thereof. In some embodiments, the changes to one or more amino acid residues that reduce an effector function of an immunoglobulin heavy chain constant region are, according to EU numbering, (a) 297A, 297Q, 297G, or 297D, (b) 279F, 279K, or 2791, (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) 33 0R, (hh) 339E or 3391, (ii) 343I or 343V, (jj) 373A, 373G, or 373S, (kk) 376E, 376W, or 376Y, (11) 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) 1235E, (vv) 1234A and 1235A, (ww) 1234A, 1235A, and G237A, (xx) 1234A, 1235A, and P329G, (yy) 1234F, 1235E, and P331S, (zz) 1234A, 1235E, and G237A, (aaa), 1234A, 1235E, G237A, and P331S, (bbb) 1234A, 1235A, G237A, P238S, H268A, A330S, and P331S, (ccc) 1234A, 1235A, and P329A, In one embodiment, 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: (ddd) G236R and 1328R, (eee) G237A, (fff) F241A, (ggg) V264A, (hhh) D265A, (iii) D265A and N297A, (jjj) D265A and N297G, (kkk) D270A, (111) A3301, (mmm) P331A or P331S, or (nnn) E233P, (ooo) 1234A, 1235E, G237A, A330S, and P331S, or (ppp) any combination of (a) through (ppp).These include, per EU numbering: 1234A, 1235E, G237A, A330S, and P331S. In certain embodiments, the amino acid changes to one or more amino acid residues that alter binding of the immunoconjugate to fetal Fc receptor (FcRn) shorten the serum half-life of the immunoconjugate. In certain embodiments, the changes to one or more amino acid residues that alter binding of the immunoconjugate to fetal 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, and combinations thereof, per EU numbering. In certain embodiments, the changes to one or more amino acid residues that alter binding of the immunoconjugate to fetal Fc receptor (FcRn) are to amino acid residues selected from the list consisting of 253, 254, 310, 435, 436, and combinations thereof, per EU numbering. In certain embodiments, the changes to one or more amino acid residues that alter binding of the immunoconjugate to fetal 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, per EU numbering. In certain embodiments, the changes to one or more amino acid residues that alter binding of the immunoconjugate to fetal Fc receptor (FcRn) are to amino acid residues selected from the list consisting of I253A, S254A, H310A, H435Q, Y436A, and combinations thereof, per EU numbering. In certain embodiments, the changes to one or more amino acid residues that alter binding of the immunoconjugate to fetal Fc receptor (FcRn) are to amino acid residues selected from the list consisting of I253A, H310A, H435Q, and combinations thereof, per EU numbering. 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 some embodiments, the immunoconjugate has a serum half-life of less than 72 hours. In some 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. In some embodiments, the antigen-binding region is linked to the immunoglobulin heavy chain constant region by a human IgG hinge region. In some embodiments, the chelator is a radioisotope chelator. In some embodiments, the chelator 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. In some embodiments, the chelator is DOTA. In some embodiments, the chelator is DOTAGA. In some embodiments, the chelator is Py4Pa. In some embodiments, the chelator is linked directly to the antigen-binding region and / or the immunoglobulin heavy chain constant region. In certain embodiments, the chelator is linked to the antigen binding region and / or the immunoglobulin heavy chain constant region by a linker. In certain embodiments, the linker is selected from 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (va1-cit), alanine-phenylalanine (a1a-phe), p-aminobenzyloxycarbonyl (PAB), and those resulting from conjugation with a linker reagent, N-succinimidyl 4-(2-pyridylthio)pentanoate (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), which form the linker moiety 4-mercaptopentanoic acid. In certain embodiments, the linker is polyethylene glycol (PEG), a polyethylene glycol polymer (PEG),and S-2-(4-isothiocyanatobenzyl) (SCN). In some embodiments, the linker is PEG5. In some embodiments, the linker is SCN. In some embodiments, the chelator is a linker chelator selected from the list consisting of FP-Ad-PEG5-DOTAGA, p-SCN-Bn-DOTA, p-SCN-Ph-Et-Py4Pa, and TFP-Ad-PEG5-Ac-Py4Pa. In some embodiments, the chelator is TFP-Ad-PEG5-DOTAGA. In some embodiments, the chelator is p-SCN-Bn-DOTA. In some embodiments, the chelator is p-SCN-Ph-Et-Py4Pa. In some embodiments, the chelator is TFP-Ad-PEG5-Ac-Py4Pa. In some embodiments, the chelator is linked to the antigen binding region and / or immunoglobulin heavy chain constant region at a ratio of 1:1 to 8:1. In some embodiments, the chelator is linked to the antigen binding region and / or immunoglobulin heavy chain constant region at a ratio of 1:1 to 6:1. In some embodiments, the chelator is linked to the antigen binding region and / or immunoglobulin heavy chain constant region at a ratio of 2:1 to 6:1. In some embodiments, the immunoconjugate further comprises a radioisotope. In some embodiments, the radioisotope is an alpha emitter. In some 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 some embodiments, the radioisotope is 225-Ac. In some embodiments, the radioisotope is a beta emitter. In some embodiments, the radioisotope is a beta emitter selected from 177-Lu, 90-Y, 67-Cu, and 153-Sm. In some embodiments, the molecular weight of the immunoconjugate is between 60 and 110 kDa. In some embodiments, the molecular weight of the immunoconjugate is between 60 and 90 kDa. In some embodiments, the molecular weight of the immunoconjugate is between 65 and 90 kDa. In some embodiments, the molecular weight of the immunoconjugate is between 70 and 90 kDa. In some 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.

[0009] Further described herein is a method for producing an immunoconjugate, comprising loading the immunoconjugate with a radioisotope. In some embodiments, the radioisotope is an α-emitter. In some 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 some embodiments, the radioisotope is 225-Ac. In some embodiments, the radioisotope is a β-emitter. In some embodiments, the radioisotope is a β-emitter selected from 177-Lu, 90-Y, 67-Cu, and 153-Sm. In some embodiments, the radioisotope is 177-Lu.

[0010] Further described herein are methods for treating cancer or tumor in an individual, comprising administering an immunoconjugate to the individual, thereby treating the cancer or tumor. In some embodiments, the individual is a human. In some embodiments, the cancer or tumor is a solid cancer or tumor. In some embodiments, the cancer or tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer. In some embodiments, the method comprises administering 0.5 μCi to 30.0 μCi per kilogram to the individual. In some embodiments, the cancer or tumor expresses an antigen specifically bound by the immunoconjugate.

[0011] Further described herein are immunoconjugates for use in methods for treating cancer or tumors in individuals. In some embodiments, the individual is a human. In some embodiments, the cancer or tumor is a solid cancer or tumor. In some embodiments, the cancer or tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer. In some embodiments, 0.5 μCi to 30.0 μCi per kilogram is administered to the individual. In some embodiments, the cancer or tumor expresses an antigen specifically bound by the immunoconjugate.

[0012] Further described herein are methods for killing cancer cells in an individual, comprising administering an immunoconjugate to the individual, thereby killing the cancer cells. In some embodiments, the individual is a human. In some embodiments, the cancer cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells. In some embodiments, the method comprises administering to the individual 0.1 μCi to 30.0 μCi per kilogram. In some embodiments, the method comprises administering to the individual 10 mCi to 75 mCi per square meter of body surface area. In some embodiments, the cancer cells express an antigen specifically bound by the immunoconjugate.

[0013] Further described herein is the use of an immunoconjugate in a method for killing cancer cells in an individual. In some embodiments, the individual is a human. In some embodiments, the cancer cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells. In some embodiments, the method comprises administering 0.5 μCi to 30.0 μCi per kilogram to the individual. In some embodiments, the cancer cells express an antigen specifically bound by the immunoconjugate.

[0014] Further described herein is a method for delivering a radioisotope to cancer or tumor cells of an individual, comprising administering an immunoconjugate to the individual, thereby delivering the radioisotope to the cancer or tumor cells. In some embodiments, the individual is a human. In some embodiments, the cancer or tumor cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells. In some embodiments, the method comprises administering 0.5 μCi to 30.0 μCi per kilogram to the individual. In some embodiments, the cancer cells express an antigen specifically bound by the immunoconjugate.

[0015] Further described herein are immunoconjugates for use in methods for delivering radioisotopes to cancer or tumor cells in an individual. In some embodiments, the individual is a human. In some embodiments, the cancer or tumor cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells. In some embodiments, the cancer or tumor cells express an antigen specifically bound by the immunoconjugate.

[0016] Further described herein is a method for imaging a tumor in an individual, comprising administering an immunoconjugate to the individual. In some embodiments, the individual is a human. In some embodiments, the cancer or tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer. In some embodiments, the tumor expresses an antigen specifically bound by the immunoconjugate.

[0017] Further described herein are immunoconjugates for use in methods for imaging tumors in an individual. In some embodiments, the individual is a human. In some embodiments, the cancer or tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer. In some embodiments, the tumor expresses an antigen specifically bound by the immunoconjugate.

[0018] Nucleic acids encoding immunoconjugates are further described herein. In some embodiments, an expression vector comprises the nucleic acid. In some embodiments, a cell comprises the nucleic acid or the expression vector. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a CHO cell.

[0019] In some embodiments, the subject radioisotope-delivery platforms have a molecular size (e.g., 60 kDa to 110 kDa) large enough to substantially reduce off-target toxicity, particularly renal damage (e.g., from the α-emitting isotope cargo), yet small enough 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 provides preferential clearance by the liver as opposed to the kidney, protecting the kidney from radiation toxicity.

[0020] In some embodiments, the subject radioisotope-delivery platforms are useful for safe and effective in vivo targeted delivery of alpha emitters, in part by reducing certain adverse effects caused by platforms having half-lives greater than 5 days and / or molecular weights less than 60 kDa.

[0021] In some embodiments, the subject radioisotope-delivery platforms are useful for safe and effective in vivo targeted delivery of alpha emitters, in part by exhibiting reduced loss of targeting ability due to radiolysis compared to other possible delivery platforms.

[0022] In some embodiments, the subject radioisotope-delivery platforms are useful for safe and effective in vivo targeted delivery of alpha emitters, in part by exhibiting increased stability in manufacture at temperatures required for certain radiolabeling processes (e.g., high temperature chelation with certain chelators) compared to other possible delivery platforms that use antibody fragments.

[0023] In one embodiment, the present invention provides an immunoconjugate for delivering an alpha-emitting radioisotope in vivo. In one embodiment, the immunoconjugate can also deliver other atoms in vivo. In one embodiment, the immunoconjugate can deliver an imaging metal (e.g., 111-In, 89-Zr, 64-Cu, 68-Ga, or 134-Ce) in vivo.

[0024] In one embodiment, the immunoconjugate comprises an antibody construct and a chelating agent and has a molecular weight of 60 to 110 kDa, preferably 60 to 100 kDa, preferably 60 to 90 kDa, preferably 65 to 90 kDa, preferably 70 to 90 kDa. The chelating agent is capable of chelating an alpha-radioisotope such that the antibody construct is linked to the alpha-radioisotope.

[0025] At least one of the variant constant regions in the immunoconjugate has at least one FcRn-binding mutation. In a preferred embodiment, each of the two variant constant regions of the immunoconjugate has at least one FcRn-binding mutation, and the FcRn-binding mutations are the same or different.

[0026] 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.

[0027] 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 linked to the radioisotope chelating moiety. In one embodiment, the chelator further comprises a linker between the functional group and the radioisotope chelating moiety.

[0028] 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.

[0029] In one embodiment, the present invention provides a pharmaceutical composition comprising a radioimmunoconjugate of the present invention and a pharmaceutically acceptable carrier.

[0030] In one embodiment, the present invention provides a method for delivering an alpha-emitting radioisotope to cancer cells in vivo in a patient, comprising administering to the patient a radioimmunoconjugate or pharmaceutical composition of the present invention, In one embodiment, the patient is a human patient.

[0031] In one embodiment, the present invention provides a method of inhibiting the growth of cancer cells, the method comprising contacting the cancer cells with a radioimmunoconjugate of the present invention. In one embodiment, the cancer cells are in vivo in a patient. In one embodiment, the method comprises administering to the patient a pharmaceutical composition of the present invention. In one embodiment, the patient is a human patient.

[0032] In one embodiment, the present invention provides a method of killing cancer cells, comprising contacting the cancer cells with a radioimmunoconjugate of the present invention. In one embodiment, the cancer cells are in vivo in a patient. In one embodiment, the method comprises administering to the patient a pharmaceutical composition of the present invention. In one embodiment, the patient is a human patient.

[0033] In one embodiment, the present invention provides a method of treating cancer in a patient in need thereof, comprising administering to the patient a radioimmunoconjugate or pharmaceutical composition of the present invention. In one embodiment, the patient is a human patient.

[0034] In one embodiment, the present invention provides a targeted imaging complex comprising an immunoconjugate of the present invention and further comprising an imaging metal. In one aspect, the present invention provides a targeted imaging complex comprising an antibody construct of the immunoconjugate of the present invention and further comprising an imaging metal. In one embodiment, the imaging metal is a radioisotope. In one embodiment, the imaging metal is selected from the group consisting of 111-In, 89-Zr, 64-Cu, 68-Ga, and 134-Ce. In one embodiment, the imaging metal is selected from the group consisting of 111-In, 89-Zr, 64-Cu, 68-Ga, and 134-Ce. In one embodiment, the imaging metal is 111-In. In one embodiment, the imaging metal is covalently attached to the immunoconjugate or antibody construct. In one embodiment, the imaging metal is associated with a chelator of the immunoconjugate. In one embodiment, the present invention provides a method for determining the location of cancer cells in vivo in a patient, comprising administering to the patient a targeted imaging complex of the present invention. In one embodiment, the patient is a human patient.

[0035] In one embodiment, the present invention provides a kit for preparing a radiopharmaceutical of the present invention, comprising an immunoconjugate of the present invention. In one embodiment, the present invention provides a kit comprising a radioimmunoconjugate of the present invention. In one embodiment, the present invention provides a kit for preparing a pharmaceutical composition of the present invention, comprising an immunoconjugate of the present invention. In one embodiment, the present invention provides a kit for preparing a pharmaceutical composition of the present invention, comprising a radioimmunoconjugate of the present invention. In one embodiment, the present invention provides a kit comprising a pharmaceutical composition of the present invention.

[0036] In some embodiments, the immunoconjugates or radioimmunoconjugates of the invention comprise a dimerization domain or motif. In some further embodiments, the dimerization domain or motif is in the hinge region and / or the variant constant region.

[0037] In some embodiments, the immunoconjugate or radioimmunoconjugate or pharmaceutical composition of the invention has a half-life in human serum of less than 96 hours. In some further embodiments, the half-life in human serum is less than 72 hours. In some further embodiments, the half-life is less than 48, 36, 24, and / or 12 hours. In some embodiments, the half-life is 4 to 8 hours, 6 to 12 hours, 8 to 16 hours, 12 to 24 hours, or 24 to 48 hours.

[0038] In one aspect, the invention provides a radioimmunoconjugate comprising an immunoconjugate of the invention and further comprising a beta particle emitter, such as, for example, 177-Lu, 90-Y, 67-Cu, or 153-Sm, hi one aspect, the invention provides a pharmaceutical composition comprising such a radioimmunoconjugate.

[0039] In one aspect, the invention provides a radioimmunoconjugate comprising an immunoconjugate of the invention and further comprising an α particle emitter and a β and / or γ particle emitter, hi one aspect, the invention provides a pharmaceutical composition comprising such a radioimmunoconjugate.

[0040] In some embodiments, kits of the invention include reagents or pharmaceutical devices in addition to an immunoconjugate, radioimmunoconjugate or pharmaceutical composition of the invention.

[0041] In some embodiments, the kits of the present invention are immunoassay kits for specifically detecting an antigen in a biological sample, the immunoassay kit comprising (a) an immunoconjugate, radioimmunoconjugate, or targeted imaging complex and / or compositions thereof described herein, and (b) instructions for detecting the immunoconjugate, radioimmunoconjugate, or targeted imaging complex.

[0042] In another aspect, the present invention provides an isolated nucleic acid encoding an antigen-binding arm or a component thereof provided herein. In one aspect, the present invention provides an isolated nucleic acid encoding an antigen-binding region of an immunoconjugate herein. In one aspect, the present invention provides an isolated nucleic acid encoding a VHH polypeptide of an immunoconjugate herein. In one aspect, the present invention provides an isolated nucleic acid encoding a hinge region of an immunoconjugate herein. In one aspect, the present invention provides an isolated nucleic acid encoding a variant constant region of an immunoconjugate herein. In one aspect, the present invention provides an isolated nucleic acid encoding a VHH polypeptide of an immunoconjugate herein and a hinge region of an immunoconjugate herein. In one aspect, the present invention provides an isolated nucleic acid encoding a VHH polypeptide of an immunoconjugate herein, a hinge region of an immunoconjugate herein, and a variant constant region of an immunoconjugate herein.

[0043] In another aspect, the invention provides a vector comprising a nucleic acid provided herein. In some embodiments, the vector is an expression vector.

[0044] In another aspect, the present invention provides methods of using the immunoconjugates, radioimmunoconjugates, targeted imaging complexes, or pharmaceutical compositions of the invention. In some embodiments, the present invention provides methods of treating a disease, disorder, or condition comprising administering to a patient in need thereof a pharmaceutically effective amount of the radioimmunoconjugate or pharmaceutical composition herein.

[0045] In some embodiments, the methods of the present invention comprise administering to a subject in need thereof any of the radioimmunoconjugates or pharmaceutical compositions described herein. In some further embodiments, the methods are for inhibiting the growth of and / or killing cancer cells or tumors.

[0046] In some embodiments, the use of an immunoconjugate or radioimmunoconjugate described herein is provided for the manufacture of a medicament for treating a disease, disorder, or condition in a subject, such as, for example, cancer.

[0047] In another aspect, the present invention provides a process for making a radioimmunoconjugate or pharmaceutical composition of the invention, the process comprising radiolabeling the immunoconjugate with a suitable isotope, e.g., an alpha or beta particle emitter.

[0048] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description and appended claims. The above-described elements of the present invention may be individually combined or freely removed to create other embodiments of the present invention, without any reference hereinafter to such combinations or removals. [Brief explanation of the drawings]

[0049] [Figure 1A] 1A and 1B show the binding of anti-HER2 and anti-D113 VHH-Fc constructs. [Figure 1B] 1A and 1B show the binding of anti-HER2 and anti-D113 VHH-Fc constructs. [Figure 2A] Figures 2A, 2B and 2C show the binding of anti-HER2 and anti-D113 VHH-Fc constructs to cells expressing HER2 and / or D113. [Figure 2B] Figures 2A, 2B and 2C show the binding of anti-HER2 and anti-D113 VHH-Fc constructs to cells expressing HER2 and / or D113. [Figure 2C] Figures 2A, 2B and 2C show the binding of anti-HER2 and anti-D113 VHH-Fc constructs to cells expressing HER2 and / or D113. [Figure 3A] 3A and 3B show the internalization of anti-HER2 and anti-D113 VHH-Fc constructs in cells expressing HER2 and D113. [Figure 3B] 3A and 3B show the internalization of anti-HER2 and anti-D113 VHH-Fc constructs in cells expressing HER2 and D113. [Figure 4] FIG. 4 shows the self-interaction data for anti-HER2 and anti-D113 VHH-Fc constructs. [Figure 5] FIG. 5 shows a diagram of the chemical synthesis of the linker molecule. [Figure 6] FIG. 6 shows a diagram of the chemical synthesis of the linker molecule. [Figure 7A] Figures 7A, 7B and 7C show the immunoreactive fractions of the different VHH-Fc constructs. [Figure 7B] Figures 7A, 7B and 7C show the immunoreactive fractions of the different VHH-Fc constructs. [Figure 7C] Figures 7A, 7B and 7C show the immunoreactive fractions of the different VHH-Fc constructs. [Figure 8] FIG. 8 shows a comparison of imaging with 111In-labeled VHH-Fc compared to the biodistribution of 225Ac-labeled VHH-Fc. [Figure 9A] Figures 9A, 9B, 9C and 9D show the biodistribution of labeled anti-HER2 VHH-Fc constructs over time. [Figure 9B] Figures 9A, 9B, 9C and 9D show the biodistribution of labeled anti-HER2 VHH-Fc constructs over time. [Figure 9C] Figures 9A, 9B, 9C and 9D show the biodistribution of labeled anti-HER2 VHH-Fc constructs over time. [Figure 9D] Figures 9A, 9B, 9C and 9D show the biodistribution of labeled anti-HER2 VHH-Fc constructs over time. [Figure 10A] Figures 10A, 10B and 10C show the tumor to tumor tissue ratio of labeled anti-HER2 VHH-Fc constructs. [Figure 10B] Figures 10A, 10B and 10C show the tumor to tumor tissue ratio of labeled anti-HER2 VHH-Fc constructs. [Figure 10C]Figures 10A, 10B and 10C show the tumor to tumor tissue ratio of labeled anti-HER2 VHH-Fc constructs. [Figure 11] FIG. 11 shows the biodistribution of labeled anti-HER2 VHH-Fc constructs. [Figure 12] FIG. 12 shows the systemic clearance of 111In-labeled VHH-Fc (H101) and VHH-Fc variants (H105, H107, and H108). [Figure 13] FIG. 13 shows the biodistribution of labeled anti-D113 VHH-Fc constructs over time. [Figure 14] FIG. 14 shows the biodistribution of labeled anti-D113 VHH-Fc constructs. [Figure 15A] Figures 15A and 15B show the biodistribution of 225Ac-labeled anti-HER2 (15A) and anti-D113 (15B) VHH-Fc constructs. [Figure 15B] Figures 15A and 15B show the biodistribution of 225Ac-labeled anti-HER2 (15A) and anti-D113 (15B) VHH-Fc constructs. [Figure 16A] Figures 16A, 16B and 16C show the results of toxicity studies performed with 225Ac-labeled anti-HER2 VHH-Fc constructs. [Figure 16B] Figures 16A, 16B and 16C show the results of toxicity studies performed with 225Ac-labeled anti-HER2 VHH-Fc constructs. [Figure 16C] Figures 16A, 16B and 16C show the results of toxicity studies performed with 225Ac-labeled anti-HER2 VHH-Fc constructs. [Figure 17] FIG. 17 shows the immunoreactive fractions of different anti-DD13 VHH-Fc constructs loaded with 177Lu. [Figure 18] FIG. 18 shows the chemical structures of certain linker-chelators described herein.

[0050] (Detailed Description of the Invention) The present invention will now be described in more detail using illustrative and 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.

[0051] In particular, in embodiments, the present invention 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 platforms of the present invention provide shorter half-lives compared to conventional IgG, but longer half-lives than smaller monomeric antibody fragment formats. In some embodiments, the subject radioisotope delivery platforms possess a molecular size large enough (e.g., 60 kDa to 110 kDa) to substantially reduce target exotoxin, particularly renal damage (e.g., from α- or β-emitting isotope cargoes), yet small enough to increase tissue penetration compared to conventional IgG, maintaining target specificity and increasing the probability of a first decay event in the target tissue. In some embodiments, the present radioisotope delivery platforms are useful for safe and effective targeted delivery of radioisotopes (such as α- or β-emitters) in vivo, 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 in vivo targeted delivery of radioisotopes (such as α- 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 in vivo targeted delivery of radioisotopes (such as α- or β-emitters) in part by exhibiting increased stability in manufacturing at temperatures required for certain radiolabeling processes (e.g., high temperature chelation with certain chelators) compared to other possible delivery platforms that use antibody fragments.

[0052] Immunoconjugates In one aspect, the present invention provides immunoconjugates that specifically bind to target antigens with high affinity. In some embodiments, the present invention 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 an affinity of less than 1 mM, less than 100 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 0.01 nM, or less than 0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 K of M D The antibody binds to a specific antigen and / or epitope with an affinity characterized by:

[0053] The immunoconjugates described herein can serve as platforms for radioisotope delivery. Provided herein are radioisotope delivery platforms with relatively short half-lives (e.g., less than 1 or 2 weeks, but greater than 2 to 8 hours).

[0054] In one embodiment, an immunoconjugate of the present disclosure comprises a) an antigen-binding region and b) an immunoglobulin heavy chain constant region. 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 chelator. 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 chelator. 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 chelator, wherein the immunoconjugate has a molecular weight of 60 to 110 kDa.

[0055] In one embodiment, an immunoconjugate of the present disclosure comprises a) a VHH antigen-binding region and b) an immunoglobulin heavy chain constant region. In one embodiment, an immunoconjugate of the present disclosure comprises a) a VHH antigen-binding region, b) an immunoglobulin heavy chain constant region, and c) a chelator. In one embodiment, an immunoconjugate of the present disclosure comprises a) a VHH antigen-binding region, b) an immunoglobulin heavy chain constant region, and c) a radioisotope chelator. In one embodiment, an immunoconjugate of the present disclosure comprises a) a VHH antigen-binding region, b) an immunoglobulin heavy chain constant region, and c) a radioisotope chelator, wherein the immunoconjugate has a molecular weight of 60 to 110 kDa.

[0056] In one embodiment, an immunoconjugate of the present disclosure comprises a) a VHH antigen-binding region and b) an immunoglobulin Fc region, collectively referred to as aVHH-Fc. In one embodiment, an immunoconjugate of the present disclosure comprises a) a VHH antigen-binding region, b) an immunoglobulin Fc region, and c) a chelating agent. In one embodiment, an immunoconjugate of the present disclosure comprises a) a VHH antigen-binding region, b) an immunoglobulin Fc region, and c) a radioisotope chelating agent. In one embodiment, an immunoconjugate of the present disclosure comprises a) a VHH antigen-binding region, b) an immunoglobulin Fc region, and c) a radioisotope chelating agent, and the molecular weight of the immunoconjugate is 60 to 110 kDa.

[0057] 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, 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, 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, b) a variant immunoglobulin Fc region, and c) a radioisotope chelator, wherein the immunoconjugate has a molecular weight of 60 to 110 kDa. 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.

[0058] In some embodiments, the radioisotope delivery platform has a size greater than about 60 kDa to avoid certain toxicities from the α-radioisotope cargo, such as off-target nephrotoxicity. 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 to 110 kDa due to its dimeric structure of two individual antigen-binding arms, each having a hinge region and a VHH polypeptide fused to 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.

[0059] 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 of the antigen-binding arms 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, and the hinge region of the arm is covalently linked to the variant constant region of the arm, such that the hinge region is interposed between and thereby links the antigen-binding region and the variant constant region in the antigen-binding arm.

[0060] In a preferred embodiment, at least one of the two antigen-binding regions in the immunoconjugate consists of one or two variable heavy chain (VHH) polypeptides. In a preferred embodiment, at least one of the two antigen-binding regions consists of one VHH polypeptide. In a preferred embodiment, each of the two antigen-binding regions of the immunoconjugate consists of one VHH polypeptide, and the VHH polypeptides are the same or different.

[0061] In one embodiment, the antigen-binding regions of the immunoconjugate bind to the same antigen. In one embodiment, the antigen-binding regions of the immunoconjugate bind to different antigens. In one embodiment, the antigen-binding regions of the immunoconjugate are the same. In one embodiment, the antigen-binding regions of the immunoconjugate are different. In one embodiment, the antigen-binding region of each antigen-binding arm consists of one or two VHH polypeptides.

[0062] In one embodiment, the antigen-binding region of one antigen-binding arm consists of two VHH polypeptides, and the antigen-binding region of the other antigen-binding arm does not contain a VHH polypeptide. In one embodiment, the two antigen-binding arms bind to the same antigen. In one embodiment, the two antigen-binding arms bind to different antigens. In one embodiment, the two VHH polypeptides are the same. In one embodiment, the two VHH polypeptides are different. In one embodiment, the immunoconjugate is bispecific.

[0063] In one embodiment, the antigen-binding region of one antigen-binding arm consists of one VHH polypeptide, and the antigen-binding region of the other antigen-binding arm consists of two VHH polypeptides. In one embodiment, the two antigen-binding arms bind to the same antigen. In one embodiment, the two antigen-binding arms bind to different antigens. In one embodiment, the three VHH polypeptides are the same. In one embodiment, two of the three VHH polypeptides are the same and different from the third VHH polypeptide. In one embodiment, the three VHH polypeptides are different. In one embodiment, the immunoconjugate is bispecific.

[0064] In one embodiment, the antigen-binding region of each antigen-binding arm of the immunoconjugate consists of one VHH polypeptide. In one embodiment, the VHH polypeptides bind to the same antigen. In one embodiment, the VHH polypeptides bind to different antigens. In one embodiment, the VHH polypeptides are the same. In one embodiment, the VHH polypeptides are different. In one embodiment, the immunoconjugate is bispecific.

[0065] antigen binding region The antigen-binding region confers specificity to the immunoconjugate and may suitably comprise a small antigen-binding polypeptide. Such a small antigen-binding polypeptide offers advantages such as reducing the overall size of the immunoconjugate molecule, allowing tumor penetration and labeling. The small antigen-binding polypeptide may lack certain regions essential for binding, such as the light chain constant region, heavy chain constant region, CH1 region, or hinge region. In some embodiments, the antigen-binding region may lack the light chain variable region. In some embodiments, the small antigen-binding region may have a molecular weight of 10 kDa to 40 kDa.

[0066] 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.

[0067] The antigen-binding region may comprise a VHH polypeptide, an scFv polypeptide, or a VNAR polypeptide. In some embodiments, the antigen-binding region comprises a VHH polypeptide. In some embodiments, the antigen-binding region comprises an ScFv polypeptide. In some embodiments, the antigen-binding region comprises a VNAR polypeptide. In some embodiments, the antigen-binding region is humanized.

[0068] The antigenic region may include specificity for an antigen selected by one of skill in the art to achieve a desired function, e.g., targeting a particular cancer, tumor, or cell type suitable for treatment with the described immunoconjugate or radioimmunoconjugate. As described herein, the antigen-binding region may be an antibody fragment or format known in the art. Intact antibodies can be engineered to fit various small antigen-binding region formats (e.g., scFv) described herein. The antigen-binding region may specifically bind to a tumor antigen (e.g., an antigen specifically expressed or enriched in cancerous cells). In some embodiments, the tumor antigen is Her2, Trop2, CEA, NaPi2b, uPAR, CDCP1, MUC-1, MUC-16, CEACAM-5, MR-1, Fn14, MAGE-3, NY-ESO-1, EGFR, PDGFR, IGF1R, CSF-1R, PSMA, PSCA, STEAP-1, FAP, TEM8, 5T4, VEGFR, NRP1, CD4+, CD5+, CD6+, CD8+, CD9+, CD10+, CD11+, CD12+, CD13+, CD14+, CD15+, CD16+, CD17+, CD18+, CD19+, CD20+, CD21+, CD22+, CD23+, CD24+, CD19+, CD25+, CD19+, CD25+, CD19+, CD25+, CD19+, CD25+, CD19+, CD26+, CD19+, CD27+, CD19+, CD28+, CD29+, CD30+, CD31+, CD41+, CD19+, CD29+, CD29+, CD29+, CD31+, CD41+, CD19+, CD28+, CD29+, CD29+, CD31+, CD41+, CD19 19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD39, CD44, CD47, CD52, CD70, CD71, CD74, CD79B, CD132, CD133, CD138, CD166, CD205, CD276, ROR1, ROR2, glypican 3, Trail receptor 2 (DR5), PD-11, mesothein, bombesin, EpCAM, DARPP, CSPG4, galectin-3, integrin αvβ1, integrin αvβ3, integrin The tumor antigens include αvβ5, integrin αvβ6, integrin α5β1, integrin α3, integrin α5, integrin β6, nectin-4, Wnt activation inhibitory factor 1, D113, transferrin receptor, folate receptor α, tissue factor, BCMA, c-Met, 1IV-1, AX1, AFP, ENPP3, C1DN6 / 9, DPEP3, RNF43, 1RRC15, PTK7, P-cadherin, F1T3, EphA2, MTI-MMP, CXCR6, GD2, or smoothened antigen (Smo). In some embodiments, the tumor antigen includes human epidermal growth factor receptor 2 (HER2), Δ-like ligand 3 (D113), folate receptor α (FO1R1), or Wnt activation inhibitory factor 1 (WAIF1). In some embodiments, the tumor antigen includes HER2.In some embodiments, the tumor antigen comprises D113. In some embodiments, the tumor antigen comprises FO1R1. In some embodiments, the tumor antigen comprises WAIF1. In some embodiments, the tumor antigen comprises TROP2. In some embodiments, the tumor antigen comprises EGFR. In some embodiments, the tumor antigen comprises PSA. In some embodiments, the tumor antigen comprises MUC-1. In some embodiments, the tumor antigen comprises CEA. In some embodiments, the tumor antigen comprises NY-ESO-1.

[0069] In certain embodiments, the antigen-binding region of the immunoconjugate comprises a sequence that is at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 20 and that binds to HER2.

[0070] In one embodiment, the antigen-binding region of the immunoconjugate comprises a) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, b) a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and c) a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23.

[0071] In one embodiment, the antigen-binding region of the immunoconjugate comprises a sequence that is at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 30 and that binds to D113.

[0072] In one embodiment, the antigen-binding region of the immunoconjugate comprises: a) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31; b) a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32; and c) a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33.

[0073] In some embodiments, the immunoconjugates of the invention comprise, for example, an autonomous V H domains (e.g., of camelid, murine, or human origin), single domain antibody domains (sdAb), camelid-derived heavy chain antibody domains (V H H fragment or V H domain fragment), Camelidae V HH 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, "camelized" or "camelised" scaffolds are H Domains, heavy chain and C H Fd fragment consisting of I domain, single-chain FV-C H 3. Minibodies, Fc antigen binding domains (Fcabs), scFv-Fc fusions, multimerized scFv fragments (diabodies, triabodies, tetrabodies), disulfide-stabilized antibody variable (Fv) fragments (dsFv), V1, V2 H , C1 and C H Disulfide-stabilized antigen-binding (Fab) fragments consisting of I domains, 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 H fragments, bispecific tandem scFv fragments, bispecific nanobodies, bispecific minibodies, and proteins or polypeptides comprising genetically engineered counterparts of any of the foregoing that retain the paratope and target antigen binding function.

[0074] 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 a homodimer.

[0075] In one aspect, the present invention provides an antibody construct (alone or in an immunoconjugate, radioimmunoconjugate, or targeted imaging conjugate, each of the present invention) 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.

[0076] In some embodiments, the antibody construct, immunoconjugate, radioimmunoconjugate, or targeted imaging complex 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.

[0077] 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, e.g., 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-1) comprising three CDRs, i.e., 1CDR1, 1CDR2, and 1CDR3, and (b) a heavy chain variable region (HVR-H) comprising three CDRs, i.e., hCDR1, hCDR2, and hCDR3. In some embodiments, the antibody construct is chimeric or humanized.

[0078] In some embodiments, immunoconjugates of the invention comprise antibody constructs comprising an antigen-binding domain that is an antibody fragment, for example, but not limited to, an Fv, Fab, Fab', scFv, HcAb fragment, VHH fragment, sdAb fragment, diabody, or F(ab')2 fragment. In some further embodiments, immunoconjugates of the invention comprise multimers of two or more antibody fragments, such as, for example, homodimers or heterodimers comprising two antibody fragments each capable of binding to an antigen with specificity and high affinity, and each comprising a heavy chain variable region (HVR-H) comprising three CDRs, i.e., hCDR1, hCDR2, and hCDR3.

[0079] heavy chain constant region 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 offers advantages such as optimizing and adjusting serum half-life, allowing for the addition of additional sites for conjugating chelating or cytotoxic agents, and purifying the immunoconjugate using standard processes and methods. The addition of a heavy chain constant region also increases size, which may shift the catabolism and elimination of the immunoconjugate from the kidney to the liver. This may provide a safety advantage, particularly for radioimmunoconjugates, since the kidney is more sensitive to radiation than the liver. Changes affecting effector function or serum half-life can be made to residues present in the heavy chain constant region involved in fetal Fc receptor (FcRn) binding. Binding to FcRn generally contributes to an increase in the half-life of immunoglobulin Fc-containing molecules, and therefore, reduced FcRn binding 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 some embodiments, the immunoglobulin constant region comprises or consists of an Fc region. In some embodiments, the immunoglobulin heavy chain constant region comprises an immunoglobulin CH2 domain, an immunoglobulin CH3 domain, or an immunoglobulin CH2 and CH3 domains. In some 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, preventing or reducing endogenous immune responses to the immunoconjugate. In some 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.

[0080] 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 fetal Fc receptors (FcRn). 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 fetal Fc receptors (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 fetal Fc receptors (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 fetal Fc receptors (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 fetal Fc receptors (FcRn).

[0081] Changes to the heavy chain constant region of the immunoconjugate can reduce an effector function associated with the heavy chain constant region, such as 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, the 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 cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or a combination thereof. In certain embodiments, the changes to one or more amino acid residues in the immunoglobulin heavy chain constant region that reduce an effector function are, per EU numbering, (a) 297A, 297Q, 297G, or 297D; (b) 279F, 279K, or 2791; (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, (1) 236F or 236R, (m) 238A, 238E, 238G, 238H, 238i, 238V, 238W, or 238Y, (n) 248A, (o) 254D, 254E, 254G, 254H, 2541, 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, 2671, or 267K, (u) 268K, (v) 269N or 269Q, (w) 270A, 270G, 270M, or 270N, (x) 271T, (y) 272N , (z) 292E, 292F, 292G, or 2921, (aa) 293S, (bb) 301W, (cc) 304E, (dd) 311E, 311G, or 311S, (ee) 316F, (ff) 328V, (gg) 33or, (hh) 339E or 3391, (ii) 3431 or 343V, (jj) 373A, 373G, or 373S, (kk) 376E, 376W, or 376Y, (11) 380D,(mm) 382D or 382P, (nn) 385P, (oo) 424H, 424M, or 424V, (pp) 4341, (qq) 438G, (rr) 439E, 439H, or 439Q, (ss) 440A, 440D, 440E, 440F, 440M, 440T, or 440V, (tt) K322A, (uu) 1235E, (vv) 1234A and 1235A, (ww) 1234A, 1235A, and G237A, (xx) 1234A, 1235A, and P329G, (yy) 1234F, 1235E, and P331S, (zz) 1234A, 1235E, and G237A, (aaa) 1234A, 1235E, G237A, and P331S, (bbb) 12 34A, 1235A, G237A, P238S, H268A, A330S, and P331S, (ccc) 1234A, 1235A, and P329A, (ddd) G236R and 1328R, (eee) G237A, (fff) F241A, (ggg) V264A, (hhh) D265A, (iii) D265A and N297A, (j (jj) D265A and N297G, (kkk) D270A, (111) A3301, (mmm) P331A or P331S, or (nnn) E233P, (ooo) 1234A, 1235E, G237A, A330S, and P331S, or (ppp) any combination of (a) through (ooo). In one embodiment, the changes to one or more amino acid residues that reduce an effector function of an immunoglobulin heavy chain constant region include 1234A, 1235E, G237A, A330S, and P331S, per EU numbering.

[0082] 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 fetal 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 fetal 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, and combinations thereof, per EU numbering. In certain embodiments, the change that alters or reduces binding of the immunoconjugate to fetal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of 253, 254, 310, 435, 436, and combinations thereof, per EU numbering. In certain embodiments, the change that alters or reduces binding of the immunoconjugate to fetal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of 1253A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof, per EU numbering. In certain embodiments, the change that alters or reduces binding of the immunoconjugate to fetal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of 1253A, S254A, H310A, H435Q, Y436A, and combinations thereof, per EU numbering. In certain embodiments, the change that alters or reduces binding of the immunoconjugate to fetal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of 1253A, H310A, H435Q, and combinations thereof, per EU numbering. In certain embodiments, the change that alters or reduces binding of the immunoconjugate to fetal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of H310A, H435Q, and combinations thereof, per EU numbering.

[0083] 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, and the heavy chain constant region comprises a 1253A substitution per EU numbering.

[0084] 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 the same sequence as 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, and the heavy chain constant region comprises a S254A substitution per EU numbering.

[0085] 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 the same sequence as 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, and the heavy chain constant region comprises an H310A substitution per EU numbering.

[0086] 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 the same sequence as 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, and the heavy chain constant region comprises an H435Q substitution per EU numbering.

[0087] 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 the same sequence as 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, and the heavy chain constant region comprises a Y436A substitution per EU numbering.

[0088] 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 per EU numbering.

[0089] 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 1234A, 1235E, G237A, A330S, and P331S substitutions per EU numbering.

[0090] 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 the following substitutions per EU numbering: a1234A, 1235E, G237A, H310A, A330S, and P331S.

[0091] 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 the same sequence as SEQ ID NO: 9, wherein the heavy chain constant region comprises the following substitutions per EU numbering: 1234A, 1235E, G237A, H435Q, A330S, and P331S.

[0092] 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 the same sequence as SEQ ID NO: 10 per EU numbering.

[0093] 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.

[0094] In one embodiment, at least one of the variant constant regions in the immunoconjugate has at least one FcRn-binding mutation. In a preferred embodiment, each of the two variant constant regions of the immunoconjugate has at least one FcRn-binding mutation, and the FcRn-binding mutations are the same or different.

[0095] 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, hi certain embodiments, the immunoconjugate has a serum half-life of about 12 hours to about 120 hours. In certain embodiments, the immunoconjugate is incubated 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, or 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 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. The immunoconjugate has a serum half-life of about 72 to about 120 hours, about 84 to about 96 hours, about 84 to about 108 hours, about 84 to about 120 hours, about 96 to about 108 hours, about 96 to about 120 hours, or about 108 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 up to 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.

[0096] 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 9 days, about 3 to about 10 days, about 4 to about 5 days, about 4 to about 6 days, about 4 to about 7 days, or about 4 to about 8 days. The immunoconjugate may have a serum half-life of about 4 to about 9 days, about 4 to about 10 days, about 5 to about 6 days, about 5 to about 7 days, about 5 to about 8 days, about 5 to about 9 days, about 5 to about 10 days, about 6 to about 7 days, about 6 to about 8 days, about 6 to about 9 days. The immunoconjugate may have a serum half-life of about 6 to about 10 days, about 7 to about 8 days, about 7 to about 9 days, about 7 to about 10 days, about 8 to about 9 days, about 8 to about 10 days, or 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 up to 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.

[0097] In some embodiments, the heavy chain constant region has a molecular weight of about 10 kDa to about 25 kDa. In some 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 some 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 some embodiments, the heavy chain constant region has a molecular weight of at least about 10 kDa, about 15 kDa, or about 20 kDa. In some embodiments, the heavy chain constant region has a molecular weight of at most about 15 kDa, about 20 kDa, or about 25 kDa.

[0098] In some embodiments, the immunoconjugate of the present invention comprises a linker or hinge region, which is a polypeptide that connects the antigen-binding region to the heavy chain constant region or mutant constant region of the present invention. Natural and synthetic hinge regions that connect immunoglobulin components are well known in the art and can be used in the present invention. See, for example, US 8,067,548 and its references.

[0099] In one embodiment, the hinge regions of the immunoconjugates are the same. In one embodiment, the hinge regions of the immunoconjugates are different.

[0100] The antigen-binding region and the heavy chain constant region (with or without the altered amino acid sequence) can be linked by a suitable hinge or linker sequence. In some 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 include or comprise an IgG1 or IgG4 hinge region. In some embodiments, the hinge region is an IgG1 hinge region. In some embodiments, the hinge region is an IgG1 hinge region with a C220S substitution according to EU numbering. Suitable hinge regions include Wu et al., “Mu1timerization of a chimeric anti-CD20 sing1e-chain Fv-Fc fusion protein is mediated through variab1e domain exchange,” Protein Engineering, Design and Se1ection, Vo1ume 14, Issue 12, December 2001, Pages 1025-1033; Shu et al. a1,“Secretion of a sing1e-gene-encoded immunog1obu1in from mye1oma ce11s.”Proceedings of the Nationa1 Academy of Sciences Sep 1993,90(17)7995-7999;Davis et a1.,“Abatacept binds to the Fc receptor CD64 but does not mediate comp1ement-dependent cytotoxicity or antibody-dependent ce11u1ar cytotoxicity.”J Rheumatol. 2007 Nov;34(11):2204-10. Suitable hinges may also include non-IgG-based polypeptide linkers. The linker amino acid sequence may primarily include amino acid residues such as Gly, Ser, Ala, or Thr.The linker peptide should be of an appropriate length to link the two molecules in such a way that they assume the correct conformation relative to each other 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 can include AAEPKSS, AAEPKSS, GGGG, or GGGGDKTHTCPPCP. Alternatively, various non-proteinaceous 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.

[0101] 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 a to about 70 kDa, about 65 kDa to about 75 kDa, about 65 kDa to about 80 kDa, about 65 kDa to about 90 kDa, about 65 kDa to about 100 kDa, about 65 kDa to about 110 kDa, about 65 kDa to about 120 kDa, about 70 kDa to about 75 kDa, about 70 kDa to about 80 kDa, about 70 kDa to about 90 kDa, about 70 kDa to about 100 kDa, about 70 kDa to about 110 kDa, about 70 kDa to about 120 kDa, about 75 kDa to about 80 kDa, about 75 kDa to about 90 kDa, about 75 kDa to about 100 kDa, about 75 kDa to about 110 kDa, about 75 kDa to about 120 kDa, about 80 kDa to about 90 kDa, about 80 kDa to about The immunoconjugate has a molecular weight of about 100 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 about 60 kDa, 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. 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.

[0102] 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.

[0103] 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 more radioresistant organs such as the target tissue (i.e., tumor or cancerous tissue) or the liver.

[0104] 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 one embodiment, the kidneys are exposed to 5 Gy or less per treatment.

[0105] In some embodiments, the immunoconjugates of the present disclosure provide a total radiation exposure per treatment measured in Gray (Gy). In some embodiments, bone marrow is exposed to 4 Gy or less per treatment. In some embodiments, bone marrow is exposed to 3 Gy or less per treatment. In some embodiments, bone marrow is exposed to 2 Gy or less per treatment. In some embodiments, bone marrow is exposed to 1.5 Gy or less per treatment. In some embodiments, bone marrow is exposed to 1.0 Gy or less per treatment. In some embodiments, bone marrow is exposed to 0.5 Gy or less per treatment.

[0106] 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.

[0107] In certain embodiments, the immunoconjugates of the present disclosure provide an increased radiation dose in the tumor compared to the blood when measured as the 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.

[0108] In certain embodiments, the immunoconjugates of the present disclosure provide an increased radiation dose in the tumor compared 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.

[0109] In certain embodiments, the 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.

[0110] In some embodiments, the present invention contemplates variants of the immunoconjugates of the present invention comprising an Fc region that retain some, but not all, effector functions, making them desirable candidates for applications in which in vivo half-life of the immunoconjugate is important but 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 FcγγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγγRIII, whereas monocytes express FcγγRI, FcγγRII, 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 to assess 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 for Flow Cytometry (Cell Technology, Inc., Mountain View, CA); and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) 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 C1ynes et al. Proc Natl Acad Sci USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the immunoconjugate is unable to bind C1q and thus lacks CDC activity (see, e.g., C1q- and C3c-binding E1ISA in WO2006 / 029879 and WO2005 / 100402). To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., B1ood 101:1045-1052 (2003); Cragg, MS and MJG1ennie, B1ood 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)).

[0111] Immunoconjugates with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (US 7,332,581).

[0112] Immunoconjugates may have altered effector function by including the following changes per EU numbering: 1234A, 1235E, G237A, A330S, and P331S, which reduce Fc receptor binding. See, e.g., US 8,613,926 or Andersson C, Wenander et al., "Rapid-onset c5aR and mechanistic effects of anti-C5aR treatment in the mouse coagulation-induced arthritis mode." C1in Exp Immunol. 2014 Ju1;177(1):219-33.

[0113] Certain immunoconjugate variants have been described with improved or diminished binding to FcRs (see, e.g., US Pat. No. 6,737,056; WO 2004 / 056312; Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0114] In some embodiments, changes are made to the Fc region that result in altered (i.e., improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in US 6,194,551; WO 1999 / 051642; Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0115] Antibodies with increased half-life and improved binding to fetal Fc receptors (FcRn), which are involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976); Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934. These antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more Fc region residues, i.e., 434 or 435, e.g., Fc region residues N434A or R435A (US 7,371,826). See also Duncan and Winter, Nature 322:738-40 (1988); US Pat. No. 5,648,260; US Pat. No. 5,624,821; and WO 1994 / 029351 for other examples of Fc region variants.

[0116] To increase the serum half-life of an antibody, a salvage receptor binding epitope can be incorporated into the antibody (e.g., antibody fragment), as described, for example, in U.S. Patent No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

[0117] As will be appreciated by those skilled in the art, certain teachings herein apply to the antibody constructs, targeted imaging complexes, immunoconjugates, and radioimmunoconjugates of the invention, although reference will be made herein to only one or two such compositions (e.g., immunoconjugates) as non-limiting examples. All such uses are encompassed by the present invention.

[0118] 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.

[0119] In one embodiment, the chelator of the immunoconjugate is covalently linked to the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof. In one embodiment, the chelator is directly covalently linked to the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof (e.g., without a spacer, stretcher, or linker). In one embodiment, the chelator is covalently linked to the antigen-binding arm via a linker that is covalently linked to the chelator and covalently linked 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.

[0120] In some embodiments, immunoconjugates are formed by site-specific binding of chelator-linkers to specific amino acid or glycan residues. In some embodiments, site-specific conjugation involves the directed functionalization of specific lysine residues in framework regions with chelator-linkers. In other embodiments, these residues can be functionalized with a different reactive functional group, which then reacts with the chelator-linker in a second step to provide the immunoconjugate. In some embodiments, the reactive functional group is thiopropionate.

[0121] 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 as secondary reactive groups to which chelator-linkers are conjugated to provide immunoconjugates.

[0122] 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.

[0123] In some embodiments, the chelator-linker is attached to a 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 yield an immunoconjugate.

[0124] In some embodiments, the chelator-linker is attached by modifying one or more N-glycans with reactive functional groups by the action of glycosidase, followed by conjugation of the chelator-linker to that 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.

[0125] In one embodiment, the immunoconjugate comprises multiple chelating agents, which may be the same or different.

[0126] In one embodiment, an immunoconjugate having multiple chelators has multiple chelators attached to the same antigen-binding arm.

[0127] In one embodiment, the immunoconjugates having more than 1 chelator and more than 11 chelators have 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.

[0128] In one embodiment, the chelating agent 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 chelating agent further comprises a linker between the functional group and the radioisotope chelating moiety.

[0129] 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.

[0130] In preferred embodiments, the chelator of the immunoconjugate does not bind to the antigen-binding region in the antigen-binding arm of the immunoconjugate.

[0131] In one embodiment, the chelator of the immunoconjugate is non-covalently associated with the antigen-binding arm. In a preferred embodiment, the chelator is not associated with the antigen-binding region in the antigen-binding arm of the immunoconjugate.

[0132] 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.

[0133] In some embodiments, described herein is an immunoconjugate conjugated to a chelator. In some embodiments, the chelator is a radioisotope chelator. In some embodiments, the radioisotope chelator 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 some embodiments, the radioisotope chelator is DOTA. In some embodiments, the radioisotope chelator is DOTAGA. In some embodiments, the radioisotope chelator is Py4Pa. In some embodiments, the radioisotope is a radioisotope, wherein the radioisotope chelator is directly conjugated to the antigen-binding region and / or the immunoglobulin heavy chain constant region. In some embodiments, the radioisotope chelator is conjugated to the antigen-binding region or the immunoglobulin heavy chain constant region via a linker. In certain embodiments, the linker is selected from 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (va1-cit), alanine-phenylalanine (a1a-phe), p-aminobenzyloxycarbonyl (PAB), and those resulting from conjugation with a linker reagent: 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 some embodiments, the linker is selected from polyethylene glycol (PEG), polyethylene glycol polymer (PEG), and S-2-(4-isothiocyanatobenzyl) (SCN). In some embodiments, the linker is PEG5. In some embodiments, the linker is SCN.In certain embodiments, the radioisotope chelator is a linker-chelator selected from the list consisting of TFP-Ad-PEG5-DOT AGA, p-SCN-Bn-DOTA, p-SCN-Ph-Et-Py4Pa, and TFP-Ad-PEG5-Ac-Py4Pa.

[0134] 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 some 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 some 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.

[0135] In some embodiments, immunoconjugates of the invention comprise a linker for linking, for example, the antigen-binding arm to a chelating agent (interchangeably referred to as a "chelator") or radioisotope or cargo (e.g., a cytotoxin). The linker may comprise one or more linker moieties. In some embodiments, immunoconjugates of the invention are engineered to have a terminal lysine available for conjugation to a chelator or linker.

[0136] For example, a bifunctional chelator can be used to conjugate a radioisotope to the radioisotope delivery platform of the present invention to produce an immunoconjugate of the present invention (see, for example, 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.

[0137] 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 foregoing. Such chelating agents are suitable for coordinating metal ions such as α and β radionuclides.

[0138] In some embodiments, the chelator of the immunoconjugate or radioimmunoconjugate of the invention 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 1, et al. Bioconjugate Chem. ASAP (2020)), 3,2-HOPO (Wickstroem K, et al. a1. Int, J. Rad. Onc. 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 Brechbie1 MW Q, J. Nuc1. Med. Mol. Imaging 52:166 (2008)).

[0139] In some embodiments, the chelator of the immunoconjugate or radioimmunoconjugate of the invention 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 (Thie NA, et al. Angew. Chem. Int. Ed. 56:1 (2017)), crown (Yang H, et al. Chem. J. 26:11435 (2020)), p-SCN-Ph-Et-Py4Pa (Ill, et al. Bioconjugate Chem. ASAP (2020)), 3,2-HOPO (Wickstroem K, etal. Int, J. Rad. Onc. Biol. Phys. 105:410 (2019).) (for reviews of these and other bifunctional chelators, see, e.g., Price EW and Orvig C Chem. Soc. Rev., 2014, 43:260 (2014) and Brechbie1 MW Q, J. Nuc1. Med. Mol. Imaging 52:166 (2008)).

[0140] 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); Chappel I, et al., Bioconjug Chem 11:510 (2000); Chappel I, 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)).

[0141] In some embodiments, the chelating agent is a chelating agent suitable for chelation of α-emitters. Some chelating agents 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.

[0142] 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 alpha(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-,1,4,7,10-tetramethyl 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-carboxypyridine) (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; CHXoctapa 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; ...6,6'-(((oxybis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid; and combinations thereof.

[0143] In certain embodiments, the chelating agent is a chelating agent suitable for chelation of beta or gamma emitters. 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""-pentaacetic acid; H4OctapaN,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid; H2Dedpa1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane; H6phospaN,N'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane; TTHAtriethylenetetramine-N,N,N',N'',N"',N""-hexaacetic acid; DO2PTetraazacyclododecanedimethanephosphonic acid;HP-DO3A Hydroxypropyltetraazacyclododecanetriacetic acid; EDTA Ethylenediaminetetraacetic acid; DTPA Diethylenetriaminepentaacetic acid; DTPA-BMA Diethylenetriaminepentaacetic acid-bismethylamide; HOPO Octadentate Hydroxypyridinone; 3,2,3-1I(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-carboxamide); 3, 2-HOPON,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((carboxamide) Dimethyl)azanediyl))bis(methylene))dipicolinic acid;Neunpa-NCS=6,6'-((((4-isothiocyanatophenethyl)azanediyl)bis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid;Octapa6,6'-((ethane-1,2-diylbis((carboxymethyl)(azanediyl))bis(methylene))dipicolinic acid;Octox2,2'-(ethane-1,2-diylbis(((8-hydroxyquinolin-2-yl)methyl)(azanediyl))diacetic acid;P yPa6,6'-(((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.

[0144] Alternatively, or in addition, an isothiocyanate linker containing a lysine residue, such as p-SCN-Bn-DOTA, may be used in the immunoconjugates of the invention.

[0145] Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("va1-cit" or "vc"), alanine-phenylalanine ("a1a-phe"), p-aminobenzyloxycarbonyl ("PAB"), and those resulting from conjugation with a linker reagent: Examples include the N-succinimidyl 4-(2-pyridylthio)pentanoate-forming linker moiety 4-mercaptopentanoic acid ("SPP"), the N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylic acid-forming linker moiety 4-((2,5-dioxopyrrolidin-1-yl)methyl)cyclohexanecarboxylic acid ("SMCC" also referred to herein as "MCC"), the 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfanyl)butanoate-forming linker moiety 4-mercaptobutanoic acid ("SPDB"), N-succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB"), ethyleneoxy-CHCHO as one or more repeating units ("EO," "PEO," or "PEG"), and the like. Additional linker components are known in the art, and some are described herein. A variety of linker moieties are known in the art, some of which are described below.

[0146] In some embodiments, the linker is SCN. In some 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 some embodiments, the chelator is TFP-Ad-PEG5-DOTAGA. In some embodiments, the chelator is p-SCN-Bn-DOTA. In some embodiments, the chelator is p-SCN-Ph-Et-Py4Pa. In some embodiments, the chelator is TFP-Ad-PEG5-Ac-Py4Pa. Such linkers are shown in Figure 18.

[0147] The linker may be a "cleavable linker" that facilitates the release of the drug 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); U.S. Patent No. 5,208,020) may be used.

[0148] In certain embodiments, the linker is as shown in the following formula (Formula I):

[0149] [ka]

[0150] wherein A is a Stretcher unit, a is an integer from 0 to 1, W is an amino acid unit and w is an integer from 0 to 12, Y is a Spacer unit and y is 0, 1 or 2, and Ab, D, and p are defined above for Formula I. Exemplary embodiments of such multiple linkers are described in US20050238649.

[0151] In some embodiments, a linker component may comprise a "stretcher unit" that links the immunoconjugate to another linker component or a drug moiety. An exemplary stretcher unit is shown below (where the wavy line indicates the site of covalent attachment to the immunoconjugate):

[0152] [ka]

[0153] In some embodiments, the linker can be conjugated to the antibody via a cysteine ​​bridging functional group, such as ThioBridge® or DBM (dibromomaleimide). These linkers can act to re-stabilize 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)). An exemplary re-bridging stretcher element is shown below (where the wavy line indicates the covalent attachment site to the immunoconjugate):

[0154] [ka]

[0155] 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.

[0156] 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 the 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.

[0157] A "self-immolative" spacer unit allows for release of the drug moiety without a separate hydrolysis step. In some 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 created 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 some 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. Further examples of self-immolative spacer units 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 amide (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.

[0158] 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.

[0159] [ka] wherein Q is a drug that 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.

[0160] In some embodiments, the immunoconjugate comprises a linker, such as a dendritic linker, for covalent attachment of multiple drug moieties to an antibody 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, if a cysteine-engineered antibody has only one reactive cysteine ​​thiol group, multiple drug moieties can be attached via the dendritic linker.

[0161] Examples of linker moieties and combinations thereof are shown below, which are also suitable for use in the above formula.

[0162] [ka]

[0163] Further non-limiting examples of linkers include those described in WO2015095953.

[0164] Linker components, including stretcher, spacer, and amino acid units, can be synthesized by methods known in the art, such as those described in US20050238649.f. Variations of the immunoconjugates of the present invention.

[0165] Radioimmunoconjugates In one embodiment, the present invention 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.

[0166] The immunoconjugates of the present disclosure can be loaded with a radioisotope for therapeutic or diagnostic effects. In some embodiments, the chelating agent can further comprise a radioisotope. In some embodiments, the radioisotope is an alpha emitter. In some 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 some embodiments, the radioisotope is 225-Ac. In some embodiments, the radioisotope is a beta emitter. In some embodiments, the radioisotope is a beta emitter selected from 177-Lu, 90-Y, 67-Cu, and 153-Sm.

[0167] Also described herein is a method for producing a radioimmunoconjugate, comprising loading or complexing the immunoconjugate of the present disclosure with a radioisotope. In some embodiments, the radioisotope is an α-emitter. In some 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 some embodiments, the radioisotope is 225-Ac. In some embodiments, the radioisotope is a β-emitter. In some embodiments, the radioisotope is a β-emitter selected from 177-Lu, 90-Y, 67-Cu, and 153-Sm.

[0168] In one aspect, the present invention provides a radioimmunoconjugate comprising an immunoconjugate of the present invention and an α-emitting isotope. In one embodiment, the α-emitting isotope of the radioimmunoconjugate is 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 α-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.

[0169] In some embodiments, the immunoconjugates of the present invention are combined with a radioisotope to provide radioimmunoconjugates of the present invention. 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, or 153-Sm. 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, or thus comprises, both an alpha emitter and a beta emitter. In some embodiments, the radioisotope is selected for use in imaging with radioactive materials, such as, for example, 68-Ga, 64-Cu, 89-Zr, 111-In, or 134-Ce.

[0170] The immunoconjugates and radioimmunoconjugates of the invention may contain other cargoes or payloads, in addition to radioisotopes, including, for example, various cytotoxic agents such as small molecule chemotherapeutic agents, cytotoxic antibiotics, alkylating agents, antimetabolites, topoisomerase inhibitors, and / or tubulin inhibitors. For example, the immunoconjugates of the invention may be used to deliver non-radioisotopic cytotoxins to target cells. 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.

[0171] In some embodiments, the radioimmunoconjugates of the present invention 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.

[0172] In some embodiments, the radioimmunoconjugates of the present invention 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.

[0173] 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.

[0174] 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.

[0175] Further embodiments of immunoconjugates, antigen-binding regions, and heavy chain variable regions are described below.

[0176] In some embodiments, the immunoconjugate comprises a dimerization domain or motif. In some further embodiments, the dimerization domain or motif is in a variant constant region, linker, or hinge region.

[0177] Those skilled in the art can engineer multimeric immunoconjugates of the invention using approaches and methods known in the art, for example, engineered cysteine ​​residues can form covalent bonds, thereby stabilizing spontaneously assembling multimeric structures (see, e.g., Glockshuber R et al., Biochemistry 29:1362-7 (1990)). For example, introduction of cysteine ​​residues at specific positions can be achieved by, for example, the following amino acid residues: GGGGC and SGGGGC (Tai M et al., Biochemistry 29:8024-30 (1990); Caron P et al., J Exp Med 176:1191-5 (1992); Shopes B, J Immunol 148:2918-22 (1992); Adams G et al., Cancer Res 53:4026-34 (1993); McCartney J et al., Protein Eng 18:301-14 (1994); Perisic O et al., Structure 2:1217-26 (1994); George A et al., Proc Natl Acad Sci USA 92:8358-62 (1995); Tai M et al., Cancer Res(Suppl)55:5983-9(1995); Olafsen T et al., Protein Eng Des Sel 17: 21-7(2004)), can be used to create disulfide-stabilized structures such as Cys-diabodies, scFv' multimers, VHH multimers, VNAR multimers, and IgNAR multimers.

[0178] Alternatively, two or more polypeptide chains can be linked using polypeptide domains that self-associate or multimerize with each other (see, e.g., US 6,329,507). For example, the addition of a carboxy-terminal multimerization domain has been used to construct multivalent proteins containing immunoglobulin domains, such as scFvs, autonomous VH domains, VHHSs, VNARSs, and IgNARs. Examples of self-association domains known to those skilled in the art include immunoglobulin constant domains (e.g., knob-and-hole, electrostatic steering, and IgG / IgA chain exchange), immunoglobulin Fab chains (e.g., (Fab-scFv)2 and (Fab'scFv)2), immunoglobulin Fc domains (e.g., (scdiabody-Fc)2, (scFv-Fc)2, and scFv-Fc-scFv), immunoglobulin CHX domains, immunoglobulin CH1-3 regions, immunoglobulin CH3 domains (e.g., (scdiabody-CH3)2, LD minibodies, and Flex-minibodies), immunoglobulin CH4 domains, CHCL domains, amphipathic These include a helix bundle (e.g., scFv-HLX), a helix-turn-helix domain (e.g., scFv-dHlx), a coiled-coil structure containing leucine zipper and cartilage oligomeric matrix protein (e.g., scZIP), a cAMP-dependent protein kinase (PKA) dimerization and docking domain (DDD) in combination with an A-kinase anchor protein (AKAP) anchor domain (AD) (also referred to as "dock and lock" or "DNL"), streptavidin, a verotoxin B multimerization domain, a tetramerization region derived from p53, and a barnase-barstar interaction domain (Pack P,Pluckthun A,Biochemistry 31:1579-84(1992);Holliger P et al.,Proc Natl Acad Sci USA 90:6444-8(1993);Kipriyanov S et al.,Hum Antibodies Hybridomas 6:93-101(1995);de Kruif J,Logtenberg T,J Biol Chem 271:7630-4(1996);Hu S et al.,Cancer Res 56:3055-61(1996);Kipriyanov S et al.,Protein Eng 9:203-11(1996);Rheinnecker M et al.,J Immunol 157:2989-97(1996);Tershkikh A et al.,Proc Natl Acad Sci USA 94:1663-8(1997); Muller K et al., FEBS Lett 422:259-64(1998); Cloutier S et al., Mol Immunol 37:1067-77(2000); Li S et al., Cancer Immunol Immunother 49:243-52(2000); Schmiedl A et al., Protein Eng 13:725-34(2000);Schoonjans R et al.,J Immunol 165:7050-7(2000);Borsi L et al.,Int,J Cancer 102:75-85(2002);Deyev S et al.,Nat Biotechnol 21:1486-92(2003);Wong W,Scott,J,Nat Rev Mol Cell Biol 5:959-70(2004);Zhang J et al.,J Mol Biol 335:49-56(2004);Baillie G et al.,FEBS Letters 579:3264-70(2005);Rossi E et al.,Proc Natl Acad Sci USA 103:6841-6(2006);Simmons D et al.,J Immunol Methods 315:171-84 (2006); Braren I et al., Biotechnol Appl Biochem 47:205-14 (2007); Expr Purif 65:77-82(2009); Bell A et al.,Cancer Lett 289:81-90(2010);Iqbal U et al.,Br J Pharmacol 160:1016-28(2010);Asano R et al.,FEBS J 280:4816-26(2013);Gil D,Schrum A,Adv Biosci Biotechnol 4:73-84 (2013)). .

[0179] Those skilled in the art can use various scFv-based polypeptide interactions known in the art to engineer the multimeric immunoconjugates of the present invention, such as scFv-based dimeric, trimeric, and tetrameric complexes. For example, the length of the linker in the scFv can affect the spontaneous assembly of non-covalently based multimeric multivalent structures. Generally, a linker of 12 amino acids or less, including the absence of any linker, favors intermolecular domain swapping over intrachain domain pairing (see, for example, Dolezal O et al., Protein Eng 16: 47-56 (2003)), thereby promoting the multimerization of scFv-containing polypeptides or proteins into high molecular weight species. However, scFvs without any linker or with a linker having an exemplary length of 15 amino acid residues can multimerize (Whitlow M et al., Protein Eng 6:989-95(1993); Desplancq D et al., Protein Eng 1:1027-33(1994); Whitlow M et al., Protein Eng 7,1017-26(1994); Alfthan K et al., Protein Eng 8:725-31(1995)). Those skilled in the art can identify the multimeric structures produced and / or purified using techniques known in the art and / or described herein.

[0180] In some embodiments, amino acid sequence variants of the immunoconjugates described herein are contemplated. For example, it may be desirable to improve the binding affinity, stability, and / or other biological properties of the immunoconjugates of the present invention (e.g., altering the half-life or therapeutic window, reducing immunogenicity, or increasing ease of manufacture). Amino acid sequence variants of the immunoconjugates can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the immunoconjugate or by synthesis of the desired immunoconjugate or polypeptide. Such modifications include, for example, fusion of immunoglobulin domains or polypeptide sequences, i.e., substitution of hinge, linker, and / or chelator moieties; or substitution of radioisotopes. Such modifications include, for example, deletion and / or insertion and / or substitution of residues within the amino acid sequence of the immunoconjugate. Any combination of fusion, deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct has the desired characteristics, e.g., a specific binding affinity level for antigen binding, a specific level of KD, and / or a specific level of Koff.

[0181] Antigen-binding antibody fragments and sets of CDRs are provided herein.Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with full-length natural antibodies (e.g., full-length camelid VHH IgG2 or IgG3).Certain fragments may lack amino acid residues or domains that are not essential for the desired biological activity of the antibody or to reduce the overall size of the immunoconjugate of the present invention.

[0182] In some embodiments, variants of the immunoconjugates of the present invention are made larger by incorporating additional structures. In some embodiments, the immunoconjugate is linked to a heterologous or easily detectable moiety. In some further embodiments, the linkage comprises a proteinaceous fusion. In some further embodiments, the heterologous moiety is a cytotoxic agent. In some embodiments, a carboxy-terminal lysine residue is added to provide a site-specific binding site. Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion includes an immunoconjugate with an N-terminal methionyl residue. Other insertional variants of immunoconjugate molecules include fusions to the N- or C-terminus of the immunoconjugate to enzymes (e.g., for ADEPT) or polypeptides that increase the serum half-life of the immunoconjugate.

[0183] Nucleic acids encoding the immunoconjugates of the invention can be modified to produce chimeric or fusion immunoconjugate polypeptides, for example, by substituting human heavy and light chain constant domains (CH and CO sequences) for homologous murine sequences (U.S. Pat. No. 4,816,567, and Morrison, et al., Proc Natl Acad Sci USA 81:6851 (1984)). Alternatively, by fusing the immunoglobulin coding sequence with all or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide). Non-immunoglobulin polypeptide sequences can replace the constant domains of the immunoconjugate, or they can replace the variable domains of one antigen-binding site of the immunoconjugate to create a chimeric bivalent immunoconjugate containing one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

[0184] Mutations in the antibody constructs used as antigen-binding domains in the invention described herein can be made using, for example, any of the techniques and guidelines for conservative and non-conservative mutations described in U.S. Patent No. 5,364,934. Mutations can be substitutions, deletions, or insertions of one or more codons encoding the immunoconjugate or polypeptide, resulting in a change in the amino acid sequence compared to the native sequence antibody or polypeptide. Optionally, the mutations involve the substitution of at least one amino acid in one or more of the domains of the immunoconjugate with any other amino acid. Guidance for determining which amino acid residues can be inserted, substituted, or deleted without adversely affecting the desired activity can be found by comparing the sequence of the immunoconjugate with that of a homologous known protein molecule and minimizing the number of amino acid sequence changes made in areas of high homology. Amino acid substitutions can result from substituting one amino acid for another with similar structural and / or chemical properties, e.g., substituting serine for leucine, i.e., conservative amino acid substitutions. Insertions or deletions can optionally range from about 1 to 5 amino acids. Tolerable variations can be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for the activity exhibited by the full-length or mature native sequence.

[0185] In certain embodiments, conservative substitutions of interest are set forth in Tables B and C, including under the heading of preferred substitutions. If such a substitution results in a change in biological activity, it is designated as an exemplary substitution in Table C, or a more substantial change is introduced and the output screened, as further described below with respect to amino acid classes.

[0186] [Table 1]

[0187] Substantial changes in the function or immunological identity of the immunoconjugates of the invention are achieved by selecting substitutions that differ significantly in their effect on (a) the structure of the polypeptide backbone, e.g., sheet or helical structure, in the region of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chain. Naturally occurring residues are divided into groups based on common side chain properties. (1) Hydrophobic: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophilic: Cys, ser, thr; (3) Acidic: asp, glu; (4) Basic: asn, gin, his, lys, arg; (5) residues that influence chain orientation: Gly, pro; and (6) Aromatics: trp, tyr, phe.

[0188] Non-conservative substitutions involve exchanging a member of one of these classes for another. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.

[0189] Mutations can be made using methods known in the art, such as, for example, oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13: 4331 (1986); Zoller et al., Nucl. Acids Res., 10: 6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34: 315 (1985)), restriction-selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317: 415 (1986)), or other known methods can be performed on cloned DNA to produce DNA molecules encoding the immunoconjugate variants of the present invention.

[0190] In some embodiments, immunoconjugate variants with one or more amino acid substitutions are provided. Targeted sites for substitutional mutagenesis include the HVRs and FRs of immunoglobulin variable domains and within immunoglobulin constant domains. Amino acid substitutions can be introduced into a desired immunoconjugate, and the product can be screened for a desired activity, such as improved / retained antigen binding, reduced / retained immunogenicity, improved / retained antibody-dependent cellular cytotoxicity (ADCC), improved / retained complement-dependent cytotoxicity (CDC), improved / retained target inhibition, and / or improved / retained antibody-dependent cell-mediated phagocytosis (ADCP). Similarly, amino acid substitutions can be introduced into a desired immunoconjugate, and the product can be screened for activity, such as reduced or eliminated ADCC, CDC, target inhibition, and / or ADCP.

[0191] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and / or substantially retain certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently produced using, for example, phage-display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0192] Alterations (e.g., substitutions) can be made in HVRs, for example, to improve immunoconjugate affinity. Such alterations can be made in HVR "hotspots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in SDRs (a-CDRs), and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The secondary library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (for example, 4 to 6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0193] In some embodiments, substitutions, insertions, or deletions can occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the immunoconjugate to bind to the antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity can be made in HVRs. Such changes can be outside of HVR "hot spots" or SDRs. In some embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0194] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution.

[0195] Alternatively, or in addition, a crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact and adjacent residues can be targeted or eliminated as candidates for substitution. Mutants can be screened to determine whether they contain the desired properties.

[0196] In some embodiments, immunoconjugates of the invention comprise antibody constructs comprising humanized immunoglobulin domains (used herein as antigen-binding regions).

[0197] Humanized forms of non-human (e.g., camelid, mouse, or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., 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 cases, 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, humanized antibodies 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 of human immunoglobulin consensus sequences. A humanized antibody can 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); Presta, Curr. Op. Struct. Biol., 2:593-6 (1992)).

[0198] Methods for humanizing non-human antibodies are well known in the art. Generally, humanized antibodies have one or more amino acid residues introduced into them 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 essentially be performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody according to the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988)). Such "humanized" antibodies are thus 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.

[0199] 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). This 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 changes in the acceptor framework sequence.

[0200] Any cysteine ​​residue not involved in maintaining the proper conformation of the immunoconjugates of the invention can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bond(s) can be added to an immunoconjugate of the invention to improve its stability, particularly where the antibody is an antibody fragment such as an Fv or VHH fragment.

[0201] In some embodiments, it may be desirable to generate 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 located at accessible sites of the immunoconjugate and can be used to conjugate the immunoconjugate to 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, all eight of the heavy chain (EU numbering), and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies can be produced, for example, as described in US Pat. No. 7,521,541.

[0202] One skilled in the art will appreciate that amino acid changes may alter post-translational processes of the immunoconjugate, such as changing the number or position of glycosylation sites or altering membrane anchoring properties.

[0203] 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. For purposes herein, "altering the native glycosylation pattern" is intended to mean the deletion of one or more carbohydrate moieties found in the parent immunoconjugate of the invention (either by removing the underlying glycosylation site or by deleting glycosylation by chemical and / or enzymatic means), and / or the addition of one or more glycosylation sites not present in the native sequence immunoconjugate of the invention. Furthermore, this phrase includes qualitative changes in the glycosylation of the native protein, involving a change in the nature and proportion of the various carbohydrate moieties present.

[0204] Glycosylation of antibodies and other polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0205] Addition or deletion of glycosylation sites to an immunoconjugate can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites. Addition of glycosylation sites to an immunoconjugate of the present invention is conveniently achieved by altering the amino acid sequence to include one or more of the above tripeptide sequences (for N-linked glycosylation sites). Alterations can also be made by adding or substituting one or more serine or threonine residues to the sequence of the original immunoconjugate of the present invention (for O-linked glycosylation sites). The immunoconjugates of the present invention can be optionally altered through changes at the DNA level, particularly by mutating the DNA encoding the immunoconjugate of the present invention at preselected bases to produce codons that translate into the desired amino acids.

[0206] If the immunoconjugate contains an Fc region, the carbohydrate attached thereto can be varied. Natural antibodies produced by mammalian cells typically contain biantennary oligosaccharides, 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 oligosaccharides 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 oligosaccharides in the immunoconjugates of the present invention can be made to generate immunoconjugate variants with specific improved properties.

[0207] Another means of increasing the number of carbohydrate moieties on the immunoconjugates of the invention is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, for example, in WO 87 / 05330, published September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0208] Removal of carbohydrate moieties present on the immunoconjugates of the present invention can be accomplished chemically or enzymatically, or by mutational substitution of codons encoding amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and are described, for example, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of various endo- and exo-glycosidases, as described by Enzymok, 138:350 (1987).

[0209] In some embodiments, immunoconjugate variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such immunoconjugates can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (Eu numbering of Fc region residues). However, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylated variants may have improved ADCC function (see, e.g., US2003 / 0157108; US2004 / 0093621). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include the following: US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 01 15614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 01 10704; US2004 / 01 10282; US2004 / 0109865; WO2003 / 0851 19; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031 140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lecl3 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US2003 / 0157108; WO2004 / 056312, Adams et al., see especially Example 11), and knockout cell lines, such as α1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); WO2003 / 085107).

[0210] Further provided are immunoconjugate variants having bisected oligosaccharides, in which the biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such immunoconjugate variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878; US6,602,684; US2005 / 0123546. Also provided are immunoconjugate variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such immunoconjugate variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 030087; WO1998 / 058964; and WO1999 / 022764.

[0211] Immunoconjugate Derivatives and Other Modifications Covalent modification of the immunoconjugates of the present invention is included within the scope of the present invention. One type of covalent modification involves reacting targeted amino acid residues of the immunoconjugates of the present invention with an organic derivatizing agent that can react with selected side chains or N- or C-terminal residues of the immunoconjugate. Derivatization with bifunctional agents is useful, for example, for crosslinking the immunoconjugates of the present invention to a water-insoluble support matrix or surface for use in methods for purifying the immunoconjugates of the present invention, and vice versa. Commonly used cross-linking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, such as esters with 4-azidosalicylic acid, homobifunctional imidoesters including disuccinimidyl esters (e.g., 3,3'-dithiobis(succinimidyl propionate)), bifunctional maleimides (e.g., bis-N-maleimido-1,8-octane), and drugs (e.g., methyl-3-[(p-azidophenyl)dithio]propioimidate).

[0212] 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.

[0213] 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 derivatizing 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, prolyl 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 may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the immunoconjugate may vary, and when multiple polymers are attached, they may be the same or different molecules. In general, the number and / or type of polymers used for derivatization may 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.

[0214] PEG-derivatized immunoconjugates of the present invention can include 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 to a chelator 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.

[0215] Methods for producing immunoconjugates of the present invention The present invention provides compositions comprising one or more of the above embodiments or immunoconjugates described herein. In another aspect, the present invention 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 invention, expression vectors comprising the aforementioned nucleic acids, and host cells comprising the aforementioned expression vectors.

[0216] In another aspect, the present invention provides host cells comprising the nucleic acids and / or vectors provided herein. In some embodiments, the host cells of the present invention are isolated or purified. In some embodiments, the host cells of the present invention are in cell culture medium. The nucleic acids, expression vectors, and host cells of the present invention can be used to produce compositions comprising one or more of the immunoconjugates of the present invention. In some embodiments, the host cells are eukaryotic. In some embodiments, the host cells are mammalian. In some embodiments, the host cells are Chinese hamster ovary (CHO) cells. In some embodiments, the host cells are prokaryotic. In some embodiments, the host cells are Escherichia coli.

[0217] Exemplary techniques for producing immunoconjugates and radioimmunoconjugates of the invention for use in accordance with the methods of the invention are described below. In some embodiments, the invention provides processes for making immunoconjugates of the invention, comprising culturing host cells 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, e.g., an α- or β-particle emitter.

[0218] Production and Identification of Antigen-Binding Domains, Immunoconjugates, and Nucleic Acids The antigen-binding domain useful as the antigen-binding region herein can be identified in either monoclonal and / or polyclonal antibodies.The DNA encoding monoclonal antibodies can be easily isolated and sequenced using conventional procedures.Once isolated, DNA can be placed into an expression vector, and then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to achieve the synthesis of monoclonal antibodies in recombinant host cells (see, for example, Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol Revs. 130:151-188 (1992)).

[0219] In some embodiments, the antigen-binding domain of the immunoconjugate of the present invention or a fragment thereof is isolated by screening a phage library containing phage displaying various fragments of antibody variable regions (Fv, scFv, or VHH) fused to a phage coat protein. Such a phage library is screened for binding to a desired target antigen or epitope. Clones expressing Fv fragments, scFv, or VHH capable of binding to the desired antigen are adsorbed to the antigen and thus separated from non-binding clones in the library. Binding clones can then be eluted from the antigen and further enriched by additional cycles of antigen adsorption / elution.

[0220] In some embodiments, antibodies or antibody fragments thereof are isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J Mol Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies using phage libraries. Subsequent publications describe the production of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nucleic Acids Res. 21:2265-2266 (1993)). The variable domains can be functionally displayed on phage either as single-chain Fv (scFv) fragments in which the VH and VL are covalently linked via a short, flexible peptide, or as Fab fragments in which they are each fused to a constant domain and interact non-covalently, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994).

[0221] Repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding clones as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Naive libraries for screening can be constructed from non-immunized sources to provide high-affinity antibodies against the antigen (see, e.g., Griffiths et al., EMBO J. 12:725-734 (1993)). Another example is a naive library constructed synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions to achieve rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

[0222] Screening of the library can be achieved by various techniques known in the art. For example, the target antigen can be used to coat the wells of an adsorption plate, or expressed on host cells immobilized on an adsorption plate, or used in cell sorting, or conjugated to biotin for capture by streptavidin-coated beads, or any other method for panning the display library. The selection of antibodies with slow dissociation kinetics (and strong binding affinity) can be facilitated by using long washes and monovalent phage display, as described in Bass et al., Proteins, 8:309-314 (1990) and WO1992 / 09690, and low antigen coating density, as described in Marks et al., Biotechnol., 10:779-783 (1992).

[0223] Techniques for screening cDNA libraries are well known in the art. Libraries can be screened using probes (such as oligonucleotides of at least about 20 to 80 bases) designed to identify the gene of interest or the protein encoded by it. Screening of cDNA or genomic libraries with selected probes can be carried out using standard procedures, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means for isolating genes encoding the immunoconjugates of the invention is the use of PCR techniques (Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)).

[0224] DNA encoding the immunoconjugates of the present invention can be obtained from a cDNA library prepared from tissue believed to harbor the immunoconjugate mRNA of the present invention and express it at detectable levels. Thus, human immunoconjugates of the present invention can be conveniently obtained from a cDNA library prepared from human tissue. Genetic immunoconjugates of the present invention can also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis). In some embodiments, the desired polynucleotide sequence encoding an antibody can be isolated and sequenced from antibody-producing cells, such as hybridoma cells.

[0225] Sequences identified in such library screening methods can be deposited and compared and aligned with other known sequences available in public databases, such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within a defined region of a molecule or over the entire sequence can be determined using methods known in the art and described herein. Any antibody CDR or heavy chain variable fragment of the present invention can be obtained by designing an appropriate antigen screening procedure to select a phage clone of interest, followed by constructing an antibody clone using the variable domain and / or CDR sequences from the phage clone of interest and an appropriate constant region (Fc) sequence as described in Kabat et al., 1991, supra.

[0226] Immunoconjugate Production; Host Cells and Expression Vectors of the Invention The following description primarily relates to producing the antibody constructs of the present invention by culturing cells transformed or transfected with a vector-containing immunoconjugate of a nucleic acid encoding the antibody construct. It is, of course, contemplated that alternative methods well known in the art may be used to prepare the antibody constructs of the present invention. For example, the appropriate amino acid sequence or portions thereof may be produced 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 can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using an Applied Biosystems peptide synthesizer (Foster City, CA) using the manufacturer's instructions. Various portions of the immunoconjugates of the present invention can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired immunoconjugate of the present invention.

[0227] 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 may encode an amino acid sequence comprising the VH and / or VL amino acid sequence (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 nucleic acids 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. 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 a eukaryotic cell, 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 invention is provided, the method comprising culturing a host cell comprising nucleic acid encoding an antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0228] For recombinant production of the immunoconjugates of the present invention, for example, nucleic acids encoding the antibody constructs 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 easily 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 invention can be prepared by various 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 variant or non-variant antibody constructs.

[0229] Engineering host cells for immunoconjugate production Host cells are transfected or transformed with the expression or cloning vectors described herein for producing the immunoconjugates of the present invention 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.

[0230] Suitable host cells for cloning or expressing the nucleic acid and vector encoding immunoconjugate include prokaryotic or eukaryotic cells as described herein.For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector function are not required.For the expression of antibody fragments and polypeptides in bacteria, see, for example, US5,648,237; US5,789,199; US5,840,523; 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, immunoconjugates can be isolated from bacterial cell paste in soluble fractions and further purified.

[0231] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for immunoconjugate-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns (see, e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004); Li et al., Nat. Biotech. 24:210-215 (2006)).

[0232] Suitable host cells for the expression of glycosylated immunoconjugates are also derived from multicellular organisms (e.g., invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains suitable for use with insect cells, particularly for transfection of Spodoptera frugiperda cells, have been identified. Plant cell cultures can also be used as hosts (see, e.g., US 5,959,177; US 6,040,498; US 6,420,548; US 7,125,978; and US 6,417,429).

[0233] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells described in Graham et al., J Gen Viral. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MOCK; buffalo rat liver cells (BRL 3A)); human lung cells (W138); human hepatocytes (Hep 02); mouse mammary tumor (MMT 060562); TRI cells, e.g., as described in Mather et al., Annals NYAcad. Sci. 383:44-68 (1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFK CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77:4216 (1980)); and myeloma cell lines such as YO, NSO, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for immunoconjugate production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0234] Methods for eukaryotic cell transfection and prokaryotic cell transformation, which involve introducing DNA into a host so that the DNA is replicable, either extrachromosomally or as a chromosomal integrant, are well known to those skilled in the art and include, for example, CaCl2, CaPO4, liposome-mediated, polyethylene glycol / DMSO, and electroporation. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Calcium treatment using calcium chloride, as described by Sambrook et al., supra, or electroporation, are commonly used for prokaryotes. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene, 23:315 (1983) and WO 89 / 05859, published June 29, 1989. For mammalian cells lacking such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) can be used. A general description of mammalian cell host system transfection is described in U.S. Patent No. 4,399,216. Transformation into yeast is typically performed according to the methods of Van Solingen et al., J. Back, 130:946 (1977) and Hsiao et al., Proc Natl Acad Sci USA 76:3829 (1979). However, other methods for introducing DNA into cells, such as nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, such as polybrene and polyornithine, can also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).

[0235] prokaryotic host cells Suitable prokaryotes include, but are not limited to, archaea and eubacteria, such as gram-negative or gram-positive organisms, e.g., Enterobacteriaceae, such as E. coli. Various strains of E. coli are publicly available, such as the K12 strains MM294 (ATCC 31,446); X1776 (ATCC 31,537); W3110 (ATCC 27,325), and K5772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae, such as Escherichia coli, Enterobacteriaceae, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans, and Shigella, as well as Bacillus, such as Bacillus subtilis and Bacillus licheniformis (e.g., B. licheniformis 41P, disclosed in DD 266,710, published April 12, 1989), Pseudomonas, such as Pseudomonas aeruginosa, Rhizobia, Vitreoscilla, Paracoccus, and Streptomyces. These examples are illustrative and not limiting. E. coli strain W3110 is a common host strain for recombinant DNA production fermentations and is therefore one advantageous host or parent host. Preferably, the host cell secretes minimal amounts of proteolytic enzymes.For example, strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) can be altered to introduce genetic mutations into genes encoding proteins endogenous to the host. Examples of such hosts include E. coli W3110 strain 1A2, which has the complete genotype tonA; E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kanr; and E. coli W3110 strain 27C7, which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT rbs7 ilvG. Examples of suitable strains include E. coli W3110 strain 37D6 with kanr; E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin-resistant degP deletion mutation; E. coli W3110 strain 33D3 with the genotype W3110 ΔfhuA(ΔtonA)ptr3 lac Iq lacL8 ΔompT Δ(nmpc-fepE)degP41 kanR (U.S. Pat. No. 5,639,635); and E. coli strains with mutant periplasmic proteases as disclosed in U.S. Pat. No. 4,946,783, issued August 7, 1990. Other strains, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli λ1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608), and their derivatives, are also suitable. These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above bacteria with defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). In general, the appropriate bacterium must be selected taking into account the replication ability of the replicon in the bacterial cell.For example, Escherichia coli, Serratia, or Salmonella species can be suitably used as hosts when well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon. Typically, the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors can desirably be incorporated into the cell culture. Alternatively, in vitro methods of cloning, such as PCR or other nucleic acid polymerase reactions, are suitable.

[0236] Full-length antibodies, antibody fragments, and antibody fusion proteins can be produced in bacteria, especially if glycosylation and Fc effector functions are not required. Full-length antibodies have a longer half-life in circulation. Production in E. coli is faster and more cost-effective. For the expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237; US 5,789,199, and US 5,840,523, which describe translation initiation regions (TIRs) and signal sequences to optimize expression and secretion. After expression, the immunoconjugate is isolated from the E. coli cell paste in the soluble fraction and can be purified, for example, through a protein A or G column depending on the isotype. Final purification can be performed similarly to the process for purifying antibodies expressed in, for example, CHO cells.

[0237] eukaryotic host cell In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for immunoconjugates of the coding vectors of the present invention. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290:140 (1981); EP 139,383 published May 2, 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al., Bio / Technology, 9:968-75 (1991)), such as K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al. al.Bacteriol.,154(2):737-742(1983)), K.fragilis(ATCC12,424), K.bulgaricus(ATCC16,045), K.wickeramii(ATCC24,178), K.waltii(ATCC56,500), K.drosophilarum(ATCC36,906; Van den Berg et al.,Bio / Technology,8:135(1990)), K.thermotolerans, and K.marxianus;yarrowia(EP402,226);Pichia pastoris(EP183,070;Sreekrishna et al.,J.Basic Microbiol., 28:265-278 (1988)); Candida; Trichoderma rhesus (EP 244,234); Neurospora crassa (Case et al., Proc Natl Acad Sci USA 76:5259-5263 (1979)); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published October 31, 1990); as well as filamentous fungi, e.g., Neurospora, Penicillium, Tolypocladium (WO 91 / 00357 published January 10, 1991), and Aspergillus hosts, e.g., A.nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289 (1983)); Tilbum et al., Gene, 26:205-221 (1983); Yelton et al., Proc Natl Acad Sci USA 81:1470-1474 (1984) and A. niger (Kelly and Hynes, EMBO J., 4:475-479 (1985)). Methylotropic yeasts are suitable herein and include yeasts capable of growing on methanol selected from the genera consisting of, but not limited to, Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeast is provided in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

[0238] Suitable host cells for the expression of glycosylated immunoconjugates of the present invention are derived from multicellular organisms.Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, and plant cells such as cotton, corn, potato, soybean, petunia, tomato, and tobacco cell cultures.A large number of baculovirus strains and mutants have been identified, as well as corresponding permissible insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as viruses herein in accordance with the present invention, particularly for transfection of Spodoptera frugiperda cells.

[0239] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); the human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0240] Host cells are transformed with the above-described expression or cloning vectors for producing the immunoconjugates of the present invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences.

[0241] Selection and use of replicable vectors For recombinant production of the radioisotope delivery platform of the present invention, the nucleic acid (e.g., cDNA or genomic DNA) encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or expression. DNA encoding the immunoconjugate is easily 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 the 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.

[0242] The vector can be, for example, in the form of a plasmid, cosmid, virus particle, or phage. The appropriate nucleic acid sequence can be inserted into the vector 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. The construction of an appropriate vector containing one or more of these components uses standard ligation techniques known to those skilled in the art.

[0243] The immunoconjugates of the present invention can be recombinantly produced not only directly but also as fusion polypeptides with heterologous polypeptides, which may be signal sequences or other polypeptides having a specific cleavage site at the N-terminus of the mature protein or polypeptide. Generally, the signal sequence may be a component of the vector, or it may be part of the immunoconjugate-encoding DNA of the present invention that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion, the signal sequence may be, for example, the yeast invertase leader, α-factor leader, or the like. (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 published April 4, 1990), or a signal described in WO 90 / 13646 published November 15, 1990. In mammalian cell expression, mammalian signal sequences can be used to direct protein secretion, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.

[0244] Cultivation of host cells producing radioisotope delivery platforms The host cells used to produce the immunoconjugates of the present invention may be cultured in a variety of media and culture conditions.

[0245] Prokaryotic host cell culture Prokaryotic cells used to produce the polypeptides of the present invention are grown in a medium known in the art and suitable for culturing the selected host cells. Examples of suitable media include Luria Broth (LB) and necessary nutritional supplements. In some embodiments, the medium also contains a selection agent selected based on the construction of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium to grow cells expressing an ampicillin resistance gene.

[0246] Any necessary supplements other than carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations introduced alone or in mixture with another supplement or medium, such as a complex nitrogen source. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol, and dithiothreitol.

[0247] Prokaryotic host cells are cultured at a suitable temperature. For example, for E. coli growth, preferred temperatures range from about 20° C. to about 39° C., more preferably from about 25° C. to about 37° C., and even more preferably about 30° C. The pH of the medium can be any pH ranging from about 5 to about 9, depending primarily on the host organism. For E. coli, the pH is preferably from about 6.8 to about 7.4, more preferably about 7.0.

[0248] When an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for the activation of the promoter.In one aspect of the present invention, the PhoA promoter is used to control the transcription of polypeptide.Therefore, transformed host cells are cultured in phosphate-limited medium for induction.In some embodiments, the phosphate-limited medium is CRAP medium (see, for example, Simmons et al., J.Immunol.Methods (2002), 263:133-47).As known in the art, various other inducers can be used depending on the vector construct used.

[0249] In one embodiment, the expressed polypeptide of the present invention is secreted into the periplasm of the host cell and recovered therefrom. Protein recovery typically involves disrupting the microorganism, generally by means such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transported into the culture medium and isolated therein. The cells can be removed from the culture, and the culture supernatant can be filtered and concentrated for further purification of the produced protein. The expressed polypeptide can be further isolated and identified using well-known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

[0250] In one aspect of the present invention, immunoconjugates are produced in large quantities by fermentation processes. A variety of large-scale fed-batch fermentation procedures are available for the production of recombinant proteins. Large-scale fermentations have a capacity of at least 1000 liters, preferably about 1,000 to 100,000 liters. These fermentors use impellers to distribute oxygen and nutrients, particularly glucose (the preferred carbon / energy source). Small-scale fermentation generally refers to fermentation in fermentors with a volumetric capacity of about 100 liters or less, and can range from about 1 liter to about 100 liters.

[0251] In fermentation processes, induction of protein expression typically begins after cells have been grown under appropriate conditions to a desired density, e.g., an OD550 of about 180 to 220, at which point the cells are in early stationary phase. Various inducers, known in the art and described above, can be used depending on the vector construct used. Cells can be grown for a shorter period before induction. Cells are usually induced for about 12 to 50 hours, although longer or shorter induction times may be used.

[0252] Various fermentation conditions can be modified to improve the production yield and quality of the polypeptide of the present invention. For example, to improve the proper assembly and folding of the secreted immunoconjugate polypeptide, an additional vector overexpressing a chaperone protein, such as Dsb protein (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (a peptidyl prolyl cis, trans-isoform with chaperone activity), can be used to co-transform the host prokaryotic cell. Chaperone proteins have been demonstrated to promote the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J Bio Chem 274:19601-5; U.S. Patent No. 6,083,715; U.S. Patent No. 6,027,888; Bollmann and Pluckthun (2000) J. Biol. Chem. 275:17100-5; Rramm and Pluckthun (2000) J. Biol. Chem. 275:17106-13; Arie et al. (2001) Mol. Microbiol. 39:199-210.

[0253] To minimize proteolysis of expressed heterologous proteins (especially those that are sensitive to proteolysis), certain host strains lacking proteolytic enzymes can be used in the present invention. For example, host cell strains can be modified to introduce genetic mutations into genes encoding known bacterial proteases, such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Several E. coli protease-deficient strains are available and are described, for example, in Joly et al. (1998), supra; U.S. Patent No. 5,264,365; U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0254] In one embodiment, an E. coli strain that is deficient in a proteolytic enzyme and transformed with a plasmid that overexpresses one or more chaperone proteins is used as a host cell in the expression system of the present invention.

[0255] eukaryotic host cell culture Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma) are suitable for culturing the host cells. Additionally, see Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90 / 03430; WO87 / 00195; or U.S. Patent Re. Any of the media described in 30,985 can be used as a culture medium for host cells. Any of these media can be supplemented, as needed, with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN™), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements can also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature and pH, will be those previously used with the host cells selected for expression and will be apparent to those skilled in the art.

[0256] Purification of immunoglobulin-derived structures of the invention The immunoconjugates of the present invention 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 invention can be disrupted by various physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysing agents.

[0257] It may be desirable to purify the immunoconjugates of the present invention from recombinant cell proteins or polypeptides. The following procedures are examples of suitable purification procedures: 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; Protein A Sepharose columns to remove contaminants such as IgG; and metal chelate columns that bind epitope-tagged forms of the immunoconjugates of the present invention. 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 step selected will depend, for example, on the nature of the production process used and the particular immunoconjugate of the present invention being produced.

[0258] 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 approximately 30 minutes. The cell paste 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.

[0259] 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., 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.

[0260] After any preliminary purification steps, the mixture containing the immunoconjugate of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography at a pH of about 2.5 to 4.5, generally using an elution buffer with a low salt concentration (e.g., about 0 to 0.25 M salt).

[0261] Immunoconjugates (including antibody-drug conjugates (ADCs)) In a further aspect of the invention, an immunoconjugate of the invention according to any of the above embodiments or as described herein is conjugated to a heterologous moiety or agent (including any further exogenous substance as described herein), e.g., as described below.

[0262] In one embodiment, the present invention provides an immunoconjugate comprising an antibody construct of the present invention conjugated to one or more therapeutic agents or radioisotopes.

[0263] In some embodiments, the immunoconjugate comprises an antibody construct described herein conjugated to a radioactive atom to form a radioconjugate. As described herein, a variety of radioisotopes are available for the production of the radioconjugates of the present invention.

[0264] Immunoconjugates or antibody construct conjugates can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate H), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(pdiazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies (see, for example, WO1994 / 11026). The linker can be a "cleavable linker" that facilitates the release of cytotoxic drugs in cells. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (see, for example, Chari et al., Cancer Res. 52:127-131 (1992); US 5,208,020).

[0265] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared using crosslinker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and commercially available sulfo-SMPB and SVSB (succinimidyl-(4-vinylsulfone)benzoate) (available, for example, from Pierce Biotechnology, Inc., Rockford, IL, US).

[0266] As will be appreciated by those skilled in the art, the particular methods described above are also useful for preparing radioimmunoconjugates and targeted imaging complexes (notwithstanding the textual references to only immunoconjugates or antibody constructs), and such preparative methods are also encompassed by the present invention.

[0267] Immunoconjugation 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.

[0268] 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., the 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 the antibody platform or immunoconjugate for radioisotope conjugation.

[0269] 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).

[0270] In some embodiments, peptides can be biosynthesized or synthesized by chemical amino acid synthesis using appropriate amino acid precursors containing, for example, fluorine-19 instead of hydrogen. In some embodiments, a radiolabel can be incorporated into the peptide. In some embodiments, a radiolabel can be linked to the peptide. Iodine-123 can be incorporated using the IODOGEN method (Fraker et al. (1978) Biochem Biophys Res Commun. 80:49-57). "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.

[0271] 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 invention can be characterized for their physical / chemical properties and / or biological activity by various assays known in the art. Immunoconjugates of the present invention 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.

[0272] 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 invention can be quantified using methods known in the art, such as quantitative ELISA, quantitative Western blot, surface plasmon resonance assay, and / or Scatchard analysis.

[0273] In one embodiment, the K D is measured using a radiolabeled antigen ELISA performed with the immunoconjugate. D is measured by using a surface plasmon resonance assay using a BIACORE®-2000 or BIACORE®-3000 instrument (BIAcore, Inc., Piscataway, NJ), for example, by using an immobilized antigen CM5 chip at 25°C and 10 response units.

[0274] 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) as an immunoconjugate of the invention (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)).

[0275] The epitope and / or contact residues within the antigen to which the immunoconjugate of the invention 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).

[0276] Pharmaceutical compositions and formulations of the present invention As will be recognized by those skilled in the art, certain teachings herein below apply to the immunoconjugates and radioimmunoconjugates of the present invention, but notwithstanding specific textual reference to one type of the invention, such applications are encompassed in their entirety by the present invention.

[0277] In another aspect, the present invention provides compositions comprising an immunoconjugate or radioimmunoconjugate of the invention. The present invention further provides pharmaceutical compositions and formulations comprising at least one immunoconjugate of the invention and at least one pharmaceutically acceptable excipient or carrier. In some embodiments, the pharmaceutical formulation comprises (1) an immunoconjugate or radioimmunoconjugate of the invention, and (2) a pharmaceutically acceptable carrier.

[0278] The immunoconjugate or radioimmunoconjugate may be formulated in any form suitable for delivery to target cells / tissues. Pharmaceutical formulations of the immunoconjugates of the present invention may be prepared in the form of a lyophilized formulation or aqueous solution by mixing such immunoconjugates having the desired purity with one or more pharmaceutically acceptable carriers, diluents, and / or excipients (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers, diluents, and excipients are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, sterile water, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., 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); low molecular weight (less than about 10 residues) polysaccharides; 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).

[0279] Pharmaceutical formulations to be used for in vivo administration are generally sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0280] 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.

[0281] 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.

[0282] 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, and such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0283] The active ingredient can also be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacylate) microcapsules, prepared by coacervation technology or interfacial polymerization, respectively, in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[0284] In some embodiments, the immunoconjugate can 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 can be 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. Patent Nos. 4,485,045 and 4,544,545; and WO1997 / 38731, published October 23, 1997. Particularly useful liposomes can be produced by reverse-phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. A chemotherapeutic agent is 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.

[0285] 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.

[0286] Methods of using immunoconjugates and radioimmunoconjugates and compositions thereof In one aspect, the invention provides a method of treating a disease, disorder, or condition in a patient in need thereof, 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 invention 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.

[0287] 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.

[0288] In one embodiment, the immunoconjugates or radioimmunoconjugates or compositions of the present invention 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 immunoconjugates or radioimmunoconjugates or compositions of the present invention can be administered to humans for therapeutic purposes. Furthermore, the immunoconjugates or radioimmunoconjugates or compositions of the present invention can be administered to non-human mammals expressing a target antigen with which the immunoconjugate or radioimmunoconjugate cross-reacts (e.g., primates, pigs, rats, or mice) for veterinary purposes or as animal models of human disease. Regarding the latter, such animal models can be useful for evaluating the therapeutic efficacy of the immunoconjugates or radioimmunoconjugates or compositions of the present invention (e.g., testing dosages and time courses of administration).

[0289] The immunoconjugates or radioimmunoconjugates or compositions of the invention (and any additional therapeutic agents or adjuvants) can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, as well as, if desired for localized treatment, intralesional administration. 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.

[0290] The immunoconjugates or radioimmunoconjugates or compositions of the invention are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered 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 invention are administered to human patients according to known methods, for example, as a bolus or by continuous infusion over a period of time, by 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 invention is preferred.

[0291] For the prevention or treatment of a disease, 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 invention will depend on the type of disease being treated, the severity and course of the disease, whether the immunoconjugate or radioimmunoconjugate or composition of the present invention is administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to the immunoconjugate or radioimmunoconjugate or composition, as defined above, and the discretion of the attending physician. The immunoconjugate or radioimmunoconjugate or composition of the present invention is suitably 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 pg / 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 the 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 weekly maintenance doses of about 2 mg / kg of the immunoconjugate or radioimmunoconjugate or composition of the present invention. However, other dosing regimens may also be useful. A typical daily dose may range from about 1 pg / kg to 100 mg / kg or more, depending on the factors mentioned 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.

[0292] The dosage and administration schedule can be selected and adjusted based on the level or tolerance of the disease in the subject, which can be monitored during the course of treatment.The conjugate of the present invention can be administered once a day, once a week, once a week, less than once a day, less than once a month, less than once 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 the symptoms of the disease.Administration can be continued at any of the intervals disclosed until the symptoms of the tumor or cancer being treated are alleviated.Administration can be continued after alleviation or relief of symptoms is achieved, and such alleviation or relief is prolonged by such continued administration.

[0293] In some embodiments, an effective amount of the immunoconjugate or radioimmunoconjugate or composition may be provided as a single dose.

[0294] The immunoconjugates and radioimmunoconjugates of the present invention can be used in combination with conventional and / or novel treatment or therapeutic methods, or separately as monotherapy. In some embodiments, the immunoconjugates and radioimmunoconjugates of the present invention can be used with one or more radiosensitizing agents. Such agents include any agent that can increase the sensitivity of cancer cells to radiation therapy. In other embodiments, the immunoconjugates and radioimmunoconjugates of the present invention can be used in combination with novel and / or conventional agents that can enhance the biological effects of radiation therapy. Irradiation of tumors can induce various biological outcomes that can be exploited by combining the immunoconjugates and radioimmunoconjugates of the present invention with agents that target relevant pathways. In some embodiments, such agents can reduce tumor angiogenesis, inhibit local invasion and metastasis, prevent regrowth, enhance the immune response, deregulate cellular energy, reduce cell population, alter tumor metabolism, increase tumor damage, or reduce DNA repair. In certain embodiments, agents for use in combination with the immunoconjugates and radioimmunoconjugates of the present invention may include DDR inhibitors, e.g., PARP, ATR, ChkI, or DNA-PK; or survival signaling inhibitors, e.g., mTOR, PI3k, NF-kB; or anti-hypoxia agents, e.g., HIF-1-α, CAP, or UPR; or metabolic inhibitors, e.g., MCT1, MCT4 inhibitors; or immunotherapeutic agents, e.g., anti-CTLA4, anti-PD-1; or inhibitors of growth factor signaling, e.g., EGFR or MAPK inhibitors; or anti-invasive agents, e.g., kinase inhibitors, chemokine inhibitors, or integrin inhibitors; or anti-angiogenic agents, e.g., VEGF inhibitors.

[0295] The immunoconjugates and radioimmunoconjugates of the present invention can (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 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.

[0296] By way of example, an immunoconjugate that inhibits tumor cell growth is one that results in measurable growth inhibition of tumor cells (e.g., cancer cells). In one embodiment, an immunoconjugate or radioimmunoconjugate of the invention 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 from about 20% to about 50%, and even more preferably by more than 50% (e.g., from about 50% to about 100%), compared to a suitable control, which is typically tumor cells that have not been treated with the immunoconjugate or radioimmunoconjugate being tested.

[0297] 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 may be provided in a total protein amount of up to 100 mg, for example less than 60 mg, or 5 mg to 45 mg, or 0.1 pg / kg to 1 mg / kg patient body weight, for example 1 pg / kg to 1 mg / kg patient body weight total protein amount, or 10 pg / kg to 1 mg / kg patient body weight, or 100 pg / kg to 1 mg / kg patient body weight, or 0.1 pg / kg to 100 pg / kg patient body weight, or 0.1 pg / kg to 50 pg / kg patient body weight, or 0.1 pg / kg to 10 pg / kg patient body weight, or 0.1 pg / kg to 40 pg / kg patient body weight, or 1 pg / kg to 40 pg / kg patient body weight, or 0.1 mg / kg to 1.0 mg / kg patient body weight, such as 0.2 mg / kg patient body weight to 0.6 mg / kg patient body weight.

[0298] 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 may be administered at 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 0.5mg / kg to about 30mg / kg, about 1mg / kg to about 2mg / kg, about 1mg / kg to about 5mg / kg, about 1mg / kg to about 10mg / kg, about 1mg / kg to about 3mg / kg, about 1mg / kg to about 4mg / kg, about 1mg / kg to about 5mg / kg, about 1mg / kg to about 10mg / kg, about 1mg / kg to about 20mg / kg, about 1mg / kg to about 30mg / kg, about 2mg / kg to about 5mg / kg, about 2mg / kg to about 10mg / kg g, about 2mg / kg to about 3mg / kg, about 2mg / kg to about 4mg / kg, about 2mg / kg to about 5mg / kg, about 2mg / kg to about 10mg / kg, about 2mg / kg to about 20mg / kg, about 2mg / kg to about 30mg / kg, about 5mg / kg to about 10mg / kg, about 5mg / kg to about 3mg / kg, about 5mg / kg to about 4mg / kg, about 5mg / kg to about 5mg / kg, about 5mg / kg to about 10mg / kg, about 5mg / kg to about 20mg g / kg, about 5 mg / kg to about 30 mg / kg, about 10 mg / kg to about 3 mg / kg, about 10 mg / kg to about 4 mg / kg, about 10 mg / kg to about 5 mg / kg, about 10 mg / kg to about 10 mg / kg, about 10 mg / kg to about 20 mg / kg, about 10 mg / kg to about 30 mg / kg, about 3 mg / kg to about 4 mg / kg, about 3 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 g / 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 is administered at a dose of 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. .

[0299] In some embodiments, the method comprises administering an effective amount of a radioimmunoconjugate comprising 225-Ac that is 0.01 to 0.1 mCi, or 0.1 mCi to 1.0 mCi, or 1.0 mCi to 2.0 mCi, or 2.0 mCi to 4.0 mCi.

[0300] In some embodiments, the methods comprise administering an effective amount of a radioimmunoconjugate comprising 225-Ac at 0.1 pCi / kg to 2.0 pCi / kg of the subject's body weight, or 0.1 pCi / kg to 1.0 pCi / kg of the subject's body weight, or 1.0 pCi / kg to 3.0 pCi / kg of the subject's body weight, or 3.0 pCi / kg to 10.0 pCi / kg of the subject's body weight, or 10.0 pCi / kg to 20.0 pCi / kg of the subject's body weight, or 10.0 pCi / kg to 30.0 pCi / kg of the subject's body weight.

[0301] In some embodiments, the effective amount of 225-Ac is from about 0.1 microcuries to about 20 microcuries. In some embodiments, the effective amount of 225-Ac is from about 0.1 microcuries to about 0.2 microcuries, from about 0.1 microcuries to about 0.5 microcuries, from about 0.1 microcuries to about 1 microcurie, from about 0.1 microcuries to about 2 microcuries, from about 0.1 microcuries to about 3 microcuries, from about 0.1 microcuries to about 4 microcuries, from about 0.1 microcuries to about 5 microcuries, from about 0.1 microcuries to about 10 microcuries, from about 0.1 microcuries to about 20 microcuries, from about 0.2 microcuries to about 0.5 microcuries, from about 0.2 microcuries to about 1 microcurie, 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 some 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. In embodiments where the radioactive isotope of the radioimmunoconjugate is 111-In, the effective amount is, for example, 15.0 mCi (i.e., the amount of In-111 administered to the subject delivers a total body radiation dose of less than 15.0 mCi).

[0302] 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.

[0303] According to embodiments in which the radioactive isotope of the radioimmunoconjugate is 111-In, an 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. 1.0mCi to 5.0mCi, 1.0mCi to 10.0mCi, 1.0mCi to 15.0mCi, 1.0mCi to 20.0mCi, 2.0mCi to 3.0mCi, 3.0mCi to 4.0mCi, 4.0mCi to 5.0mCi, 5.0mCi to 10.0mCi, 5.0mCi to 15.0mCi, 5.0mCi to 20.0mCi, 6.0mCi to 14.0mCi, 7.0mCi to 13.0mCi, 8.0mCi to 12.0mCi, 9.0mCi to 11.0mCi, or 10.0mCi to 15.0mCi.

[0304] 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, 1.0 mCi, 10.0 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.

[0305] According to embodiments in which the radioactive isotope of the radioimmunoconjugate is 225-Ac, an effective amount is, for example, less than 30.0 pCi / kg (i.e., the amount of 225-Ac administered to a subject delivers a radiation dose of less than 30.0 pCi per kilogram of the subject's body weight).

[0306] According to embodiments in which the radioactive isotope of the radioimmunoconjugate is 225-Ac, effective amounts are 30 pCi / kg, 25 pCi / kg, 20 pCi / kg, 17.5 pCi / kg, 15.0 pCi / kg, 12.5 pCi / kg, 10.0 pCi / kg, 9 pCi / kg, 8 pCi / kg, 7 pCi / kg, 6 pCi / kg, 5 pCi / kg, 4.5 pCi / kg, 4.0 pCi / kg. g, 3.5 pCi / kg, 3.0 pCi / kg, 2.5 pCi / kg, 2.0 pCi / kg, 1.5 pCi / kg, 1.0 pCi / kg, 0.9 pCi / kg, 0.8 pCi / kg, 0.7 pCi / kg, 0.6 pCi / kg, 0.5 pCi / kg, 0.4 pCi / kg, 0.3 pCi / kg, 0.2 pCi / kg, 0.1 pCi / kg, or less than 0.05 pCi / kg.

[0307] According to embodiments in which the radioactive isotope of the radioimmunoconjugate is 225-Ac, an effective amount is from 0.05 pCi / kg to 0.1 pCi / kg, 0.1 pCi / kg to 0.2 pCi / kg, 0.2 pCi / kg to 0.3 pCi / kg, 0.3 pCi / kg to 0.4 pCi / kg, 0.4 pCi / kg to 0.5 pCi / kg, 0.5 pCi / kg to 0.6 pCi / kg, 0.6 pCi / kg to 0.7 pCi / kg, 0.7 pCi / kg to 0.8 pCi / kg, 0.8 pCi / kg to 0.9 pCi / kg, 0.9 ... pCi / kg to 0.9 pCi / kg, 0.9 pCi / kg to 1.0 pCi / kg, 1.0 pCi / kg to 1.5 pCi / kg, 1.5 pCi / kg to 2.0 pCi / kg, 2.0 pCi / kg to 2.5 pCi / kg, 2.5 pCi / kg to 3.0 pCi / kg, 3.0 pCi / kg to 3.5 pCi / kg, 3.5 pCi / kg to 4.0 pCi / kg, 4.0 pCi / kg to 4.5 pCi / kg, or 4.5 pCi / kg to 5.0 pCi / kg.

[0308] According to embodiments in which the radioactive isotope of the radioimmunoconjugate is 225-Ac, an effective amount is 0.05 pCi / kg, 0.1 pCi / kg, 0.2 pCi / kg, 0.3 pCi / kg, 0.4 pCi / kg, 0.5 pCi / kg, 0.6 pCi / kg, 0.7 pCi / kg, 0.8 pCi / kg, 0.9 pCi / kg, 1.0 pCi / kg, 1.5 pCi / kg, 2.0 pCi / kg, 2.5 pCi / kg, 3.0 pCi / kg, 3.5 pCi / kg, 4.0 pCi / kg, 4.5 pCi / kg, 5.0 pCi / kg, 6.0 pCi / kg, 6.5 pCi / kg, 7.0 pCi / kg, 7.5 pCi / kg, 8.0 pCi / kg, 8.5 pCi / kg, 9.0 pCi / kg, 9.5 pCi / kg, 10.0 pCi / kg, 10.5 pCi / kg, 11.0 pCi / kg, 12.0 pCi / kg, 13.0 pCi / kg, 14.0 pCi / kg, 15.0 pCi / kg, 16.0 pCi / kg, 17.0 pCi / kg, 18.0 pCi / kg, 19.0 pCi / kg, 20.0 pCi / kg, 21.0 pCi / kg, 22.0 pCi / kg, 23.0 pCi / kg, 24.0 pCi / kg, 25.0 pCi / kg, 26.0 pCi / kg, 27.0 pCi / kg, i / kg, 3.0pCi / kg, 3.5pCi / kg, 4.0pCi / kg or 4.5pCi / kg, 5.0pCi / kg, 6.0pCi / kg, 7.0pCi / kg, 8.0pCi / kg, 9.0pCi / kg, 10.0pCi / kg, 12.5pCi / kg, 15.0pCi / kg, 17.5pCi / kg, 20.0pCi / kg, 25pCi / kg, or 30pCi / kg.

[0309] In embodiments where the radioisotope of the radioimmunoconjugate is 177-Lu, an effective amount is from 0.1 uCi to 100 mCi per square meter of body surface area.

[0310] In embodiments where the radioactive isotope of the radioimmunoconjugate is 177-Lu, the effective amount is from 1 mCi to 100 mCi per square meter of body surface area, hi certain embodiments, the effective amount is from about 1 per square meter to about 100 per square meter. In certain embodiments, an effective amount is from about 1 per square meter to about 5 per square meter, from about 1 per square meter to about 10 per square meter, from about 1 per square meter to about 15 per square meter, from about 1 per square meter to about 20 per square meter, from about 1 per square meter to about 75 per square meter, from about 1 per square meter to about 100 per square meter, from about 5 per square meter to about 10 per square meter, from about 5 per square meter to about 15 per square meter, from about 5 per square meter to about 20 per square meter, from about 5 per square meter to about 25 per square meter, from about 5 per square meter to about 75 per square meter, from about 5 per square meter to about 100 per square meter, from about 10 per square meter to about 15 per square meter, from about 10 per square meter to about 50 per square meter. The preferred range is about 20 per square meter, about 10 per square meter to about 25 per square meter, about 10 per square meter to about 75 per square meter, about 10 per square meter to about 100 per square meter, about 15 per square meter to about 20 per square meter, about 15 per square meter to about 25 per square meter, about 15 per square meter to about 75 per square meter, 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 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, about 75 per square meter, or about 100 per square meter.In some embodiments, an 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 some embodiments, an 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.

[0311] According to certain aspects of the invention, preparations of the radioimmunoconjugates of the invention or compositions thereof (e.g., pharmaceutical compositions) may comprise a radiolabeled fraction (radioimmunoconjugate) and an unlabeled fraction (immunoconjugate), wherein the ratio of labeled to unlabeled may be from about 1:1000 to 1:1.

[0312] Additionally, pharmaceutical compositions can be provided as single-dose compositions tailored to a particular patient, i.e., as patient-specific therapeutic compositions, where the amount of labeled and unlabeled immunoconjugate (labeled immunoconjugate, for clarity, is the same as radioactive immunoconjugate 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 the patient in a single treatment session, such that little or no composition remains in the vial after administration.

[0313] Currently, cancer treatments, depending on the stage of the cancer, include one or a combination of surgery to remove cancerous tissue, radiation therapy, and chemotherapy. Treatment using the radioimmunoconjugates of the present invention (interchangeably "radiolabeled immunoconjugates") 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 invention is useful for alleviating target antigen-expressing cancers at the time of initial diagnosis or recurrence of the disease.

[0314] In some embodiments, determining whether a cancer is suitable for treatment by the methods disclosed herein comprises 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 embodiment, target antigen overexpression is analyzed by immunohistochemistry (IHC). Paraffin-embedded tissue sections from tumor biopsies are subjected to IHC assays and follow target antigen staining intensity criteria. Alternatively, or in addition, FISH assays such as INFORM® (sold by Ventana, Arizona, USA) or PATHVISION® (Vysis, Illinois, USA) can be performed on formalin-fixed, paraffin-embedded tumor tissue to determine the degree of target antigen overexpression in the tumor (if present).

[0315] Overexpression or amplification of a target antigen can be assessed using in vivo detection assays, for example, by administering a molecule (e.g., an antibody construct or immunoconjugate of the invention) 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.

[0316] 2. Use of Immunoconjugates and Radioimmunoconjugates of the Invention to Kill Cells The immunoconjugates or radioimmunoconjugates of the present invention can be used, for example, in vitro, ex vivo, and in vivo methods. In one aspect, the present invention provides a method for inhibiting cell growth or proliferation either in vivo or in vitro, comprising exposing cells to the immunoconjugates or radioimmunoconjugates of the present invention under conditions that allow the immunoconjugates or radioimmunoconjugates to bind to their target antigen. The immunoconjugates or radioimmunoconjugates of the present invention 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.

[0317] In one aspect, the present invention provides a method for killing antigen-expressing cells, comprising contacting the cells with an immunoconjugate or radioimmunoconjugate of the present 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).

[0318] In one aspect, the immunoconjugates or radioimmunoconjugates of the invention are used to treat or prevent cell proliferative disorders. In certain embodiments, the cell proliferative disorder comprises a solid tumor cancer. Solid tumor cancers are cancers involving abnormal tissue masses, e.g., carcinomas and sarcomas. In certain other embodiments, the cell proliferative disorder comprises a liquid tumor cancer or a blood cancer; interchangeably, such cancers are present in bodily fluids (e.g., leukemia and lymphoma). 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 comprises a solid tumor cancer. Solid tumor cancers are cancers involving abnormal tissue masses, e.g., carcinomas and sarcomas. In certain other embodiments, the cell proliferative disorder comprises a liquid tumor cancer or a blood cancer; interchangeably, such cancers are present in bodily fluids (e.g., leukemia and lymphoma).

[0319] In one aspect, the invention provides a method for treating a cell proliferative disorder comprising administering to an individual an effective amount of an immunoconjugate or radioimmunoconjugate of the invention.

[0320] In addition to direct cell killing of target cells expressing the cell surface antigen specifically bound by the immunoconjugates or radioimmunoconjugates of the present invention, the immunoconjugates or radioimmunoconjugates of the present invention 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, information gathering, and / or diagnostic functions. Non-cytotoxic, or optionally toxic, variants of the immunoconjugates or radioimmunoconjugates of the present invention 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.

[0321] 3. Use of the Immunoconjugates, Radioimmunoconjugates, and Targeted Imaging Complexes of the Invention for Antigen Detection, In Vivo Imaging, Diagnostics, and Prognostics As described herein, in some embodiments, the antibody constructs, immunoconjugates, radioimmunoconjugates, and targeted imaging complexes of the present invention have a variety of non-therapeutic uses. In some embodiments, the compositions of the present invention can be used to identify patient populations predicted to benefit from a particular therapeutic approach or modality (e.g., treatment with an immunoconjugate or radioimmunoconjugate of the present invention). In some embodiments, the compositions of the present invention can be useful for staging target antigen-expressing cancers (e.g., by radioactive imaging) or as prognostic indicators of disease progression. In some embodiments, the compositions are also useful for in vitro detection and quantitation of target epitopes, for example, in ELISA or Western blots, and for purifying or immunoprecipitation of target antigens from cell or tissue samples.

[0322] In some embodiments, the immunoconjugates or radioimmunoconjugates of the present invention are used in methods for detecting the presence or level of antigens, for example, in vitro in biological samples or in vivo using imaging techniques.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 immunoconjugates or radiolabeled immunoconjugates of the present invention are used for detection, they can contain radioactive atoms, such as 99m-Tc or 111-In, for scintigraphy studies.

[0323] The labeled immunoconjugates of the present invention are useful as imaging biomarkers and probes for various biomedical and molecular imaging methods and techniques, such as (i) MRI (magnetic resonance imaging); (ii) MicroCT (computed tomography); (iii) SPECT (single-photon emission computed tomography); (iv) PET (positron emission tomography) Chen et al., Bioconjugate Chem. 15:41-9 (2004); (v) bioluminescence; (vi) fluorescence; and (vii) ultrasound. Immunoscintigraphy is an imaging procedure in which a radiolabeled antibody is administered to an animal or human patient and photographs are taken of the site in the body where the antibody is localized (US6528624). Imaging biomarkers can be objectively measured and evaluated as indicators of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions.

[0324] Another aspect of the present invention is a method for determining the presence of a target antigen in a sample suspected of containing the target antigen, comprising exposing the sample to an immunoconjugate that binds to the target antigen and determining binding of the immunoconjugate to the target antigen in the sample, the presence of such binding indicating the presence of the target antigen in the sample. Optionally, the sample may contain cells (which may be cancer cells) suspected of expressing the target antigen. The immunoconjugate used in this method may optionally be detectably labeled, attached to a solid support, etc.

[0325] Another embodiment of the present invention is directed to a method of 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 complex between the immunoconjugate and the target antigen in the test sample, wherein the formation of a complex 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.

[0326] In some embodiments, the immunoconjugates of the present invention, including compositions comprising those described above and / or provided herein, are useful for detecting the presence of a target antigen, for example, in vivo or in a biological sample. The immunoconjugates of the present invention can be used in a variety of different assays, including, but not limited to, ELISA, bead-based immunoassays, and mass spectrometry.

[0327] In some embodiments, the immunoconjugates of the present invention are useful for quantifying the amount of a target antigen in a sample. In some embodiments, the biological sample is a biological fluid such as whole blood or components of whole blood, 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, urine, and other components of the body that may contain the target antigen of interest. In various embodiments, the sample is a bodily 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 one or more therapeutic antibodies that bind to the same target antigen.

[0328] In some embodiments, the sample is from a mammal. In some embodiments, the sample is from a human subject, for example, when measuring antigen expression in a clinical sample. In some embodiments, the biological sample is from a clinical patient or a patient treated with a treatment / therapy (e.g., an antibody therapy targeting the same target antigen). In some embodiments, the biological sample is serum or plasma. In some embodiments, the biological sample is serum from a clinical patient. In some embodiments, the biological sample is a biopsy. In some embodiments, the biological sample is a biopsy 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.

[0329] In some embodiments, compositions comprising "labeled" immunoconjugates are provided. Labels include, but are not limited to, labels or moieties that are directly detected (such as fluorescent, chromophore, electron-dense, chemiluminescent, and radioactive labels), as well as moieties such as enzymes or ligands that are indirectly detected, for example, via an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, rare earth chelates or fluorophores such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, e.g., firefly luciferase and bacterial luciferase, luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, J3-galactosidase, glucoamylase, lysozyme, sugar oxidases, e.g., glucose oxidase, galactose oxidase, glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase conjugated to enzymes that use hydrogen peroxide to oxidize dye precursors, such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals.

[0330] Conventional methods can be used to covalently bind these labels to protein or polypeptide.For example, by using coupling agents such as dialdehyde, carbodiimide, dimaleimide, bis-imidate, bis-diazotized benzidine, etc., immunoconjugate or antibody construct of the present invention can be tagged with above-mentioned fluorescent label, chemiluminescent label and enzyme label (see, for example, US3,645,090 (enzymes); US3,940,475 (fluorimetry); Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014-1021 (1974); Pain et al., J.Immunol.Methods, 40:219-230 (1981); Nygren, J.Histochem and Cytochem, 30:407-412 (1982)).

[0331] Conjugation of such labels, including enzymes, to immunoconjugates or antibody constructs is a standard procedure for those skilled in the art of immunoassay technology (see, for example, O'Sullivan et al. "Methods for the Preparation of Enzyme-Antibody Conjugates for Use in Enzyme Immunoassay," in Methods in Enzymology, ed. J. Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, New York, 1981), pp. 147-166). Suitable commercially available labeled antibodies can also be used.

[0332] Finally, after adding the labeled immunoconjugate, the amount of bound immunoconjugate is determined by washing away excess unbound labeled immunoconjugate, then measuring the amount of bound label using a detection method appropriate for the label, and correlating the measured amount with the amount of the immunoconjugate of interest in the biological sample. For example, in the case of an enzyme, the amount of color developed and measured is a direct measurement of the amount of the immunoconjugate of interest present. Specifically, when HRP is the label, color can be detected using the substrate TMD, using a reading wavelength of 450 nm and a reference wavelength of 620 or 630 nm.

[0333] In one example, after washing the enzyme-labeled secondary antibody against the unlabeled immunoconjugate from the stationary phase, the immobilized capture reagent is incubated with a substrate for the enzyme to develop and measure color or chemiluminescence, and the concentration of the antibody of interest is then calculated by comparing the color or chemiluminescence produced by a parallel run of the immunoconjugate of interest.

[0334] In some embodiments, the method involves bead-based immunoassays, ELISA assays, or mass spectrometry techniques. The mass analyzer of such a mass spectrometer includes, but is not limited to, quadrupole (Q), time-of-flight (TOF), ion trap, magnetic sector, Fourier transform ion cyclotron resonance (FT-ICR), or a combination thereof. The ion source of the mass spectrometer should primarily generate sample molecular ions or pseudo-molecular ions, as well as certain characterizable fragment ions. Examples of such ion sources include atmospheric pressure ionization sources, such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), and matrix-assisted laser desorption ionization (MALDI). ESI and MALDI are the two most commonly used methods for ionizing proteins for mass analysis of small molecules, such as by liquid chromatography-mass spectrometry (LC / MS) (Lee, M., LC / MS Applications in Drug Development (2002) J. Wiley & Sons, New York). Another example is surface-enhanced laser desorption ionization (SELDI). SELDI is a surface-based ionization technique that enables high-throughput mass spectrometry. SELDI is typically used to analyze complex mixtures of proteins and other biomolecules. SELDI uses chemically reactive surfaces, such as "protein chips," to interact with analytes, e.g., proteins, in solution. Such surfaces selectively interact with and immobilize the analytes. Thus, analytes of the invention can be partially purified on the chip and then rapidly analyzed in a mass spectrometer. Throughput can be increased by providing multiple reactive moieties at different sites on the substrate surface.

[0335] In another aspect, the present invention provides a method for detecting an antigen in a biological sample, comprising: (a) contacting the biological sample with an immunoconjugate described herein to allow the formation of the immunoconjugate; and (b) detecting or measuring the level of immunoconjugate bound to the sample. In some embodiments, the immunoconjugate is immobilized on a solid support. In some embodiments, the immobilized immunoconjugate is conjugated to biotin and bound to a streptavidin-coated microtiter plate.

[0336] 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 invention comprises 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 invention. The label or package insert indicates that the composition is used to treat cancer. The label or package insert further comprises instructions for administering the immunoconjugate composition to a cancer patient. Additionally, the article of manufacture may further comprise a second container containing 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.

[0337] In another aspect, the present invention 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 or diagnostic composition) provided herein. Another aspect of the present invention is a kit useful for a variety of purposes, such as, for example, killing target antigen-expressing cells; detecting target antigen-expressing cells; or quantifying, purifying, or immunoprecipitating target antigens from cells.

[0338] In some embodiments, the kit of the present invention 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 invention or a composition comprising the same (e.g., as 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 isolation and purification of the target antigen, the kit may contain an immunoconjugate of the present invention bound to beads (e.g., Sepharose beads). The present invention provides kits comprising antibodies for detecting and / or quantifying a target antigen in vitro, for example, by ELISA or Western blot. In some embodiments, the capture reagent (e.g., an immunoconjugate of the present invention) is coated or attached to a solid material (e.g., a bead, a microtiter plate, or a comb). The detectable antibody can be a labeled antibody that is directly detected, or an unlabeled antibody that is detected by a labeled antibody against the unlabeled antibody (e.g., an antibody produced in a different species). If the label is an enzyme, the kit usually includes the substrate and cofactor required by the enzyme; if the label is a fluorophore, a dye precursor that provides the detectable chromophore; and if the label is biotin, avidin, such as avidin, streptavidin, or streptavidin conjugated with HRP or β-galactosidase by MUG.

[0339] Similar to the products of the present invention, kits of the present invention 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 invention. 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 carrying out 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 may be provided as dry powders (including excipients), usually lyophilized, which, when dissolved, provide a reagent solution having the appropriate concentration for combination with the sample to be tested.

[0340] The present invention is further illustrated by the following non-limiting examples of immunoconjugates comprising the aforementioned structures and functions, particularly VHH polypeptides, platforms with molecular weights of 60 to 110 kDa, serum half-lives of less than 96 hours, which in some embodiments exhibit enhanced stability across the temperatures required for certain radiolabeling processes compared to other antibody fragment platforms, and in some embodiments, reduced loss of targeting ability due to radiolysis compared to other possible delivery platforms. Certain Numbered Embodiments of the Present Disclosure 1. An immunoconjugate for delivering an alpha-emitting radioisotope in vivo, comprising: a) an antibody construct consisting of two antigen-binding arms, each of the antigen-binding arms independently consisting of: (i) an antigen-binding region, (ii) a hinge region, and (iii) a variant constant region; the antigen-binding region is covalently linked to the hinge region, and the hinge region is covalently linked to the variant constant region, such that the hinge region is interposed between the antigen-binding region and the variant constant region, thereby linking the antigen-binding region and the variant constant region; and at least one of the antigen-binding regions has one or two overlapping regions. and b) a chelator capable of chelating an alpha-emitting radioisotope such that the antibody construct is linked to the alpha-emitting radioisotope; and the molecular weight of the immunoconjugate is 60 to 110 kDa, 60 to 100 kDa, 60 to 90 kDa, 65 to 90 kDa, and / or 70 to 90 kDa. 2. The immunoconjugate of embodiment 1, wherein said antigen-binding regions bind to the same antigen. 3. The immunoconjugate of embodiment 1, wherein the antigen-binding regions bind to different antigens. 4. The immunoconjugate of embodiment 1 or 2, wherein the antigen-binding regions are the same. 5. The immunoconjugate of embodiment 1, 2 or 3, wherein the antigen-binding regions are different. 6. The immunoconjugate of any one of embodiments 1 to 5, wherein each antigen-binding region consists of one or two VHH polypeptides. 7. The immunoconjugate of embodiment 6, wherein each antigen-binding region consists of one VHH polypeptide. 8. The immunoconjugate of embodiment 7, wherein said VHH polypeptides bind to the same antigen. 9. The immunoconjugate of embodiment 8, wherein said VHH polypeptides are the same. 10. The immunoconjugate of embodiment 7, wherein said VHH polypeptides bind to different antigens. 11. The immunoconjugate of any one of embodiments 1 to 10, wherein the variant constant regions are the same. 12. The immunoconjugate of any one of embodiments 1 to 10, wherein the variant constant regions are different. 13. The immunoconjugate of any one of embodiments 1 to 12, wherein the hinge regions are the same. 14. The immunoconjugate of any one of embodiments 1 to 12, wherein the hinge regions are different. 15. The immunoconjugate of any one of embodiments 1 to 14, wherein at least one of said variant constant regions consists of a CH2 domain and a CH3 domain, and said CH2 domain and said CH3 domain are human antibody domains. 16. The immunoconjugate of embodiment 15, wherein each variant constant region consists of a CH2 domain and a CH3 domain, and said CH2 domain and said CH3 domain are human antibody domains. 17. The immunoconjugate of any one of embodiments 1 to 16, wherein each variant constant region has at least one FcRn-binding mutation. 18. The immunoconjugate of any one of embodiments 1 or 17, wherein at least one of said FcRn-binding mutations is selected from the group consisting of positions 251, 252, 253, 254, 255, 288, 309, 310, 312, 385, 386, 388, 400, 415, 433, 435, 436, 439 and 447. 19. The immunoconjugate of any one of embodiments 1 to 18, wherein at least one of said variant constant regions has a reduced effector function compared to IgG1. 20. The immunoconjugate of any of embodiments 1 to 19, having a serum half-life of less than 96 hours, less than 72 hours, less than 60 hours, less than 48 hours, less than 36 hours, less than 24 hours, or less than 12 hours. 21. A radioimmunoconjugate comprising an immunoconjugate according to any one of embodiments 1 to 20 and an alpha-radioactive isotope. 22. The radioimmunoconjugate of embodiment 21, wherein the alpha-emitting isotope is selected from the group consisting of 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi. 23. The radioimmunoconjugate according to embodiment 22, wherein the radioisotope is 225-Ac. 24. A pharmaceutical composition comprising a radioimmunoconjugate according to any one of embodiments 21 to 23 and a pharmaceutically acceptable carrier. 25. A method for delivering an alpha-emitting isotope to cancer cells in vivo in a patient, comprising administering to said patient the pharmaceutical composition of embodiment 24. 26. A method of inhibiting the proliferation of cancer cells, comprising contacting said cancer cells with a radioimmunoconjugate according to any one of embodiments 21 to 23. 27. A method of killing cancer cells, comprising contacting said cancer cells with a radioimmunoconjugate according to any one of embodiments 21 to 23. 28. The method of embodiment 26 or 27, wherein the cancer cells are in vivo in a patient. 29. A method for treating cancer in a patient in need thereof, comprising administering to the patient the pharmaceutical composition of embodiment 24. 30. The method of embodiment 25, 28 or 29, wherein the patient is a human patient. 31. A kit comprising an immunoconjugate according to any one of embodiments 1 to 20, or a radioimmunoconjugate according to any one of embodiments 21 to 23, or a pharmaceutical composition according to embodiment 24. 32. A kit for the preparation of a pharmaceutical composition comprising an immunoconjugate according to any one of embodiments 1 to 20. 33. A kit for the preparation of a pharmaceutical composition, comprising a radioimmunoconjugate according to any one of embodiments 21 to 23. 34. An immunoconjugate for delivering an alpha-emitting radioisotope in vivo, comprising: a) an antibody construct consisting of two antigen-binding arms, each of which independently comprises: (i) an antigen-binding region, (ii) a hinge region, and (iii) a variant constant region; the antigen-binding region is covalently linked to the hinge region, and the hinge region is covalently linked to the variant constant region, such that the hinge region is interposed between the antigen-binding region and the variant constant region, thereby linking the antigen-binding region and the variant constant region; each of the antigen-binding regions consists of a single VHH polypeptide that binds to the same antigen and has the same amino acid sequence; and the variant constant regions are covalently linked to the same VHH polypeptide. and b) an antibody construct having the same amino acid sequence as each of the variant constant regions, each of the variant constant regions consisting of a CH2 domain and a CH3 domain, each of the variant constant regions having at least one FcRn-binding mutation; the hinge regions having the same amino acid sequence; and the antigen-binding arms being covalently linked to one another; and b) a chelating agent; the chelating agent is capable of chelating an alpha-radioisotope such that the antibody construct is linked to the alpha-radioisotope; and the molecular weight of the immunoconjugate is 60 to 110 kDa, 60 to 100 kDa, 60 to 90 kDa, 65 to 90 kDa, and / or 70 to 90 kDa. 35. An immunoconjugate for delivering an alpha-emitting radioisotope in vivo, comprising: a) an antibody construct consisting of two antigen-binding arms, each of the antigen-binding arms independently comprising: (i) an antigen-binding region, (ii) a hinge region, and (iii) a variant constant region; the antigen-binding region is covalently linked to the hinge region, and the hinge region is covalently linked to the variant constant region, such that the hinge region is interposed between the antigen-binding region and the variant constant region, thereby linking the antigen-binding region and the variant constant region; the antigen-binding regions are composed of single VHH polypeptides that bind to different antigens and have different amino acid sequences; and the variant constant regions are composed of the same VHH polypeptides. and b) a chelating agent capable of chelating an alpha radioisotope such that the antibody construct is linked to the alpha radioisotope; and wherein the molecular weight of the immunoconjugate is 60 to 110 kDa, 60 to 100 kDa, 60 to 90 kDa, 65 to 90 kDa, and / or 70 to 90 kDa. 36. The immunoconjugate of embodiment 34 or 35, wherein said CH2 domain and said CH3 domain are human antibody domains. 37. The immunoconjugate of any one of embodiments 34 to 36, wherein at least one of the FcRn-binding mutations is selected from the group consisting of positions 251, 252, 253, 254, 255, 288, 309, 310, 312, 385, 386, 388, 400, 415, 433, 435, 436, 439 and 447. 38. The immunoconjugate of any one of embodiments 34 to 37, wherein said variant constant region has a reduced effector function compared to IgG1. 39. The immunoconjugate of any of embodiments 34 to 38, having a serum half-life of less than 96 hours, less than 72 hours, less than 60 hours, less than 48 hours, less than 36 hours, less than 24 hours, or less than 12 hours. 40. A radioimmunoconjugate comprising an immunoconjugate according to any one of embodiments 34 to 39 and an alpha-radioactive isotope. 41. The radioimmunoconjugate of embodiment 40, wherein the alpha-emitting isotope is selected from the group consisting of 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi. 42. The radioimmunoconjugate according to embodiment 41, wherein the radioisotope is 225-Ac. 43. A pharmaceutical composition comprising a radioimmunoconjugate according to any one of embodiments 40 to 42 and a pharmaceutically acceptable carrier. 44. A method for in vivo delivery of an alpha-emitting isotope to cancer cells in a patient, comprising administering to said patient the pharmaceutical composition of embodiment 43. 45. A method for inhibiting the proliferation of cancer cells, comprising contacting said cancer cells with a radioimmunoconjugate according to any one of embodiments 40 to 42. 46. ​​A method of killing cancer cells, comprising contacting said cancer cells with a radioimmunoconjugate according to any one of embodiments 40 to 42. 47. The method of embodiment 45 or 46, wherein the cancer cells are in vivo in a patient. 48. A method for treating cancer in a patient in need thereof, comprising administering to the patient the pharmaceutical composition of embodiment 43. 49. The method of embodiment 44, 47 or 48, wherein the patient is a human patient. 50. A kit comprising an immunoconjugate according to any one of embodiments 34 to 39, a radioimmunoconjugate according to any one of embodiments 40 to 42, or a pharmaceutical composition according to embodiment 43. 51. A kit for the preparation of a pharmaceutical composition, comprising an immunoconjugate according to any one of embodiments 34 to 39. 52. A kit for the preparation of a pharmaceutical composition, comprising a radioimmunoconjugate according to any one of embodiments 40 to 42. 53. A targeted imaging complex comprising the immunoconjugate of any one of embodiments 1 to 20 or any one of embodiments 34 to 39, and further comprising an imaging metal. 54. The targeted imaging conjugate of embodiment 53, wherein the imaging metal is 111-In. 55. The immunoconjugate of embodiment 18 or 37, wherein at least one FcRn-binding mutation is selected from the group consisting of positions 253, 254, 310, 435 and 436. 56. The immunoconjugate of embodiment 55, wherein at least one FcRn-binding mutation is selected from the group consisting of 1253A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q and Y436A.

[0341] Specific Definitions Certain specific details are described herein 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 interpreted in an 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 including "and / or" unless the content clearly dictates otherwise. Furthermore, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0342] 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. Mausubel 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.

[0343] 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.

[0344] 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.

[0345] 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%, ±5%, or ±1%.

[0346] 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 the series of amino acids or amino acid residues that physically comprise a polypeptide. A "protein" is a macromolecule comprising 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 the 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.

[0347] 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, pyroglutamate, 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.

[0348] As used herein, the term "radioisotope" includes, but is not limited to, any one of alpha-emitting isotopes (interchangeably α-emitting isotopes), beta-emitting isotopes (interchangeably β-emitting isotopes), and / or gamma-emitting isotopes (interchangeably γ-emitting isotopes), such as, but not limited to, 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-Cu, 89-Zr, 137-Cs, 212-Pb, and 103-Pd.

[0349] As used herein, the term "radioimmunoconjugate" refers to a molecular complex comprising (1) an immunoconjugate according to the present invention 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 invention provides a radioimmunoconjugate comprising an α-emitting isotope and a β-emitting isotope. The term "radioconjugate" is used interchangeably herein with the term "radioimmunoconjugate." In one embodiment, the radioisotope is associated with a chelator of the radioimmunoconjugate. In one embodiment, the radioisotope is directly linked to the immunoconjugate.

[0350] As used herein, the term "immunoconjugate" refers to a molecular complex comprising at least one antigen-binding region (e.g., a variable region or 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 bound (by peptide bond or otherwise) to the antigen-binding region. In some embodiments, the immunoconjugate further comprises a chelating agent (interchangeably "chelator"). In one embodiment, the immunoconjugate comprises an antibody construct of the invention linked directly or indirectly to a cytotoxic agent or a radioisotope.

[0351] 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, intact antibodies, and functional (antigen-binding) antibody fragments thereof, such as 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, and tandem tri-scFv. Unless otherwise specified, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. The antibody can comprise a human IgG1 constant region. The antibody can comprise a human IgG4 constant region.

[0352] 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, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3), and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). "Framework region" and "FR" are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4). The exact amino acid sequence boundaries of a given CDR or FR are 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 antibody are selected from the methods that can be defined, including Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0353] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Rabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Both the Rabat and Chothia numbering schemes are based on the most common antibody region sequence lengths, with insertions accommodated by insertion letters, e.g., "30a," and deletions occurring 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.

[0354] 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 (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three CDRs (see, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated using the VH or VL domain from an antibody that binds the antigen and then screened against a library of complementary VL or VH domains, respectively (see, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0355] The antigen-binding region of the immunoconjugate described herein can be humanized. With respect to an immunoconjugate, "humanized" 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 can optionally include at least a portion of an antibody constant region derived from a human antibody.

[0356] Among the immunoconjugates provided are human immunoconjugates. A "human immunoconjugate" is an immunoconjugate having an antigen-binding region with an amino acid sequence corresponding to that of an antibody produced by a non-human source using a human or human cell, or a human antibody repertoire, including a human antibody library, or other human antibody-encoding sequence. This 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.

[0357] 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 arm directly or via one or more linkers in the compositions of the invention. The immunoconjugates of the invention 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).

[0358] The phrase "antigen-binding region" as used herein refers to the region of an immunoconjugate responsible for specific binding to an antigen, e.g., 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 a fragment thereof, as known to those skilled in the art. In one embodiment, the "antigen-binding region" of an antigen-binding arm contains 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, and which are the same or different.

[0359] The term "VHH polypeptide" as used herein encompasses natural and synthetic compositions and refers to polypeptides constituting VHH fragments known in the art, i.e., polypeptides constituting single-domain heavy chain-only variable domain fragments, or polypeptides structurally and functionally similar to VHH fragments, such structures being further described below and capable of specifically binding to 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 antigen with specificity and high affinity. In a preferred embodiment, the VHH polypeptide is a single heavy chain variable domain comprising the following arrangement: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. VHH polypeptides can be obtained, for example, as antigen-binding fragments of heavy chain-only antibodies produced 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.Please refer to PMID:9323027.

[0360] 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.

[0361] 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.

[0362] As used herein, a "linker" may also be referred to as a "linker sequence," a "spacer," a "tether sequence," or grammatical equivalents. A "linker" as referred to herein connects two distinct molecules that themselves have target binding, catalytic activity, or are naturally expressed and assembled as separate polypeptides, or that comprise distinct domains of the same polypeptide. For example, two distinct binding moieties, a heavy / light chain pair, 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 the heavy chain variable region in an scFv molecule; or they 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 proteins or protein domains, bonds via disulfide bonds, and bonds via chemical cross-linking reagents. In one aspect of this embodiment, the linker is a peptide bond produced by recombinant technology or peptide synthesis.

[0363] An antibody that "binds" to 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, which is generally a molecule of similar structure that lacks binding activity.

[0364] "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 complex or radioimmunoconjugate that binds to its target antigen has an affinity of <1 mM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 The dissociation constant (K D In some embodiments, the immunoconjugate or antibody construct or targeted imaging complex or radioimmunoconjugate of the invention binds 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.

[0365] As used herein, the term "mutated constant region" refers to a polypeptide comprising a portion of an immunoglobulin heavy chain constant region that has been modified from a native immunoglobulin amino acid sequence, preferably at one to several amino acid positions. Unless otherwise specified herein, the numbering of amino acid residues in an Fc region or constant region follows 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."

[0366] The 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 algorithm required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent 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 has been submitted, along with user documentation, to the U.S. Copyright Office, Washington, DC 20559, and 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.

[0367] 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, with, or against a given amino acid sequence B (alternatively, this may be referred to as a given amino acid sequence A having a particular % amino acid sequence identity with or containing a given amino acid sequence B) is calculated as follows: 100 times the 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 and B will not equal the % amino acid sequence identity of B and A. 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.

[0368] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction.Cyto...

Claims

1. 1. An immunoconjugate comprising: a) an antigen-binding region; b) an immunoglobulin heavy chain constant region; and c) a chelating agent, wherein said immunoconjugate has a molecular weight of between 60 and 110 kDa, and wherein said immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces binding of said immunoconjugate to fetal Fc receptors (FcRn).

2. The immunoconjugate of claim 1 , wherein the antigen-binding region comprises an scFv polypeptide or a VHH polypeptide.

3. The immunoconjugate described in claim 1 or 2, wherein the antigen-binding region specifically binds to HER2 or DLL3.

4. The immunoconjugate of any one of claims 1 to 3, wherein the immunoglobulin heavy chain constant region further comprises a change to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region, optionally wherein the changes to one or more amino acid residues that reduce an effector function of the immunoglobulin heavy chain constant region include, per EU numbering, L234A, L235E, G237A, A330S, and P331S.

5. The immunoconjugate of any one of claims 1 to 4, wherein an amino acid change to one or more amino acid residues that alters binding of the immunoconjugate to the fetal Fc receptor (FcRn) shortens the serum half-life of the immunoconjugate.

6. The immunoconjugate of any one of claims 1 to 5, wherein the alteration to one or more amino acid residues that alters binding of the immunoconjugate to fetal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of H310A, H310D, H310E, H310Q, H435A, H435Q, and combinations thereof, per EU numbering.

7. The immunoconjugate of any one of claims 1 to 5, wherein the changes to one or more amino acid residues that alter binding of the immunoconjugate to fetal Fc receptor (FcRn) are to amino acid residues selected from the list consisting of I253A, H310A, H435Q, and combinations thereof, per EU numbering.

8. The immunoconjugate described in any one of claims 1 to 7, wherein the immunoconjugate has a serum half-life of less than 72 hours.

9. The immunoconjugate of claim 1, wherein the chelating agent is an alpha-emitter chelating agent or a beta- or gamma-emitter chelating agent.

10. The immunoconjugate of claim 9, wherein the chelating agent is selected from the list consisting of DOTA, DO3A, DOTAGA, Py4Pa, Crown, Macropa, HEHA, CHXoctapa, Bispa, Noneunpa, and combinations thereof.

11. The immunoconjugate of any one of claims 1 to 10, further comprising a linker covalently linking the chelator to the immunoglobulin heavy chain constant region, the linker comprising one or more linker components selected from a stretcher unit, an amino acid unit, a spacer unit, a self-immolative spacer unit, or a combination thereof.

12. An immunoconjugate described in any one of claims 1 to 11, wherein the chelator or linker is covalently conjugated to the antigen binding region and / or the immunoglobulin heavy chain constant region at one or more lysine or cysteine ​​residues.

13. An immunoconjugate described in any one of claims 1 to 12, further comprising a radioactive isotope, wherein the radioactive isotope is an alpha emitter or a beta emitter.

14. The immunoconjugate of claim 13, wherein 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.

15. The immunoconjugate of claim 13, wherein the radioisotope is a beta emitter selected from 177-Lu, 90-Y, 67-Cu, and 153-Sm.

16. An immunoconjugate described in any one of claims 1 to 15, wherein the molecular weight of the immunoconjugate is between 60 and 90 kDa.

17. A pharmaceutical composition comprising an immunoconjugate according to any one of claims 1 to 12 and a pharma- ceutically acceptable excipient, diluent, or carrier.

18. A pharmaceutical composition comprising an immunoconjugate according to any one of claims 13 to 16 and a pharma- ceutically acceptable excipient, diluent, or carrier.

19. The pharmaceutical composition of claim 18 for use in a method for treating cancer or a tumor in an individual.

20. The pharmaceutical composition of claim 18 for use in delivering a radioisotope to cancer or tumor cells in an individual.

21. The pharmaceutical composition of claim 18 for use in a method for imaging a tumor in an individual.

22. A nucleic acid encoding an immunoconjugate described in any one of claims 1 to 8.

23. A host cell comprising the nucleic acid described in claim 22.

24. A method for producing the antigen-binding region and immunoglobulin heavy chain constant region of claim 1, comprising culturing a host cell of claim 23 under conditions suitable for expression of the antigen-binding region and the immunoglobulin heavy chain constant region, and optionally recovering the antigen-binding region and the immunoglobulin heavy chain constant region from the host cell.

25. A method for producing a radioactive immunoconjugate, comprising the step of loading or complexing an immunoconjugate described in any one of claims 9 to 12 with a radioactive isotope.

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