VHH antibody DOTA conjugates

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 immunoconjugate has a molecular weight of 60-110 kDa. The immunoconjugate can be used to deliver alpha-emitters and beta-emitters for tumor or cancer treatment.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 373,189, filed August 22, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] The excellent specificity of antibodies, such as IgG, for their antigens makes them a highly targeted platform for therapeutics. However, the typical serum half-life of at least 3 weeks for IgG limits therapeutic efficacy, especially due to prolonged exposure and chronic off-target toxicity. 225 alpha-emitting isotopes such as Ac (actinium-225), and 177 This is a disadvantage for the delivery of radioisotopes, including beta-emitting isotopes such as Lu (lutetium-177) and 90Y (yttrium-90). The emergence of engineered smaller antibody formats (e.g., monomeric scFvs, heavy-chain-only antibodies, or single-domain antibody fragments) offers the superior specificity of full-sized antibodies (e.g., IgG (approximately 150 kDa)) in smaller formats (e.g., 15–30 kDa) and much shorter serum half-lives (e.g., 30 minutes to 2 hours) (Bates A, Power, C, Antibodies (Basel) 8:28 (2019)). Unfortunately, these short half-lives do not allow sufficient time for effective target binding due to poor retention and tumor uptake. 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] 225Ac 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 cell killers, overcoming the acquired resistance observed in response to other therapies. Unfortunately, many challenges remain regarding systemic administration and achieving desired dosimetry in target versus non-target tissues as a result of decay events at different locations in vivo. The key to the application of α-emitting radionuclides as targeted therapeutics 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 generation, the time of therapeutic administration, the daughter nuclide decay pathway and half-life, circulation time, and the biodistribution and pharmacokinetics of the delivery vehicle. Unfortunately, the emission of alpha particles 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 "free" daughter nuclide that can induce multiple toxicities. See, e.g., Robertson A et al., Curr Radiopharm 11:156 (2018). Thus, 225 Nephrotoxicity caused by Ac recoil daughter nuclides (e.g., 213-Bi) has been reported to date. 225 This has been a major limitation to the therapeutic use of Ac (see, for example, 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 intervening radioactive decay can damage antibody components and target sequences, especially 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., Larsen R, Bruland O, J. Labeled Chem. Radiopharm. 36:1009-18 (1995)). At the higher specific activities required for therapeutic administration, immunoreactivity can rapidly decrease 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 α-emitting isotope-labeled antibodies was observed at doses exceeding 1,200 Gy (Zalutsky M et al., J Nucl Med. 42(10):1508-15(2001)). Therefore, identifying a suitable targeted delivery vehicle for α-emitting radioisotopes is not straightforward.

[0005] Furthermore, there are additional issues with targeted radiometric delivery platforms, including alpha- and beta-emitting radioisotopes, that require simultaneous optimization when designing such platforms, such as immunogenicity, specificity, tissue penetration, stability, ease of manufacture, and acceptable therapeutic window. Summary of the Invention

[0006] The present invention relates to immunoconjugates or radioimmunoconjugates, compositions comprising the immunoconjugates or radioimmunoconjugates, and methods of using such immunoconjugates and compositions. The immunoconjugates and compositions of the invention have many uses, for example, for delivery of radioisotopes to kill target cells (e.g., cancer cells that express the target antigen to which the radioimmunoconjugate binds), for 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 many of the 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 allowing the antibody fragment to be stored in the body long enough to exert therapeutic activity. 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. Furthermore, 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, the present specification describes an immunoconjugate comprising: a) an antigen-binding region; b) an immunoglobulin heavy chain constant region; and c) a chelating agent, wherein the immunoconjugate has a molecular weight of 60 to 110 kDa. In certain embodiments, the antigen-binding region comprises an scFv polypeptide or a VHH polypeptide. In certain embodiments, the antigen-binding region comprises an scFv polypeptide. In certain embodiments, the antigen-binding region comprises a VHH polypeptide. In certain embodiments, the antigen-binding region is humanized.

[0009] In some embodiments, Formula (I)

[0010] [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, R 1 is a chelating moiety or a radionuclide complex thereof; R 2 is the tumor-targeting moiety R 3 is a moiety capable of reacting with an amine (-NH2) or thiol (-SH) of X 1 Ha-NR a -, -NR a S(=O)2-, -NR a S(=O)NR a or one or more independently selected natural or unnatural amino acids, any free amine of the amino acid optionally being substituted with R 5 or -C(=O)R 5 and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R; R a are each independently selected from hydrogen and C1-C4 alkyl; L is -L 1 -L 2 -L 3 -L 4 -L 5 is a linker which is L 1 is unsubstituted or substituted C1-C 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, unsubstituted or substituted C-C 20 Alkenylene, unsubstituted or substituted C-C 20 Alkynylene, C4-C 20 Polyethylene glycol, -(X 3 CH2CH2) t -, unsubstituted or substituted cycloalkylene, unsubstituted or substituted heterocycloalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; X 3 are each independently O and NR 4 is selected from each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; L 2 is absent or unsubstituted or substituted C1-C 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, -C(=O)-(unsubstituted or substituted C-C 10 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 alkylene)-, -C(=O)-(CH2CH2X 3 ) m -(CH2) P -, -C(=O)NR 4 -(CH2CH2X 3 ) m -(CH2) p -, -NR 4 C(=O)-(CH2CH2X 3 ) m -(CH2) p - or -(CH2CH2X 3 ) m -(CH2) p - and R 4are each independently selected from hydrogen and C1-C6 alkyl; each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each p is independently 0, 1, or 2; L 3 is absent or is one or more independently selected groups selected from natural or unnatural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates, and any free amine (—NH) of an amino acid is optionally substituted with R 5 or -C(=O)R 5 and any free carboxylic acid of any amino acid is optionally substituted with -C(=O)NH-R5, and the substituted benzyl is independently substituted with halogen, -OH, -OR 5 , -CO2H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C1-C6 alkyl, wherein the substituted C1-C6 alkyl is selected from -OH, -CO2H, -NHR 5 , -C(=O)NHR 5 , and -NHC(=O)R 5 is replaced by L 4 is absent or unsubstituted or substituted C1-C 10 Alkylene, -C(=O)-(unsubstituted or substituted C-C 10 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 alkylene)-, -C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2X 3 ) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2X3 ) n -(CH2) q -, -(CH2CH2X 3 ) n -(CH2) q - or -(X 3 CH2CH2) n - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each q is independently 0, 1, or 2; L 5 does not exist or -C(=O)-(CH2) n -, -C(=O)NR 4 -(CH2) n -, -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2X 3 ) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -(CH2CH2X 3 ) n -(CH2) q -, -C(=O)-(X 3 CH2CH2) n - or -(X 3 CH2CH2) n - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each q is independently 0, 1, or 2; R a are each independently selected from hydrogen and C1-C4 alkyl; R b are each independently selected from hydrogen and C1-C4 alkyl; Heteroalkylene is a heteroalkylene group in which one carbon atom is -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NH)-, or -S(=O)(=NR 5 )-, -NR 5 -, -P(=O)OR b -, -NR a C(=O)-, -C(=O)NR a -, -OC(=O)NR a -, -NR a C(=O)NR a -, -NR a C(=N-CN)NR a -, -NR a C(=NR 5 )NR a -, or -NR a alkylene substituted with C(=O)O-; -L 1 -, -L 2 -, -L 3 -, -L 4 -, and -L 5 If one of - is replaced, -L 1 -, -L 2 -, -L 3 -, -L 4 -, and -L 5 - is halogen, -OH, -OR 5 , -CO2H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C1-C6 alkyl, wherein the substituted C1-C6 alkyl is selected from -OH, -CO2H, -NHR 5 , -C(=O)NHR 5 , and -NHC(=O)R 5 is replaced by R 5 are independently C1-C 10 Alkyl, C4-C 30 polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; However, L 3 If there is no R, then there is at least one R 5exists, or X 1 is -N(C1-C4 alkyl), -NR a S(=O)2-, -NR a S(=O)NR a - or one or more independently selected natural or unnatural amino acids.

[0011] In some embodiments, the immunoconjugate has Formula (II), Formula (III), or Formula (IV):

[0012] [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, R 1 is a chelating moiety or a radionuclide complex thereof; -NH-R 3 is a tumor-targeting moiety, X is absent, -O-, -S-, -S(=O)-, -S(=O)2-, or -NR a -, -C(=O)-, -NR a C(=O)-, -C(=O)NR a -, -C(=O)O-, -OC(=O)-, -OC(=O)NR a -, -NR a C(=O)NR a -, -NR a C(=S)NR a -, -NR a C(=O)O-, R a are each independently selected from hydrogen and C1-C4 alkyl; X 1 Ha-NR a -, -NR a S(=O)2-, -NR a S(=O)NR a or one or more independently selected natural or unnatural amino acids, any free amine of the amino acid optionally being substituted with R 5 or -C(=O)R 5and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R; R a are each independently selected from hydrogen and C1-C4 alkyl; L is -L 1 -L 2 -L 3 -L 4 -L 5 is a linker which is L 1 is unsubstituted or substituted C1-C 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, unsubstituted or substituted C-C 20 Alkenylene, unsubstituted or substituted C-C 20 Alkynylene, C4-C 20 Polyethylene glycol, -(X 3 CH2CH2) t -, unsubstituted or substituted cycloalkylene, unsubstituted or substituted heterocycloalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; X 3 are each independently O and NR 4 is selected from each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; L 2 is absent or unsubstituted or substituted C1-C 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, -C(=O)-(unsubstituted or substituted C-C 10 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 alkylene)-, -C(=O)-(CH2CH2X 3 ) m -(CH2) P -, -C(=O)NR 4 -(CH2CH2X3 ) m -(CH2) p -, -NR 4 C(=O)-(CH2CH2X 3 ) m -(CH2) p - or -(CH2CH2X 3 ) m -(CH2) p - and R 4 are each independently selected from hydrogen and C1-C6 alkyl; each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each p is independently 0, 1, or 2; L 3 is absent or is one or more independently selected groups selected from natural or unnatural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates, and any free amine (—NH) of an amino acid is optionally substituted with R 5 or -C(=O)R 5 and any free carboxylic acid of any amino acid is optionally substituted with -C(=O)NH-R5, and the substituted benzyl is independently substituted with halogen, -OH, -OR 5 , -CO2H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C1-C6 alkyl, wherein the substituted C1-C6 alkyl is selected from -OH, -CO2H, -NHR 5 , -C(=O)NHR 5 , and -NHC(=O)R 5 is replaced by L 4 is absent or unsubstituted or substituted C1-C 10 Alkylene, -C(=O)-(unsubstituted or substituted C-C 10 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4C(=O)-(unsubstituted or substituted C1-C 10 alkylene)-, -C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2X 3 ) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -(CH2CH2X 3 ) n -(CH2) q - or -(X 3 CH2CH2) n - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each q is independently 0, 1, or 2; L 5 does not exist or -C(=O)-(CH2) n -, -C(=O)NR 4 -(CH2) n -, -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2X 3 ) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -(CH2CH2X 3 ) n -(CH2) q -, -C(=O)-(X 3 CH2CH2) n - or -(X 3 CH2CH2) n - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each q is independently 0, 1, or 2; R a are each independently selected from hydrogen and C1-C4 alkyl; R b are each independently selected from hydrogen and C1-C4 alkyl; Heteroalkylene is a heteroalkylene group in which one carbon atom is -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NH)-, or -S(=O)(=NR 5 )-, -NR 5 -, -P(=O)OR b -, -NR a C(=O)-, -C(=O)NR a -, -OC(=O)NR a -, -NR a C(=O)NR a -, -NR a C(=N-CN)NR a -, -NR a C(=NR 5 )NR a -, or -NR a alkylene substituted with C(=O)O-; -L 1 -, -L 2 -, -L 3 -, -L 4 -, and -L 5 If one of - is replaced, -L 1 -, -L 2 -, -L 3 -, -L 4 -, and -L 5 - is halogen, -OH, -OR 5 , -CO2H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C1-C6 alkyl, wherein the substituted C1-C6 alkyl is selected from -OH, -CO2H, -NHR 5 , -C(=O)NHR 5 , and -NHC(=O)R5 is replaced by R 5 are independently C1-C 10 Alkyl, C4-C 30 polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; However, L 3 If there is no R, then there is at least one R 5 exists, or X 1 is -N(C1-C4 alkyl), -NR a S(=O)2-, -NR a S(=O)NR a - or one or more independently selected natural or unnatural amino acids.

[0013] In some embodiments, the immunoconjugate has formula (II):

[0014] [ka] or a pharmaceutically acceptable salt thereof.

[0015] In some embodiments, the immunoconjugate has formula (III):

[0016] [ka] or a pharmaceutically acceptable salt thereof.

[0017] In some embodiments, the immunoconjugate has formula (IV):

[0018] [ka] or a pharmaceutically acceptable salt thereof.

[0019] In some embodiments, R 2is the tumor-targeting moiety R 3 This is the moiety that can react with the amine (-NH2) of lysine.

[0020] In some embodiments, the compound of formula (II) has the formula (IIa):

[0021] [ka] or a pharmaceutically acceptable salt thereof, wherein -NHCH2CH2CH2CH2- is a tumor-targeting moiety R 3 is the side chain of the lysine residue.

[0022] In some embodiments, the compound of formula (III) has the formula (IIIa):

[0023] [ka] or a pharmaceutically acceptable salt thereof, wherein -NHCH2CH2CH2CH2- is a tumor-targeting moiety R 3 is the side chain of the lysine residue.

[0024] In some embodiments, R 2 is the tumor-targeting moiety R 3 It is a moiety that can react with the thiol (-SH) of cysteine.

[0025] In some embodiments, the compound of formula (IV) has the formula (IVa):

[0026] [ka] or a pharmaceutically acceptable salt thereof, wherein -NHCH2CH2CH2CH2- is a tumor-targeting moiety R 3 is the side chain of the lysine residue.

[0027] In some embodiments, the immunoconjugate has Formula (V), Formula (VI), Formula (VII), or Formula (VIII):

[0028] [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, R 1 is a chelating moiety or a radionuclide complex thereof; -SR 3 is a tumor-targeting moiety, X 1 Ha-NR a -, -NR a S(=O)2-, -NR a S(=O)NR a or one or more independently selected natural or unnatural amino acids, any free amine of the amino acid optionally being substituted with R 5 or -C(=O)R 5 and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R; R a are each independently selected from hydrogen and C1-C4 alkyl; L is -L 1 -L 2 -L 3 -L 4 -L 5 is a linker which is L 1 is unsubstituted or substituted C1-C 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, unsubstituted or substituted C-C 20 Alkenylene, unsubstituted or substituted C-C 20 Alkynylene, C4-C 20 Polyethylene glycol, -(X 3 CH2CH2) t -, unsubstituted or substituted cycloalkylene, unsubstituted or substituted heterocycloalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; X 3 are each independently O and NR 4is selected from each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; L 2 is absent or unsubstituted or substituted C1-C 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, -C(=O)-(unsubstituted or substituted C-C 10 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 alkylene)-, -C(=O)-(CH2CH2X 3 ) m -(CH2) P -, -C(=O)NR 4 -(CH2CH2X 3 ) m -(CH2) p -, -NR 4 C(=O)-(CH2CH2X 3 ) m -(CH2) p - or -(CH2CH2X 3 ) m -(CH2) p - and R 4 are each independently selected from hydrogen and C1-C6 alkyl; each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each p is independently 0, 1, or 2; L 3 is absent or is one or more independently selected groups selected from natural or unnatural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates, and any free amine (—NH) of an amino acid is optionally substituted with R 5 or -C(=O)R 5and any free carboxylic acid of any amino acid is optionally substituted with -C(=O)NH-R5, and the substituted benzyl is independently substituted with halogen, -OH, -OR 5 , -CO2H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C1-C6 alkyl, wherein the substituted C1-C6 alkyl is selected from -OH, -CO2H, -NHR 5 , -C(=O)NHR 5 , and -NHC(=O)R 5 is replaced by L 4 is absent or unsubstituted or substituted C1-C 10 Alkylene, -C(=O)-(unsubstituted or substituted C-C 10 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 alkylene)-, -C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2X 3 ) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -(CH2CH2X 3 ) n -(CH2) q - or -(X 3 CH2CH2) n - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each q is independently 0, 1, or 2; L 5 does not exist or -C(=O)-(CH2) n-, -C(=O)NR 4 -(CH2) n -, -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2X 3 ) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -(CH2CH2X 3 ) n -(CH2) q -, -C(=O)-(X 3 CH2CH2) n - or -(X 3 CH2CH2) n - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each q is independently 0, 1, or 2; R a are each independently selected from hydrogen and C1-C4 alkyl; R b are each independently selected from hydrogen and C1-C4 alkyl; Heteroalkylene is a heteroalkylene group in which one carbon atom is -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NH)-, or -S(=O)(=NR 5 )-, -NR 5 -, -P(=O)OR b -, -NR a C(=O)-, -C(=O)NR a -, -OC(=O)NR a -, -NR a C(=O)NR a -, -NR a C(=N-CN)NR a -, -NR a C(=NR 5 )NR a-, or -NR a alkylene substituted with C(=O)O-; -L 1 -, -L 2 -, -L 3 -, -L 4 -, and -L 5 If one of - is replaced, -L 1 -, -L 2 -, -L 3 -, -L 4 -, and -L 5 - is halogen, -OH, -OR 5 , -CO2H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C1-C6 alkyl, wherein the substituted C1-C6 alkyl is selected from -OH, -CO2H, -NHR 5 , -C(=O)NHR 5 , and -NHC(=O)R 5 is replaced by R 5 are independently C1-C 10 Alkyl, C4-C 30 polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; However, L 3 If there is no R, then there is at least one R 5 exists, or X 1 is -N(C1-C4 alkyl), -NR a S(=O)2-, -NR a S(=O)NR a - or one or more independently selected natural or unnatural amino acids.

[0029] In some embodiments, the immunoconjugate has formula (V):

[0030] [ka] or a pharmaceutically acceptable salt thereof.

[0031] In some embodiments, the immunoconjugate has formula (VI):

[0032] [ka] or a pharmaceutically acceptable salt thereof.

[0033] In some embodiments, the immunoconjugate has formula (VII):

[0034] [ka] or a pharmaceutically acceptable salt thereof.

[0035] In some embodiments, the immunoconjugate has formula (VIII):

[0036] [ka] or a pharmaceutically acceptable salt thereof.

[0037] In some embodiments, the compound of formula (V) has the formula (Va):

[0038] [ka] or a pharmaceutically acceptable salt thereof, where -SCH2- is a tumor-targeting moiety R 3 The thiol (-SH) is the side chain of the cysteine ​​residue in

[0039] In some embodiments, the compound of formula (VI) has formula (VIa):

[0040] [ka] or a pharmaceutically acceptable salt thereof, where -SCH2- is a tumor-targeting moiety R 3 The thiol (-SH) is the side chain of the cysteine ​​residue in

[0041] In some embodiments, the compound of formula (VI) has formula (VIa):

[0042] [ka] or a pharmaceutically acceptable salt thereof, where -SCH2- is a tumor-targeting moiety R 3 The thiol (-SH) is the side chain of the cysteine ​​residue in

[0043] In some embodiments, the compound of formula (VII) has the formula (VIIa)

[0044] [ka] or a pharmaceutically acceptable salt thereof, where -SCH2- is a tumor-targeting moiety R 3 The thiol (-SH) is the side chain of the cysteine ​​residue in

[0045] In some embodiments, the compound of formula (VIII) has the formula (VIIIa)

[0046] [ka] or a pharmaceutically acceptable salt thereof, where -SCH2- is a tumor-targeting moiety R 3 The thiol (-SH) is the side chain of the cysteine ​​residue in

[0047] In some embodiments, the tumor-targeting moiety R 3is a polypeptide that contains an antigen-binding region and an immunoglobulin heavy chain constant region, and the molecular weight of the polypeptide is 60 to 110 kDa.

[0048] In some embodiments, the antigen-binding region comprises an scFv polypeptide or a VHH polypeptide. In some embodiments, the immunoglobulin heavy chain constant region comprises an immunoglobulin CH2 domain, an immunoglobulin CH3 domain, or an immunoglobulin CH2 domain and CH3 domain. In some embodiments, the immunoglobulin heavy chain constant region is an IgA, IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the antigen-binding region is humanized, the immunoglobulin heavy chain constant region is a human immunoglobulin heavy chain constant region, or both. In some embodiments, the immunoglobulin heavy chain constant region comprises a modification 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 the neonatal Fc receptor (FcRn), or the immunoglobulin heavy chain constant region comprises a modification 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 the neonatal Fc receptor (FcRn). In some embodiments, the immunoglobulin heavy chain constant region comprises a modification to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region, or the immunoglobulin heavy chain constant region comprises a modification to one or more amino acid residues that alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn), or both. In some embodiments, the modification to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region is a modification that reduces complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or a combination thereof.

[0049] In certain embodiments, the antigen-binding region specifically binds to HER2 or DLL3. In certain embodiments, the antigen-binding region specifically binds to HER2. In certain embodiments, the antigen-binding region of the immunoconjugate 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 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 binds to HER2. In certain embodiments, the antigen-binding region specifically binds to DLL3. In certain embodiments, the antigen-binding region of the immunoconjugate 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 DLL3. 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: 30 and binds to DLL3. In certain embodiments, the immunoglobulin heavy chain constant region comprises an immunoglobulin CH2 domain, an immunoglobulin CH3 domain, or an immunoglobulin CH2 domain and CH3 domain. In certain embodiments, the immunoglobulin heavy chain constant region comprises an immunoglobulin CH2 domain and a CH3 domain. In certain embodiments, the immunoglobulin heavy chain constant region is a human immunoglobulin heavy chain constant region. In certain embodiments, the immunoglobulin heavy chain constant region is an IgA, IgG1, IgG2, IgG3, or IgG4 isotype. In certain embodiments, the immunoglobulin heavy chain constant region is an IgG1 isotype. In certain embodiments, the immunoglobulin heavy chain constant region is an IgG4 isotype. In certain embodiments, the immunoglobulin heavy chain constant region reduces an effector function of the immunoglobulin heavy chain constant region oror comprises a modification to one or more amino acid residues that alter binding of the immunoconjugate to neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises a modification 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 neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises a modification to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region. In certain embodiments, the immunoglobulin heavy chain constant region comprises a modification to one or more amino acid residues that alter binding of the immunoconjugate to neonatal Fc receptor (FcRn). In certain embodiments, the modification to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region is a modification that reduces complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or a combination thereof. In certain embodiments, the modification to one or more amino acid residues that reduces an effector function of an immunoglobulin heavy chain constant region is one of the following, according to EU numbering: (a) 297A, 297Q, 297G, or 297D; (b) 279F, 279K, or 279L; (c) 228P; (d) 235A, 235E, 235G, 235Q, 235R, or 235S; (e) 237A, 237E, 237K, 237N, or 237R; (f) 234A, 234V, or 234F; (g) 233P; (h) 328A; (i) 327Q or 327T; (j) 329A, 329G, or 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 2 54V, (p) 255N, (q) 256H, 256K, 256R, or 256V, (r) 264S, (s) 265H, 265K, 265S, 265Y, or 265A, (t) 267G, 267H, 267I, or 267K, (u) 268K, (v) 269N or 269Q, (w) 270A,270G, 270M, or 270N, (x) 271T, (y) 272N, (z) 292E, 292F, 292G, or 292I, (aa) 293S, (bb) 301W, (cc) 304E, (dd) 311E, 311G, or 311S, (ee) 316F, (ff) 328V, (gg) 330R, (hh) 339E or 339 L, (ii) 343I or 343V, (jj) 373A, 373G, or 373S, (kk) 376E, 376W, or 376Y, (ll) 380D, (mm) 382D or 382P, (nn) 385P, (oo) 424H, 424M, or 424V, (pp) 434I, (qq) 438G, (rr) 439E, 439H, or 439Q, (ss) 440A, 440D, 440E, 440F, 440M, 440T, or 440V, (tt) K322A, (uu) L235E, (vv) L234A and L235A, (ww) L234A, L235A, and G237A, (xx) L234A, L235A, and P329G, (yy) L234F, L235 E, and P331S, (zz) L234A, L235E, and G237A, (aaa), L234A, L235E, G237A, and P331S, (bbb) L234A, L235A, G237A, P238S, H268A, A330S, and P331S, (ccc) L234A, L235A, and P329A, (ddd) G236R and L328R, (eee) G237A, (fff) F241A, (ggg) V264A, (hhh) D265A, (iii) D265A and N297A, (jjj) D265A and N297G, (kkk) D270A, (lll) A330L, (mmm) P331A or P331S, or (nnn) E233P, (ooo) L234A, L235E, G237A, A330S, and P331S, or (ppp) any combination of (a)-(ppp). In certain embodiments, the modification to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region comprises L234A, L235E, G237A, A330S, and P331S according to EU numbering.Amino acid modifications to one or more amino acid residues that alter binding of the immunoconjugate to the neonatal Fc receptor (FcRn) decrease the serum half-life of the immunoconjugate. In certain embodiments, the modifications to one or more amino acid residues that alter binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are to amino acid residues selected from the list consisting of 251, 252, 253, 254, 255, 288, 309, 310, 312, 385, 386, 388, 400, 415, 433, 435, 436, 439, 447, and combinations thereof, according to EU numbering. In certain embodiments, the modifications to one or more amino acid residues that alter binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are to amino acid residues selected from the list consisting of 253, 254, 310, 435, 436, and combinations thereof, according to EU numbering. In certain embodiments, the modification to one or more amino acid residues that alter binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 1253A, 1253D, 1253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof, according to EU numbering. In certain embodiments, the modification to one or more amino acid residues that alter binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 1253A, S254A, H310A, H435Q, Y436A, and combinations thereof, according to EU numbering. In certain embodiments, the modification to one or more amino acid residues that alters binding of the immunoconjugate to neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of I253A, H310A, H435Q, and combinations thereof, according to 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 isIt has a serum half-life of less than 120 hours. In certain embodiments, the immunoconjugate has a serum half-life of less than 72 hours. In certain embodiments, the 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 certain embodiments, the antigen-binding region is linked to the immunoglobulin heavy chain constant region by a human IgG hinge region.

[0050] In certain embodiments, the chelating agent is conjugated to the antigen-binding region and / or the immunoglobulin heavy chain constant region at a ratio of 1:1 to 8:1. In certain embodiments, the chelating agent is conjugated to the antigen-binding region and / or the immunoglobulin heavy chain constant region at a ratio of 1:1 to 6:1. In certain embodiments, the chelating agent is conjugated to the antigen-binding region and / or the immunoglobulin heavy chain constant region at a ratio of 2:1 to 6:1. In certain embodiments, the immunoconjugate further comprises a radioisotope. In certain embodiments, the radioisotope is an alpha emitter. In certain embodiments, the radioisotope is 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi, and 213 In certain embodiments, the radioisotope is an alpha emitter selected from the list consisting of Bi. 225 In certain embodiments, the radioisotope is a β-emitter. In certain embodiments, the radioisotope is 177 Lu, 90 Y, 67 Cu, and 153The beta-emitter is selected from Sm. In certain embodiments, the immunoconjugate has a molecular weight of 60-100 kDa. In certain embodiments, the immunoconjugate has a molecular weight of 60-90 kDa. In certain embodiments, the immunoconjugate has a molecular weight of 65-90 kDa. In certain embodiments, the immunoconjugate has a molecular weight of 70-90 kDa. In certain embodiments, the immunoconjugate forms a dimer with another immunoconjugate. In certain embodiments, the immunoconjugate further comprises a pharmaceutically acceptable excipient or carrier. In certain embodiments, the immunoconjugate is formulated for intravenous administration.

[0051] Also described herein are methods of making an immunoconjugate, comprising loading the immunoconjugate with a radioisotope. In certain embodiments, the radioisotope is an alpha emitter. In certain embodiments, the radioisotope is 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi, and 213 In certain embodiments, the radioisotope is an alpha emitter selected from the list consisting of Bi. 225 In certain embodiments, the radioisotope is a β-emitter. In certain embodiments, the radioisotope is 177 Lu, 90 Y, 67 Cu, and 153 In certain embodiments, the radioisotope is a beta emitter selected from Sm. 177 This is Lu.

[0052] Also described herein are methods of treating cancer or tumor in an individual, the methods comprising administering an immunoconjugate to the individual, thereby treating the cancer or tumor. In certain embodiments, the individual is a human. In certain embodiments, the cancer or tumor is a solid cancer or tumor. In certain embodiments, the cancer or tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer. In certain embodiments, the method further comprises administering 0.5 μCi to 30.0 μCi per kilogram to the individual. In certain embodiments, the cancer or tumor expresses an antigen that is specifically bound by the immunoconjugate.

[0053] Also described herein are immunoconjugates for use in methods of treating cancer or tumors in individuals. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer or tumor is a solid cancer or tumor. In certain embodiments, the cancer or tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer. In certain embodiments, 0.5 μCi to 30.0 μCi per kilogram is administered to the individual. In certain embodiments, the cancer or tumor expresses an antigen that is specifically bound by the immunoconjugate.

[0054] Also described herein are methods for killing cancer cells in an individual, the methods comprising administering to the individual an immunoconjugate, thereby killing the cancer cells. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells. In certain embodiments, the method comprises administering to the individual between 0.1 μCi and 30.0 μCi per kilogram. In certain embodiments, the method comprises administering to the individual between 10 mCi and 75 mCi per square meter of body area. In certain embodiments, the cancer cells express an antigen that is specifically bound by the immunoconjugate.

[0055] Also described herein is the use of an immunoconjugate in a method for killing cancer cells in an individual. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells. In certain embodiments, the method comprises administering 0.5 μCi to 30.0 μCi per kilogram to the individual. In certain embodiments, the cancer cells express an antigen that is specifically bound by the immunoconjugate.

[0056] Also described herein are methods for delivering a radioisotope to cancer or tumor cells in an individual, the methods comprising administering an immunoconjugate to the individual, thereby delivering the radioisotope to the cancer or tumor cells. In certain embodiments, the individual is a human. In certain embodiments, the cancer or tumor cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells. In certain embodiments, the method comprises administering 0.5 μCi to 30.0 μCi per kilogram to the individual. In certain embodiments, the cancer or tumor cells express an antigen that is specifically bound by the immunoconjugate.

[0057] Also described herein are immunoconjugates for use in delivering radioisotopes to cancer cells or tumor cells in an individual. In certain embodiments, the individual is a human. In certain embodiments, the cancer cells or tumor cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells. In certain embodiments, the cancer cells or tumor cells express an antigen that is specifically bound by the immunoconjugate.

[0058] Also described herein are methods for imaging a tumor in an individual, comprising administering an immunoconjugate to the individual. In certain embodiments, the individual is a human. In certain embodiments, the cancer or tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer. In certain embodiments, the tumor expresses an antigen that is specifically bound by the immunoconjugate.

[0059] Also described herein are immunoconjugates for use in methods for imaging tumors in individuals. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer or tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer. In certain embodiments, the tumor expresses an antigen that is specifically bound by the immunoconjugate.

[0060] Nucleic acids encoding the immunoconjugates are also described herein. In certain embodiments, an expression vector comprises the nucleic acid. In certain embodiments, a cell comprises the nucleic acid or the expression vector. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a CHO cell.

[0061] In some embodiments, the subject radioisotope delivery platforms have a molecular size (e.g., 60 kDa-110 kDa) large enough to substantially reduce off-target toxicity, particularly renal damage (e.g., from α-emitting isotope cargo), and a small enough size to increase tissue penetration and the probability of a first decay event in the target tissue compared to conventional IgG while maintaining target specificity. Such a size results in preferential clearance by the liver as opposed to the kidney, protecting the kidney from radiation toxicity.

[0062] 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.

[0063] 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.

[0064] 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 specific chelators) compared to other possible delivery platforms that use antibody fragments.

[0065] In one embodiment, the present invention provides an immunoconjugate for delivering an alpha-emitting radioisotope in vivo. In one embodiment, the immunoconjugate is further capable of delivering other atoms in vivo. In one embodiment, the immunoconjugate is capable of delivering imaging metals in vivo (e.g., 111-In, 89-Zr, 64-Cu, 68-Ga, or 134-Ce).

[0066] In one embodiment, the immunoconjugate comprises an antibody construct and a chelating agent, and has a molecular weight of 60-110 kDa, preferably 60-100 kDa, preferably 60-90 kDa, preferably 65-90 kDa, preferably 70-90 kDa. The chelating agent is capable of chelating an α-emitting radioisotope such that the antibody construct is bound to the α-emitting radioisotope.

[0067] 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.

[0068] 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.

[0069] In one embodiment, the chelating agent comprises a radioisotope chelating moiety and a functional group that allows covalent binding 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.

[0070] 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.

[0071] In one embodiment, the present invention provides a pharmaceutical composition comprising a radioimmunoconjugate of the present invention and a pharmaceutically acceptable carrier.

[0072] In certain embodiments, the present invention provides a method for delivering an alpha-emitting radioisotope to cancer cells in a patient in vivo, comprising administering to the patient a radioimmunoconjugate or pharmaceutical composition of the present invention. In one embodiment, the patient is a human patient.

[0073] In one embodiment, the present invention provides a method for inhibiting the growth of cancer cells, comprising contacting the cancer cells with a radioimmunoconjugate of the present invention. In certain embodiments, 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.

[0074] In one embodiment, the present invention provides a method for killing cancer cells, comprising contacting the cancer cells with a radioimmunoconjugate of the present invention. In certain embodiments, the cancer cells are in vivo in a patient. In one embodiment, the method comprises administering a pharmaceutical composition of the present invention to the patient. In one embodiment, the patient is a human patient.

[0075] 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.

[0076] 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 an 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 111 In (indium-111), 89 Zr (zirconium-89), 64 Cu (copper-64), 68 Ga (gallium-69), and 134 In one embodiment, the imaging metal is selected from the group including Ce (Cesium-134). 111 In, 89 Zr, 64 Cu, 68 Ga, and 134In one embodiment, the imaging metal is selected from the group consisting of: 111 In one embodiment, the imaging metal is covalently bound to the immunoconjugate or antibody construct. In one embodiment, the imaging metal is associated with a chelator of the immunoconjugate. In one embodiment, the invention provides a method for determining the location of cancer cells in a patient in vivo, comprising administering to the patient a targeted imaging complex of the invention. In one embodiment, the patient is a human patient.

[0077] 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 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 radioimmuno ...

[0078] 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.

[0079] 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 4-8 hours, 6-12 hours, 8-16 hours, 12-24 hours, or 24-48 hours.

[0080] In one aspect, the invention includes an immunoconjugate of the invention, e.g., 177 Lu, 90 Y, 67 Cu, or 153 Radioimmunoconjugates are provided which further comprise a beta particle emitter, such as Sm. In one aspect, the present invention provides pharmaceutical compositions comprising such radioimmunoconjugates.

[0081] In one embodiment, the present invention provides a radioimmunoconjugate comprising an immunoconjugate of the present invention and further comprising an α particle emitter and a β and / or γ particle emitter, hi one embodiment, the present invention provides a pharmaceutical composition comprising such a radioimmunoconjugate.

[0082] In some embodiments, kits of the invention include reagents or pharmaceutical devices in addition to an immunoconjugate, radioimmunoconjugate, or pharmaceutical composition of the invention.

[0083] In some embodiments, the kits of the present invention are immunoassay kits for specifically detecting an antigen in a biological sample, and include (a) an immunoconjugate, radioimmunoconjugate, or targeted imaging complex described herein, and / or compositions thereof, and (b) instructions for detecting the immunoconjugate, radioimmunoconjugate, or targeted imaging complex.

[0084] 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.

[0085] In another aspect, the invention provides a vector comprising a nucleic acid provided herein. In some embodiments, the vector is an expression vector.

[0086] 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 a radioimmunoconjugate or pharmaceutical composition herein.

[0087] 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. For some further embodiments, the methods are for inhibiting the growth of and / or killing cancer cells or tumors.

[0088] 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.

[0089] In another aspect, the present invention provides a process for making a radioimmunoconjugate or pharmaceutical composition of the present invention, said method comprising the step of radiolabeling the immunoconjugate with a suitable isotope, e.g., an alpha particle emitter or a beta particle emitter.

[0090] 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 form other embodiments of the present invention, absent any description to the contrary. [Brief explanation of the drawings]

[0091] [Figure 1A] Binding of anti-HER2 and anti-DLL3 VHH-Fc constructs is shown. [Figure 1B] Binding of anti-HER2 and anti-DLL3 VHH-Fc constructs is shown. [Figure 2A] Binding of anti-HER2 and anti-DLL3 VHH-Fc constructs to cells expressing HER2 and / or DLL3 is shown. [Figure 2B] Binding of anti-HER2 and anti-DLL3 VHH-Fc constructs to cells expressing HER2 and / or DLL3 is shown. [Figure 2C] Binding of anti-HER2 and anti-DLL3 VHH-Fc constructs to cells expressing HER2 and / or DLL3 is shown. [Figure 3A] 1 shows the internalization of anti-HER2 and anti-DLL3 VHH-Fc constructs in cells expressing HER2 and DLL3. [Figure 3B] 1 shows the internalization of anti-HER2 and anti-DLL3 VHH-Fc constructs in cells expressing HER2 and DLL3. [Figure 4] 1 shows the self-interaction data of anti-HER2 and anti-DLL3 VHH-Fc constructs. [Figure 5] 1 shows a diagram of the chemical synthesis of the linker molecule. [Figure 6] 1 shows a diagram of the chemical synthesis of the linker molecule. [Figure 7A] The immunoreactive fractions of the different VHH-Fc constructs are shown. [Figure 7B] The immunoreactive fractions of the different VHH-Fc constructs are shown. [Figure 7C] The immunoreactive fractions of the different VHH-Fc constructs are shown. [Figure 8] 1 shows a comparison of imaging with 111In-labeled VHH-Fc compared to the biodistribution of 225Ac-labeled VHH-Fc. [Figure 9A] 1 shows the biodistribution of labeled anti-HER2 VHH-Fc constructs over time. [Figure 9B] 1 shows the biodistribution of labeled anti-HER2 VHH-Fc constructs over time. [Figure 9C] 1 shows the biodistribution of labeled anti-HER2 VHH-Fc constructs over time. [Figure 9D] 1 shows the biodistribution of labeled anti-HER2 VHH-Fc constructs over time. [Figure 10A] Tumor:non-tumor tissue ratios of labeled anti-HER2 VHH-Fc constructs are shown. [Figure 10B] Tumor:non-tumor tissue ratios of labeled anti-HER2 VHH-Fc constructs are shown. [Figure 10C] Tumor:non-tumor tissue ratios of labeled anti-HER2 VHH-Fc constructs are shown. [Figure 11] 1 shows the biodistribution of labeled anti-HER2 VHH-Fc constructs. [Figure 12] Figure 1 shows the systemic clearance of 111In-labeled VHH-Fc (H101) and VHH-Fc variants (H105, H107, and H108). [Figure 13]1 shows the biodistribution of labeled anti-DLL3 VHH-Fc constructs over time. [Figure 14] 1 shows the biodistribution of labeled anti-DLL3 VHH-Fc constructs. [Figure 15A] Figure 15 shows the biodistribution of 225Ac-labeled anti-HER2 (Figure 15A) and anti-DLL3 (Figure 15B) VHH-Fc constructs. [Figure 15B] Figure 15 shows the biodistribution of 225Ac-labeled anti-HER2 (Figure 15A) and anti-DLL3 (Figure 15B) VHH-Fc constructs. [Figure 16A] 1 shows the results of a toxicity study performed with 225Ac-labeled anti-HER2 VHH-Fc constructs. [Figure 16B] 1 shows the results of a toxicity study performed with 225Ac-labeled anti-HER2 VHH-Fc constructs. [Figure 16C] 1 shows the results of a toxicity study performed with 225Ac-labeled anti-HER2 VHH-Fc constructs. [Figure 17] Immunoreactive fractions of different anti-DDL3 VHH-Fc constructs loaded with 177Lu are shown. [Figure 18] 1 shows the chemical structures of certain linker-chelators described herein. [Figure 19A] 1 shows imaging experiments with 111In in naive mice to measure the total body clearance of radioactivity using different conjugates (see Example B-26). [Figure 19B] 1 shows imaging experiments with 111In in naive mice to measure the total body clearance of radioactivity using different conjugates (see Example B-26). DETAILED DESCRIPTION OF THE INVENTION

[0092] The present invention will now be described in more detail using exemplary, 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 complete, 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.

[0093] In particular, in embodiments, the present invention addresses many of the challenges inherent in the targeted delivery of radioisotopes in vivo through the selection and specific construction 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 (e.g., 60 kDa-110 kDa) large enough to substantially reduce off-target toxicity, particularly renal damage (e.g., from α- or β-emitting radioisotope cargoes), yet small enough to increase tissue penetration and the probability of a first decay event in the target tissue compared to conventional IgG while maintaining target specificity. In some embodiments, the subject radioisotope delivery platforms are useful for the 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), 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.

[0094] 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 a specific affinity of ≦1 μM, <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 antibody binds to a specific antigen and / or epitope with an affinity characterized by a KD of 0.01 M.

[0095] 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 more than 2 to 8 hours).

[0096] 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 chelating moiety or a radionuclide complex thereof. 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 chelating moiety or a radionuclide complex thereof, wherein the immunoconjugate has a molecular weight of 60-110 kDa.

[0097] 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 chelating moiety or a radionuclide complex thereof. 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 chelating moiety or a radionuclide complex thereof, wherein the immunoconjugate has a molecular weight of 60 to 110 kDa.

[0098] In one embodiment, an immunoconjugate of the present disclosure comprises a) a VHH antigen-binding region, b) an immunoglobulin Fc region, collectively referred to as VHH-Fc, and c) a chelating moiety or a radionuclide complex thereof. 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 moiety or a radionuclide complex thereof, wherein the immunoconjugate has a molecular weight of 60-110 kDa.

[0099] 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 chelating moiety or a radionuclide complex thereof. 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 chelating moiety or a radionuclide complex thereof, wherein the immunoconjugate has a molecular weight of 60-110 kDa. In a specific embodiment, the variant immunoglobulin Fc region comprises one or more amino acid modifications to decrease the serum or plasma half-life of the immunoconjugate.

[0100] In some embodiments, the radioisotope delivery platform has a size greater than about 60 kDa to avoid certain toxicities from alpha-emitting isotope cargoes, such as off-target nephrotoxins. In some embodiments, the radioisotope delivery platform has a size less than about 110 kDa to improve tumor penetration. In some embodiments, the radioisotope delivery platform has a size of 60-110 kDa due to its dimeric structure of two individual antigen-binding arms, each having a hinge region and a VHH polypeptide fused to a wild-type or variant constant region. In some embodiments, the variant constant region has specific amino acid substitutions relative to the wild-type Fc region to reduce half-life and / or eliminate Fc effector function.

[0101] 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, which in turn is covalently linked to the variant constant region of the arm, such that the hinge region is interposed between the antigen-binding region and the variant constant region in the antigen-binding arm, thereby linking the antigen-binding region and the variant constant region.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] antigen binding region The antigen-binding region confers specificity to the immunoconjugate and may suitably comprise a small antigen-binding polypeptide. Such small antigen-binding polypeptides offer advantages such as a reduced overall size of the immunoconjugate molecule, enabling tumor penetration and labeling. Small antigen-binding polypeptides may lack certain regions essential for binding, such as the light chain constant region, heavy chain constant region, CH1 region, or hinge region. In certain embodiments, the antigen-binding region may lack the light chain variable region. In certain embodiments, small antigen-binding regions may have a molecular weight of 10 kDa to 40 kDa.

[0108] 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 kDa to about 35 kDa, The small antigen-binding domain has a molecular weight of about 15 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 domain 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.

[0109] The antigen-binding region may comprise a VHH polypeptide, an scFv polypeptide, or a VNAR polypeptide. In certain embodiments, the antigen-binding region comprises a VHH polypeptide. In certain embodiments, the antigen-binding region comprises an ScFv polypeptide. In certain embodiments, the antigen-binding region comprises a VNAR polypeptide. In certain embodiments, the antigen-binding region is humanized.

[0110] The antigenic region may include specificity for an antigen selected by one skilled 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 certain 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, CD19, 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 (DR2R), or IL-1. 5), PD-L1, mesothelin, bombesin, EpCAM, DARPP, CSPG4, galectin-3, integrin αvβ1, integrin αvβ3, integrin αvβ5, integrin αvβ6, integrin α5β1, integrin α-3, integrin α-5, integrin β-6, nectin-4, Wnt activation inhibitor 1, DLL3, transferrin receptor, folate receptor α, tissue factor, BCMA, c-Met, LIV-1, AXL, AFP, ENPP3, CLDN6 / 9, DPEP3, RNF43, LRRC15, PTK7, P-cadherin, FLT3, EphA2, MTI-MMP, CXCR6, GD2, or smoothened antigen (Smo). In certain embodiments, the tumor antigen comprises human epidermal growth factor receptor 2 (HER2), delta-like ligand 3 (DLL3), folate receptor alpha (FOLR1), or Wnt activation inhibitory factor 1 (WAIF1).In certain embodiments, the tumor antigen comprises HER2. In certain embodiments, the tumor antigen comprises DLL3. In certain embodiments, the tumor antigen comprises FOLR1. In certain embodiments, the tumor antigen comprises WAIF1. In certain embodiments, the tumor antigen comprises TROP2. In certain embodiments, the tumor antigen expresses EGFR. In certain embodiments, the tumor antigen comprises PSA. In certain embodiments, the tumor antigen comprises MUC-1. In certain embodiments, the tumor antigen comprises CEA. In certain embodiments, the tumor antigen comprises NY-ESO-1.

[0111] 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.

[0112] In certain embodiments, the antigen-binding region of the immunoconjugate comprises a) a CDR 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.

[0113] 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: 30 and that binds to DLL3.

[0114] In certain embodiments, 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.

[0115] In some embodiments, the immunoconjugates of the invention comprise, for example, an autonomous V Hdomains (e.g., derived from camelid, mouse, or human sources), single domain antibody domains (sdAb), camelid-derived heavy chain antibody domains (V H H fragment or V H domain fragment), Camelidae V H H fragment or V H Heavy chain antibody domain derived from domain fragment, heavy chain antibody domain derived from cartilaginous fish, immunoglobulin novel antigen receptor (IgNAR), V NAR fragments, single-chain variable (scFv) fragments, nanobodies, V H A "camelized" scaffold containing the heavy chain and C domains H Single-domain Fd fragment, 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), V L , V H , C L , and C H Disulfide-stabilized antigen-binding (Fab) fragments consisting of one domain, scFvs containing disulfide-stabilized heavy and light chains (sc-dsFvs), bivalent nanobodies, bivalent minibodies, bivalent F(ab')2 fragments (Fab dimers), and bispecific tandem V H Includes synthetically engineered antibody derivatives such as 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 paratope and target antigen binding function.

[0116] 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.

[0117] In one aspect, the present invention provides an antibody construct (alone or in the context of a respective immunoconjugate, radioimmunoconjugate or targeted imaging complex of the 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.

[0118] 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.

[0119] In some embodiments, the present disclosure provides an immunoconjugate that specifically binds to an antigen. In some embodiments, the immunoconjugate comprises an antibody construct comprising a heavy chain variable region (HVR-H) comprising three CDRs: hCDR1, hCDR2, and hCDR3, for example, derived from a camelid antibody or IgNAR. In some embodiments, the immunoconjugate comprises (a) a light chain variable region (HVR-L) comprising three CDRs: lCDR1, lCDR2, and lCDR3, and (b) a heavy chain variable region (HVR-H) comprising three CDRs: hCDR1, hCDR2, and hCDR3. In some embodiments, the antibody construct is chimeric or humanized.

[0120] In some embodiments, immunoconjugates of the invention comprise antibody constructs comprising an antigen-binding domain that is an antibody fragment, including, 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: hCDR1, hCDR2, and hCDR3.

[0121] 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 can 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 catabolism and elimination of the immunoconjugate from the kidney to the liver. This can provide a safety advantage, particularly for radioimmunoconjugates, as the kidney is more sensitive to radiation than the liver. Modifications affecting effector function or serum half-life can be made to residues present in the heavy chain constant region involved in neonatal Fc receptor (FcRn) binding. Binding to FcRn generally contributes to an increase in the half-life of immunoglobulin Fc-containing molecules, and therefore, reduced FcRn binding can reduce the half-life of Fc-containing molecules. Reduced FcRn binding can provide benefits such as a reduced half-life of the immunoconjugate and, therefore, reduced subsequent toxicity due to cytotoxic agents or radioisotopes. In certain embodiments, the immunoglobulin constant region comprises or consists of an Fc region. In certain embodiments, the immunoglobulin heavy chain constant region comprises an immunoglobulin CH2 domain, an immunoglobulin CH3 domain, or an immunoglobulin CH2 and CH3 domains. In certain embodiments, the immunoglobulin heavy chain constant region comprises an immunoglobulin CH2 and CH3 domains. For treatment or imaging of human individuals, the immunoglobulin heavy chain constant region may be human, which may prevent or reduce endogenous immune responses to the immunoconjugate. In certain embodiments, the immunoglobulin heavy chain constant region is a human immunoglobulin heavy chain constant region. In specific embodiments, the immunoglobulin heavy chain constant region is of the IgA, IgG1, IgG2, IgG3, or IgG4 isotype.In certain embodiments, the immunoglobulin heavy chain constant region is of the IgG1 isotype. In certain embodiments, the immunoglobulin heavy chain constant region is of the IgG4 isotype.

[0122] The immunoglobulin heavy chain constant region may be a variant constant region comprising one or more modifications 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 modifications to one or more amino acid residues that reduce an effector function of the immunoglobulin heavy chain constant region or alter binding of the immunoconjugate to neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises modifications to one or more amino acid residues that reduce an effector function of the immunoglobulin heavy chain constant region or decrease binding of the immunoconjugate to neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises modifications to one or more amino acid residues that reduce an effector function of the immunoglobulin heavy chain constant region and decrease binding of the immunoconjugate to neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises modifications to one or more amino acid residues that reduce an effector function of the immunoglobulin heavy chain constant region. In certain embodiments, the immunoglobulin heavy chain constant region comprises a modification to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn).

[0123] Modifications to the heavy chain constant region of the immunoconjugate can reduce effector functions 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, modifications to one or more amino acid residues that reduce an effector function of an immunoglobulin heavy chain constant region are modifications that reduce complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or a combination thereof. In certain embodiments, the modification to one or more amino acid residues that reduces an effector function of an immunoglobulin heavy chain constant region is one of the following, according to EU numbering: (a) 297A, 297Q, 297G, or 297D; (b) 279F, 279K, or 279L; (c) 228P; (d) 235A, 235E, 235G, 235Q, 235R, or 235S; (e) 237A, 237E, 237K, 237 N, 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, 25 4H, 254I, 254N, 254P, 254Q, 254T, or 254V, (p) 255N, (q) 256H, 256K, 256R, or 256V, (r) 264S, (s) 265H, 265K, 265S, 265Y, or 265A, (t) 267G, 267H, 267I, or 267K, (u) 268K, (v) 269N or 269Q, (w) 270A, 270G, 270M, or 270N, (x) 271T, (y) 272N, (z) 292E, 292F, 292G, or 292I, (aa) 293S, (bb) 301W, (cc) 304E, (dd) 311E, 311G, or 311S, (ee) 316F, (ff) 328V, (gg) 330R, (hh) 339E or 339L, (ii) 343I or 343V, (jj) 373A, 373G, or 373S, (kk) 376E, 376W,or 376Y, (ll) 380D, (mm) 382D or 382P, (nn) 385P, (oo) 424H, 424M, or 424V, (pp) 434I, (qq) 438G, (rr) 439E, 439H, or 439Q, (ss) 440A, 440D, 440E, 440F, 440M, 440T, or 440V, (tt) K32 2A, (uu)L235E, (vv)L234A and L235A, (ww)L234A, L235A, and G237A, (xx)L234A, L235A, and P329G, (yy)L234F, L235E, and P331S, (zz)L234A, L235E, and G237A, (aaa), L234A, L235E, G237A, and P 331S, (bbb) L234A, L235A, G237A, P238S, H268A, A330S, and P331S, (ccc) L234A, L235A, and P329A, (ddd) G236R and L328R, (eee) G237A, (fff) F241A, (ggg) V264A, (hhh) D265A, (iii) D265A and N In a specific embodiment, the modification to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region is selected from the list consisting of L234A, L235E, G237A, A330S, and P331S, according to EU numbering.

[0124] Modifications to the heavy chain constant region of the immunoconjugate can decrease the serum half-life of the immunoconjugate. In certain embodiments, amino acid modifications that alter or decrease binding of the immunoconjugate to neonatal Fc receptor (FcRn) decrease the serum half-life of the immunoconjugate. In certain embodiments, the modification that alters or decreases binding of the immunoconjugate to neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of 251, 252, 253, 254, 255, 288, 309, 310, 312, 385, 386, 388, 400, 415, 433, 435, 436, 439, 447, and combinations thereof, according to EU numbering. In certain embodiments, the modification that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of 253, 254, 310, 435, 436, and combinations thereof, according to EU numbering. In certain embodiments, the modification that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of I253A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof, according to EU numbering. In certain embodiments, the modification that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 1253A, S254A, H310A, H435Q, Y436A, and combinations thereof, according to EU numbering. In certain embodiments, the modification that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 1253A, H310A, H435Q, and combinations thereof, according to EU numbering.In a specific embodiment, the modification that alters or decreases binding of the immunoconjugate to neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of H310A, H435Q, and combinations thereof, according to EU numbering.

[0125] 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 an I253A substitution according to EU numbering.

[0126] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 2. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 2, wherein the heavy chain constant region comprises a S254A substitution according to EU numbering.

[0127] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 3. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 3, wherein the heavy chain constant region comprises an H310A substitution according to EU numbering.

[0128] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 4. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 4, wherein the heavy chain constant region comprises an H435Q substitution according to EU numbering.

[0129] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 5. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 5, wherein the heavy chain constant region comprises a Y436A substitution according to EU numbering.

[0130] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 6. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 6, wherein the heavy chain constant region comprises H310A / H435Q substitutions according to EU numbering.

[0131] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 7. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 7, wherein the heavy chain constant region comprises L234A, L235E, G237A, A330S, and P331S according to EU numbering.

[0132] 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 a sequence identical to SEQ ID NO: 8, wherein the heavy chain constant region comprises L234A, L235E, G237A, H310A, A330S, and P331S according to EU numbering.

[0133] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 9. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence identical to SEQ ID NO: 9, wherein the heavy chain constant region comprises L234A, L235E, G237A, H435Q, A330S, and P331S according to EU numbering.

[0134] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 10. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence identical to SEQ ID NO: 10 according to EU numbering.

[0135] In one embodiment, each of the two variant constant regions has at least one FcRn-binding mutation. In one embodiment, each of the two variant constant regions has the same FcRn-binding mutation. In one embodiment, each of the two variant constant regions has a different FcRn-binding mutation.

[0136] 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.

[0137] Modifications that affect FcRn binding can decrease the serum half-life of the immunoconjugate, thus allowing one of skill in the art to select a half-life appropriate for a particular imaging or therapeutic goal. In certain embodiments, the immunoconjugate has a serum half-life of about 12 hours to about 120 hours. In certain embodiments, the immunoconjugate is administered for about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 12 hours to about 48 hours, about 12 hours to about 60 hours, about 12 hours to about 72 hours, about 12 hours to about 84 hours, about 12 hours to about 96 hours, about 12 hours to about 108 hours, about 12 hours to about 120 hours, about 24 hours to about 36 hours, about 24 hours to about 48 hours, about 24 hours to about 60 hours, about 24 hours to about 72 hours, about 24 hours to about 84 hours, about 24 hours to about 96 hours, about 24 hours to about 108 hours, about 24 hours to about 120 hours, about 36 hours to about 48 hours, about 36 hours to about 60 hours, about 36 hours to about 72 hours, about 36 hours to about 84 hours, about 36 hours to about 96 hours, about 36 hours to about 36 hours The serum half-life is 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, about 72 hours to about 120 hours, about 84 hours to about 96 hours, about 84 hours to about 108 hours, about 84 hours to about 120 hours, about 96 hours to about 108 hours, about 96 hours to about 120 hours, or about 108 hours to about 120 hours. In certain embodiments, the immunoconjugate has a serum half-life of about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours, or about 120 hours. In certain embodiments, the immunoconjugate has a serum half-life of at least about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, or about 108 hours.In certain embodiments, the immunoconjugate has a serum half-life of 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.

[0138] In certain embodiments, the immunoconjugate has a serum half-life of about 1 day to about 10 days. In certain embodiments, the immunoconjugate has a serum half-life of about 1 day to about 2 days, about 1 day to about 3 days, about 1 day to about 4 days, about 1 day to about 5 days, about 1 day to about 6 days, about 1 day to about 7 days, about 1 day to about 8 days, about 1 day to about 9 days, about 1 day to about 10 days, about 2 days to about 3 days, about 2 days to about 4 days, about 2 days to about 5 days, about 2 days to about 6 days, about 2 days to about 7 days, about 2 days to about 8 days, about 2 days to about 9 days, about 2 days to about 10 days, about 3 days to about 4 days, about 3 days to about 5 days, about 3 days to about 6 days, about 3 days to about 7 days, about 3 days to about 8 days, about It has a serum half-life of 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, about 4 to about 8 days, 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, 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 at most about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

[0139] In certain embodiments, the heavy chain constant region has a molecular weight of about 10 kDa to about 25 kDa. In certain embodiments, the heavy chain constant region has a molecular weight of about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, about 15 kDa to about 20 kDa, about 15 kDa to about 25 kDa, or about 20 kDa to about 25 kDa. In certain embodiments, the heavy chain constant region has a molecular weight of about 10 kDa, about 15 kDa, about 20 kDa, or about 25 kDa. In certain embodiments, the heavy chain constant region has a molecular weight of at least about 10 kDa, about 15 kDa, or about 20 kDa. In certain embodiments, the heavy chain constant region has a molecular weight of at most about 15 kDa, about 20 kDa, or about 25 kDa.

[0140] 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 variant constant region of the present invention. Natural and synthetic hinge regions that link immunoglobulin components are well known in the art and are available for use in the present invention. See, for example, US 8,067,548 and references therein.

[0141] In one embodiment, the hinge regions of the immunoconjugates are the same. In one embodiment, the hinge regions of the immunoconjugates are different.

[0142] The antigen-binding region and the heavy chain constant region (with or without the modified amino acid sequence) can be connected by a suitable hinge or linker sequence. In certain embodiments, the antigen-binding region is connected to the immunoglobulin heavy chain constant region by a linker amino acid sequence or a human IgG hinge region. Suitable IgG hinge regions comprise and include the hinge region of IgG1 or IgG4. In certain embodiments, the hinge region is an IgG1 hinge region. In certain embodiments, the hinge region is an IgG1 hinge region with a C220S substitution according to EU numbering. Suitable hinge regions include those described in Wu et al., "Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange," Protein Engineering, Design and Selection, Volume 14, Issue 12, December 2001, Pages 1025-1033; Shu et al., "Secretion of a single-gene-encoded immunoglobulin from myeloma cells," Proceedings of the National Academy of Sciences, September 1993, 90(17) 7995-7999; Davis et al., "Abatacept binds to the Fc receptor CD64 but does not mediate complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity," J Rheumatol. November 2007;34(11):2204-10. Suitable hinges may also include non-IgG-based polypeptide linkers. The linker amino acid sequence may include primarily the following amino acid residues: Gly, Ser, Ala, or Thr.The linker peptide must be of an appropriate length to link the two molecules in a manner that allows them to 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 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.

[0143] 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 60 kDa to about 160 kDa, about 60 kDa to about 180 kDa, about 60 kDa to about 190 kDa, about 60 kDa to about 210 kDa, about 60 kDa to about 230 kDa, about 60 kDa to about 260 kDa, about 60 kDa to about 280 kDa, about 60 kDa to about 290 kDa, about 60 kDa to about 300 kDa, about 60 kDa to about 310 kDa, about 60 kDa to about 320 kDa, about 60 kDa to about 350 kDa, about 60 kDa to about 360 kDa, about 60 kDa to about 370 kDa, about 60 kDa to about 380 kDa, about 60 kDa to about 390 kDa, about 60 kDa to about 400 kDa, about 60 kDa to about 410 kDa, about 6 5 kDa 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, Approximately 70kDa to approximately 100kDa, approximately 70kDa to approximately 110kDa, approximately 70kDa to approximately 120kDa, approximately 75kDa to approximately 80kDa, approximately 75kDa to approximately 90kDa, approximately 75kDa to approximately 100kDa, approximately 75kDa to approximately 110kDa, approximately 75kDa to approximately 120kDa, approximately 80kDa to approximately 90kDa, approximately 80kDa to approximately 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, about 80 kDa, about 120 kDa, about 90 kDa, about 100 kDa, about 110 kDa, about 100 kDa, about 120 kDa, or about 110 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.

[0144] 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.

[0145] 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 included herein. Such a safety profile may be reflected in reduced accumulation of radiation in key radiation-sensitive tissues such as the kidney and bone marrow, and / or increased accumulation of radiation in target tissues (i.e., tumor or cancerous tissue) or more radiation-resistant organs such as the liver.

[0146] In certain embodiments, the immunoconjugates of the present disclosure result in 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 no more than 8 Gy per treatment. In certain embodiments, the kidneys are exposed to no more than 5 Gy per treatment.

[0147] In certain embodiments, the immunoconjugates of the present disclosure provide a total radiation exposure per treatment measured in Gray (Gy). In certain embodiments, bone marrow is exposed to 4 Gy or less per treatment. In certain embodiments, bone marrow is exposed to 3 Gy or less per treatment. In certain embodiments, bone marrow is exposed to 2 Gy or less per treatment. In certain embodiments, bone marrow is exposed to 1.5 Gy or less per treatment. In certain embodiments, bone marrow is exposed to 1.0 Gy or less per treatment. In certain embodiments, bone marrow is exposed to 0.5 Gy or less per treatment.

[0148] In certain embodiments, the immunoconjugates of the present disclosure result in 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.

[0149] In certain embodiments, the immunoconjugates of the present disclosure provide an increased radiation dose in the tumor compared to the blood when measured as percent injected dose per gram, hi certain embodiments, the ratio of percent tumor injected dose per gram to percent blood injected dose per gram is greater than 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0150] 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.

[0151] In certain embodiments, immunoconjugates of the present disclosure result in an increased radiation dose in the liver compared to the kidney, measured as injected dose per gram, hi certain embodiments, the ratio of percent tumor injected dose per gram to percent bone marrow injected dose per gram is greater than 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0152] In some embodiments, the present invention contemplates variants of the immunoconjugates of the present invention that contain an Fc region, which retain some, but not all, effector functions, making them desirable candidates for applications in which the half-life of the immunoconjugate in vivo 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. The primary cells for mediating ADCC, NK cells, express only FcγγRIII, while 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 for assessing ADCC activity of a molecule of interest are described in US 5,500,362 (see, e.g., Hellstrom, I. et al., Proc Natl Acad Sci USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc Natl Acad Sci USA 82:1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be utilized (e.g., the ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA), and the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively / additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc Natl Acad Sci USA 95:652-656 (1998). Clq binding assays can also be performed to confirm that the immunoconjugate is unable to bind Clq and therefore lacks CDC activity (see, e.g., Clq and C3c binding ELISAs 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., Blood 101:1045-1052 (2003); Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0153] Immunoconjugates with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with substitutions of residues 265 and 297 to alanine (US 7,332,581).

[0154] Immunoconjugates may have altered effector function by including the following modifications according to EU numbering: L234A, L235E, G237A, A330S, and P331S, which reduce Fc receptor binding. See, e.g., US 8,613,926 or Andersson C, Wenander et al., "Rapid-onset clinical and mechanistic effects of anti-C5aR treatment in the mouse collagen-induced arthritis model." Clin Exp Immunol. 2014 Jul;177(1):219-33.

[0155] 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)).

[0156] In some embodiments, modifications are made in the Fc region that result in altered (i.e., improved or decreased) Clq binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in US6,194,551; WO1999 / 051642; Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0157] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is 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 with one or more internal substitutions that improve binding of the Fc region to FcRn. Such Fc variants include variants with substitutions at one or more of Fc region residues 434 or 435, for example, substitutions of 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.

[0158] To increase the serum half-life of an antibody, a salvage receptor binding epitope can be incorporated into the antibody (particularly an 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.

[0159] 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.

[0160] chelating agents As described herein, in some embodiments, a chelator (i.e., R1) is attached to a tumor-targeting moiety (e.g., a polypeptide comprising an antigen-binding region and an immunoglobulin heavy chain constant region described herein). The chelating moiety allows the tumor-targeting moiety to be loaded with an appropriate radioisotope, e.g., a beta-emitter or an alpha-emitter. The chelator may be attached to the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof. Such attachment may suitably be by 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.

[0161] In one embodiment, the chelator of the immunoconjugate is covalently bound to the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof. In one embodiment, the chelator is covalently bound directly (e.g., without a spacer, stretcher, or linker) to the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof. In one embodiment, the chelator is covalently bound to the antigen-binding arm via a linker that is covalently bound to the chelator and to the antigen-binding arm. In one embodiment, the linker is hydrophilic (e.g., a PEG chain). In one embodiment, the linker is hydrophobic (e.g., an alkyl or alkene chain). The chelator can be linked or attached to the immunoconjugate as described in Sadiki, A. et al. "Site-specific conjugation of native antibody." Antibody Therapeutics 2020, 3, 271-284.

[0162] In some embodiments, immunoconjugates are formed by site-specific conjugation of chelator-linkers directed 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, the residues can be functionalized with a different reactive functional group, which is then reacted with the chelator-linker in a second step to provide the immunoconjugate. In some embodiments, the reactive functional group is thiopropionate.

[0163] In some embodiments, non-naturally occurring cysteine ​​residues are engineered into the antibody framework as sites for thiol-directed conjugation to provide an immunoconjugate, hi some embodiments, other non-naturally occurring amino acids or amino acid sequences are engineered into the framework to serve as attachment sites for chelator-linkers or secondary reactive groups to which chelator-linkers are conjugated to provide an immunoconjugate.

[0164] In some embodiments, a non-natural amino acid containing a bridging group is engineered to provide a framework for chelator-linker attachment. In some embodiments, the non-natural amino acid contains an azide.

[0165] In some embodiments, the chelator-linker is attached to the glutamine residue via the action of a transglutaminase enzyme, while in other embodiments, a secondary reactive group is attached by transglutaminase, at which point the chelator-linker is added to yield an immunoconjugate.

[0166] In some embodiments, the chelator-linker is attached by modifying one or more N-glycans with reactive functional groups by the action of a glycosidase, 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.

[0167] In one embodiment, the immunoconjugate comprises more than one chelating agent, which may be the same or different.

[0168] In one embodiment, an immunoconjugate having more than one chelator has more than one chelator attached to the same antigen-binding arm.

[0169] In one embodiment, an immunoconjugate having more than 1 chelator and less than 11 chelators has more than 2 chelators, more than 3 chelators, more than 4 chelators, more than 5 chelators, more than 6 chelators, more than 7 chelators, more than 8 chelators, or more than 9 chelators. In one embodiment, the chelators are the same. In one embodiment, each antigen-binding arm is linked, directly or indirectly, to more than one chelator.

[0170] In one embodiment, the chelating agent comprises a radioisotope chelating moiety and a functional group that allows covalent binding 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.

[0171] 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.

[0172] In a preferred embodiment, the chelator of the immunoconjugate does not bind to the antigen-binding region in the antigen-binding arm of the immunoconjugate.

[0173] 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.

[0174] 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.

[0175] In certain embodiments, the chelating agent is a radioisotope chelating agent. In certain embodiments, the radioisotope chelating agent is selected from the list consisting of tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), α-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTAGA), or (Py4Pa). In certain embodiments, the radioisotope chelating agent is DOTA. In certain embodiments, the radioisotope chelating agent is DOTAGA. In certain embodiments, the radioisotope chelating agent is Py4Pa. In certain embodiments, the radioisotope chelating agent is directly bound to the antigen-binding region and / or the immunoglobulin heavy chain constant region. In certain embodiments, the radioisotope chelating agent is bound to the antigen-binding region or the immunoglobulin heavy chain constant region by a linker. In certain embodiments, the linker is selected from those resulting from conjugation with 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and the linker reagents: N-succinimidyl 4-(2-pyridylthio)pentanoate-forming linker moiety 4-mercaptopentanoic acid (SPP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB), polyethylene glycol (PEG), polyethylene glycol polymer (PEGn), and S-2-(4-isothiocyanatobenzyl) (SCN). In certain embodiments, the linker is selected from polyethylene glycol (PEG), polyethylene glycol polymer (PEG), and S-2-(4-isothiocyanatobenzyl) (SCN). In certain embodiments, the linker is PEG5. In certain embodiments, the linker is SCN.In certain embodiments, the radioisotope chelator is a linker-chelator selected from the list consisting of TFP-Ad-PEG5-DOTAGA, p-SCN-Bn-DOTA, p-SCN-Ph-Et-Py4Pa, and TFP-Ad-PEG5-Ac-Py4Pa.

[0176] In some embodiments, the chelator is conjugated at a predetermined ratio of polypeptide (i.e., antigen-binding region and / or immunoglobulin heavy chain constant region) to chelator. In certain embodiments, the radioisotope chelator is conjugated to the antigen-binding region and / or immunoglobulin heavy chain constant region at a ratio of 1:1 to 8:1. In certain embodiments, the radioisotope chelator is conjugated to the antigen-binding region and / or immunoglobulin heavy chain constant region at a ratio of 1:1 to 6:1. In certain embodiments, the radioisotope chelator is conjugated to the antigen-binding region and / or immunoglobulin heavy chain constant region at a ratio of 2:1 to 6:1.

[0177] For example, a bifunctional chelator is 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.

[0178] Examples of bifunctional chelators are 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), and related analogs of the foregoing. Such chelators are suitable for coordinating metal ions, such as α- and β-emitting radionuclides.

[0179] 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 L, et al. Bioconjugate Chem. ASAP (2020)), 3,2-HOPO (Wickstroem K, et al. al. 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 Brechbiel MW QJ Nucl. Med. Mol. Imaging 52:166(2008)).

[0180] In some embodiments, the chelator of the immunoconjugate or radioimmunoconjugate of the invention is selected from the group consisting of bifunctional chelators DOTA, DO3A-NHS, DOTAGA-NHS, DOTAGA-anhydride DOTAGA-TFP, p-SCN-Bn-DOTA, p-SCN-Bn-DTPA, p-SCN-Bn-CHX-A"-DTPA, p-SCN-Bn-TCMC, macropa-NCS (Thiele NA, et al. Angew. Chem. Int. Ed. 56:1 (2017)), crown (Yang H, et al. Chem. Eur. J. 26:11435 (2020)), p-SCN-Ph-Et-Py4Pa (Li L, et al. Bioconjugate Chem. ASAP (2020)), 3,2-HOPO (Wickstroem K, et al. al. Int. J. Rad. 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 Brechbiel MW QJ Nucl. Med. Mol. Imaging 52:166 (2008)).

[0181] 225 For Ac immunoconjugates, there are a variety of acyclic and cyclic ligands known in the art as suitable chelators (see, e.g., Davis I, et al., Nucl Med Biol 26:581 (1999); Chappell L, et al., Bioconjug Chem 11:510 (2000); Chappell, L, et al., Nucl Med Biol 30:581 (2003); McDevitt M, et al., Appl Radiat Isot 57:841 (2002); Gouin S, et al., Org Biomol Chem 3:453 (2005); Thiele N, et al., Angew Chem Int Ed Engl 56:14712 (2017)).

[0182] In certain embodiments, the chelator is a chelator suitable for chelation of α-emitters. Some chelators suitable for α-emitters are described in Yang et al., "Harnessing α-Emitting Radionuclides for Therapy: Radiolabeling Method Review." J Nucl Med. 2022 Jan;63(1):5-13.

[0183] In certain embodiments, chelators suitable for alpha emitter chelation include DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DO3A 1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane, DOTAGA α-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAGA anhydride (2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, Py4Pa 6,6',6'',6''''-(((pyridine-2,6-diylbis(methylene))bis(azanetriyl)-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-tetraazacyclo-octadecane-4,7,13,16-tetrayl)tetraacetic acid, Macropa 6,6'-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecane) can-7,16-diyl)bis(methylene))-dipicolinic acid, Macropa-NCS 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)-4-isothiocyanatopicolinic acid, HEHA 1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid, 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, Non ...6,6'-(((oxybis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))-dipicolinic acid, and combinations thereof.

[0184] In certain embodiments, the chelator is a chelator suitable for β- or γ-emitter chelation. In certain embodiments, the chelator suitable for β- or γ-emitter chelation is 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), DOTPA1,4,7,1 0-Tetraazacyclo-dodecane-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]hexa-decane-4,11-diacetic acid), NOTA 1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTP 1,4,7-triaza Cyclononane-1,4,7-tri(methylenephosphonic acid), TETPA 1,4,8,11-tetraazacyclo-tetradecane-1,4,8,11-tetrapropionic acid, TETA 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid, PEPA 1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N''''-pentaacetic acid, H4Octapa N,N'-Bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid, H2Dedpa 1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane, H6phospa N,N'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane, TTHA triethylene-tetramine-N,N,N',N'',N''',N'''-Hexaacetic acid, DO2P Tetraazacyclododecanedimethane-phosphonic acid, HP-DO3A Hydroxypropyltetraazacyclododecanetriacetic acid, EDTA Ethylenediaminetetraacetic acid, DTPA Diethylenetriaminepentaacetic acid, DTPA-BMA Diethylenetriaminepentaacetic acid-bismethylamide, HOPO Octadentate Hydroxypyridinone, 3,2,3-LI(HOPO)N,N'-(butane-1,4-diyl)bis(1-hydroxy-N-(3-(1-hydroxy-6-oxo-1,6-dihydro-pyridine-2-carboxamido)propyl)-6-oxo-1,6-dihydropyridine-2-carboxamido), 3,2-HOPO N,N'-(((2-(4-aminobenzyl)-3-((2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)(2-(3-hydroxy-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)-propyl)azanediyl)bis(ethane-2,1-diyl))bis(3-hydroxy-1-methyl-2-oxo -1,2-dihydropyridine-4-carboxamide), Neunpa6,6'-(((azanediylbis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))-bis(methylene))dipicolinic acid, Neunpa-NCS=6,6'-(((((4-isothiocyanatophenethyl)azanediyl)-bis(ethane-2,1-diyl))bis((carboxymethyl)azane 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, PyPa6,6'-(((pyridine-2,6-diylbis(methylene))bis ((Carboxymethyl)azanediyl))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.

[0185] In some embodiments, R 1 comprises a chelating moiety selected from the list consisting of DOTA, DO3A, DO3Apic, DOTAGA, DOTAGA anhydride, Py4Pa, Py4Pa-NCS, Crown, Macropa, Macropa-NCS, HEHA, CHXoctapa, Bispa, and Noneunpa, or a radionuclide complex thereof.

[0186] In some embodiments, R 1 comprises a chelating moiety selected from the list consisting of DOTMA, DOTPA, DO3Apic, DO3AM-acetic acid, DOTP, DOTMP, DOTA-4AMP, CB-TE2A, NOTA, NOTP, TETPA, TETA, PEPA, H4Octapa, H2Dedpa, DO2P, EDTA, DTPA-BMA, 3,2,3-LI(HOPO), 3,2-HOPO, Neunpa, Neunpa-NCS, Octapa, PyPa, porphyrin, and deferoxamine, or a radionuclide complex thereof.

[0187] In some embodiments, R 1 teeth, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A), α,α',α'',α''''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTMA), 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid (DOTPA), 2,2',2''-(10-(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, 10-((6-carboxypyridin-2-yl)methyl)-1,4,7,10-tetra-azacyclododecane-1,4,7-triacetic acid (DO3Apic), 6,6'-(((pyridine-2,6-diylbis(methylene))bis((carboxymethyl)azanediyl))-bis(methylene))dipicolinic acid (H4pypa), 6,6',6'',6''''-(((pyridine-2,6-diylbis(methylene))bis(azanetriyl))tetrakis(methylene))-tetrapicolinic acid (H4py4pa), and 3,6,9,12-tetrakis(carboxymethyl)-3,6,9,12-tetraazatetradecanedioic acid (TTHA), or a radionuclide complex thereof.

[0188] In some embodiments, R 1 has the following structure:

[0189] [ka]

[0190] [ka]

[0191] [ka]

[0192] [ka]

[0193] [ka] The chelating moiety comprises one of:

[0194] In some embodiments, R 1 contains DOTA, positions shown:

[0195] [ka] The mAb binds to a tumor-targeting moiety.

[0196] In some embodiments, R 1 contains DOTA, positions shown:

[0197] [ka] The mAb binds to a tumor-targeting moiety.

[0198] In some embodiments, R 1 contains DOTA, positions shown:

[0199] [ka] The mAb binds to a tumor-targeting moiety.

[0200] The immunoconjugates described herein may comprise one or more R 1 In some embodiments, the immunoconjugates described herein include more than one R 1 Each R 1 The parts are the same.

[0201] radionuclides In some embodiments, the radionuclide in the immunoconjugates described herein is an Auger electron-emitting radionuclide. In some embodiments, the radionuclide is an α-emitting radionuclide. In some embodiments, the radionuclide is a β-emitting radionuclide. In some embodiments, the radionuclide is a γ-emitting radionuclide. In some embodiments, the type of radionuclide used in the non-peptide targeted therapeutic compound can be tailored to the specific type of cancer, the type of targeting moiety (e.g., non-peptide ligand), etc. Radionuclides that undergo α decay emit α particles (helium ions with a +2 charge) from their nuclei. As a result of α decay, the daughter nuclide has two fewer protons and two fewer neutrons than the parent nuclide. This means that in α decay, the number of protons decreases by two and the number of nuclei decreases by four. Radionuclides that undergo β decay emit β particles (electrons) from their nuclei. During β decay, one of the neutrons changes into a proton and an electron. The proton remains in the nucleus, and the electron is emitted as a β particle. This means that in beta decay, the nucleus loses a neutron but gains a proton. In gamma decay, a nucleus in an excited (high energy) state changes to a lower energy state by emitting a gamma ray photon. There is no change in the number of protons or nuclei during gamma decay. The emission of gamma rays is often accompanied by the emission of alpha and beta particles.

[0202] Auger electrons (AE) are very low energy electrons emitted by radionuclides that decay by electron capture (EC) (e.g., 111 In (indium-111), 67 Ga (gallium-67), 99m Tc (technetium-99m), 195m Pt(Platinum-195m), 125 I (iodine-125), and 123 I (iodine-123). This energy is deposited over nanometer-micrometer distances, resulting in a high linear energy transfer that is powerful enough to cause lethal damage to cancer cells. Therefore, AE-emitting radiotherapeutic agents have great potential for the treatment of cancer.

[0203] Beta particles are electrons emitted from the nucleus. They typically have a longer range in tissue (approximately 1-5 mm) and are the most frequently used.

[0204] Alpha particles are helium nuclei (two protons and two neutrons) emitted from the nuclei of radioactive atoms. They can travel 50–100 μm in tissue, depending on the energy emitted. They are positively charged and orders of magnitude more powerful than electrons. The amount of energy deposited per path length of an alpha particle (referred to as "linear energy transfer") is approximately 400 times greater than that of an electron. This results in significantly more damage along its path than that caused by an electron. Alpha particle tracks cause an overwhelming number of complex, largely irreparable, DNA double-strand breaks. The absorbed dose required to achieve cytotoxicity is related to the number of alpha particles traversing the cell nucleus. Using this as a measure, cytotoxicity can be achieved with a 1–20 alpha particle traversal range of the cell nucleus. The resulting high potency, coupled with the short range of alpha particles (which reduces normal organ toxicity), has attracted significant interest in the development of alpha particle emitters. Typically used alpha particle emitters include: 212 Bi (bismuth-212), 212 Pb (lead-212), 213 Bi (bismuth-213), 225 Ac, 223 Ra (radium-223), and 229 Contains Th (thorium-227).

[0205] In some embodiments, the radionuclide is a diagnostic or therapeutic radionuclide.

[0206] [Table 1]

[0207] In some embodiments, the radionuclide is an Auger electron emitting radionuclide. 111 In, 67 Ga, 68 Ga,99m Tc, or 195m In some embodiments, the radionuclide is: 111 In, 67 Ga, 68 Ga, or 99m Tc is an Auger electron-emitting radionuclide.

[0208] In some embodiments, the radionuclide is an alpha-emitting radionuclide. 225 Ac, 213 Bi, 223 Ra, or 212 Pb. In some embodiments, the radionuclide is 225 In some embodiments, the radionuclide is a beta-emitting radionuclide. In some embodiments, the radionuclide is 90 Y, 177 Lu, 186 Re(186-rhenium), 188 Re (rhenium-188), 64 Cu, 67 Cu, 153 Sm (samarium-153), 89 Sr (strontium-89), 198 Au (Fri-198), 169 Er (erbium-169), 165 Dy (dysprosium-165), 99m Tc, 89 Zr, or 52 Mn (manganese-52). In some embodiments, the radionuclide is 90 Y, 177 Lu, 99m Tc, or 89 It is a beta-emitting radionuclide, Zr.

[0209] In some embodiments, the radionuclide is a gamma-emitting radionuclide. 60 Co (cobalt-60), 103 Pd (palladium-103),137 Cs (cesium-137), 169 Yb (ytterbium-169), 192 Ir (iridium-192), 212 B1, 213 Bi, or 226 Ra is a gamma-emitting radionuclide.

[0210] Linker In some embodiments, the tumor targeting moiety and the radionuclide chelator moiety R 1 and a linker is used. In some embodiments, L is a hydrophobic linker. In some other embodiments, L is a hydrophilic linker. In some embodiments, the linker is flexible. In some embodiments, the linker is rigid. In some embodiments, the linker is linear. In other embodiments, the linker is branched. In some embodiments, a branched linker has more than one R 1 In some embodiments, the linker comprises a linear structure. In some embodiments, the linker comprises a non-linear structure. In some embodiments, the linker comprises a branched structure. In some embodiments, the linker comprises a cyclic structure. In some embodiments, the linker comprises one or more linear structures, one or more non-linear structures, one or more branched structures, one or more cyclic structures, one or more flexible segments, one or more rigid segments, or a combination thereof.

[0211] The length of the linker is determined by the length of the radionuclide chelator moiety R 1and the tumor-targeting moiety, with cyclic moieties being counted by taking the shortest path around the ring. In some embodiments, the linker has a linear stretch of 1 to 100 atoms, 1 to 50 atoms, 1 to 40 atoms, 1 to 30 atoms, in other embodiments, 1 to 20 atoms, in still other embodiments, 1 to 15 atoms, in still other embodiments, 1 to 10 atoms, and in still other embodiments, 1 to 5 atoms. In some embodiments, the length of the linker is a range having a lower limit selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and an upper limit selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, and 30. In some embodiments, the length of the linker is a range having a lower limit selected from the group consisting of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and an upper limit selected from the group consisting of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0212] Linker considerations include their effect on the physical or pharmacokinetic properties of the resulting radioimmunoconjugate, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable as well as programmed degradation), rigidity, flexibility, immunogenicity, modulation of antibody binding, etc.

[0213] An optimal linker must link the carrier mAb to the radiochelator without compromising either functionality, provide a stable linkage in the circulation, and degrade under specific conditions. Stability in the circulation has proven to be a particularly important factor for success using this strategy.

[0214] In malignant tumors, the accessibility of tumor cells allows targeting by specific antibodies. Antigens expressed on tumor cells, but not on healthy cells, allow selective targeting of tumor cells while sparing non-tumor cells. mAbs tagged with short-lived radionuclides that emit short-range, high linear energy transfer particles represent an attractive means of treating tumors. These short-range particles can kill single cells without harming bystanders. High-energy alpha emitters used in radioimmunotherapy include: 211 At (astatine-211), 212 Bi, 213 Bi, and 225 This latter isotope has a long (10 day) half-life and is an in vivo isotope generator in that it decays via alpha emission by three short-lived atoms, each of which yields an alpha particle.

[0215] In some instances, tumor targeting 225 A limitation to the general use of Ac radioimmunotherapy is excessive radiation of normal tissues by the radioimmunoconjugate. With the exception of leukemia-targeting antibodies, the majority (>99%) of the injected antibody dose remains circulating in the blood or is not associated with the target tumor. The prolonged circulation time of non-targeted antibodies, typically days to weeks, results in radiation exposure of normal organs, primarily the liver and kidneys, which catabolize or retain proteins and peptides.

[0216] In the case of in vivo decay of α-emitters, a further major potential obstacle to their safe use is the sequential release of three unchelated α-emitting daughter atoms after the initial decay of the primary radionuclide. Uptake into organs such as the kidney or liver can induce toxicity. One attractive approach to overcome the long circulation time of non-targeted radioimmunoconjugates is the development of linkers that allow conditional release of the chelated radioactive payload. The introduction of a cleavage site in the link between the radiochelate and the mAb allows for the release of a low-molecular-weight radiochelate from its carrier mAb and subsequent rapid clearance of the radioconjugate after its accumulation in metabolic organs. The chelated radiometal then rapidly clears the body through the kidney, thereby reducing toxicity. This strategy has been attempted using disulfide, ester, tartrate amide, and peptide bonds (Kukis DL, et al., Cleavable linkers to enhance selectivity of antibody-targeted therapy of cancer. Cancer Biother Radiopharm. 2001;16:457-467; Quadri SM, Vriesendorp HM. Effects of linker chemistry on the pharmacokinetics of radioimmunoconjugates. QJ Nucl Med. 1998;42:250-261). The optimal linker must link the carrier mAh to the radiochelator without compromising either functionality, provide a stable linkage in circulation, and degrade under specific conditions. Stability in circulation has proven to be a particularly important factor for success using this strategy.

[0217] Peptide linkers are generally more stable in serum than esters and disulfides and have been the most successful in the design of radioimmunoconjugates and antibody-drug conjugates (ADCs). The most widely studied peptide linkers are sensitive to cathepsins. More precisely, ADCs incorporating the dipeptide valine-citrulline have been shown to enter target cells, traffic to lysosomes, and specifically release their drugs under the action of cysteine ​​proteases. The most advanced strategy for radionuclide release from radioimmunoconjugates is based on cathepsin-sensitive peptide linkers (DeNardo GL, et al., Preclinical evaluation of cathepsin-degradable peptide linkers for radioimmunoconjugates. Clin Cancer Res. 2003;9:3865S-3872S; DeNardo GL, et al. Comparison of 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-peptide-ChL6, a novel immunoconjugate with a catabolizable linker, to 2-iminothiolane-2-[p-(bromoacetamido)benzyl]-DOTA-ChL6 in breast cancer xenografts. Clin Cancer Res. 1998;4:2483-2490; DeNardo SJ, et al. Enhanced therapeutic index of Clin Cancer Res.2003;9:3938S-3944S). .In some cases, the use of cathepsin B-labile radioconjugates (e.g., glycylglycylglycyl-Lp-isothiocyanatophenylalanine-) has shown a decrease in liver dose, with a slight increase in tumor dose observed (DeNardo GL, et al., Clin Cancer Res. 2003;9:3865S-3872S; DeNardo GL, et al. Clin Cancer Res. 1998;4:2483-2490).

[0218] In some embodiments, the linker comprises one or more amino acid residues. In some embodiments, the linker comprises 1 to 3, 1 to 5, 1 to 10, 5 to 10, or 5 to 20 amino acid residues. In some embodiments, one or more amino acids of the linker are unnatural amino acids.

[0219] In some embodiments, a linker can comprise flexible and / or rigid regions. Exemplary flexible linker regions include those comprising Gly and Ser residues ("GS" linkers), glycine residues, alkylene chains, PEG chains, etc. Exemplary rigid linker regions include those comprising alpha-helix-forming sequences, proline-rich sequences, and regions rich in double and / or triple bonds.

[0220] In some embodiments, the linker comprises a peptidyl linker, which may be 3 to 20 amino acids in length, such as a single amino acid residue (e.g., polyglycine) or a combination of repeating amino acid residues to obtain a peptide linker that confers favorable pharmacokinetics.

[0221] In some embodiments, the linker comprises a peptide bond. The peptide bond may comprise an L-amino acid and / or a D-amino acid. In some embodiments, D-amino acids are preferred to minimize immunogenicity and nonspecific cleavage by background peptidases or proteases. The cellular uptake of oligo-D-arginine sequences is known to be comparable to or better than that of oligo-L-arginine.

[0222] In some embodiments, the linker is cleavable. In some embodiments, the linker is designed for cleavage under specific conditions or in a specific environment (such conditions or the environment near such targeted cells, tissues, or regions). Cleavable linkers rely on the unique properties of the cytoplasmic compartment of cells for selective release of cytotoxic drugs. Such linkers mainly include chemically cleavable linkers that respond to low pH (acid-labile linkers) or reducing environments (disulfide linkers), and enzymatically cleavable linkers (peptide linkers or β-glucuronide linkers) that are sensitive to the action of specific lysosomal enzymes.

[0223] In some embodiments, the linker is cleavable under physiological conditions. In some embodiments, the linker is cleavable under intracellular conditions. In some embodiments, the linker is chemically cleavable. In some embodiments, the linker is an enzymatically cleavable (e.g., protease-sensitive, peptidase-sensitive) linker. In some embodiments, the linker is pH-sensitive, i.e., sensitive to hydrolysis at a specific pH value. For example, a pH-sensitive linker may be hydrolyzable under acidic conditions. For example, the linker may be an acid-labile linker that is hydrolyzable in lysosomes (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic acid amide, orthoester, acetal, ketal, etc.). Such linkers may be relatively stable under neutral pH conditions, e.g., under blood pH conditions, but are unstable at pHs below 7.0, e.g., pH 6.5 to 4.5 (approximate pH of lysosomes and / or endosomes).

[0224] In some embodiments, the linker is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the linker is cleaved by a glycosidase, such as a glucuronidase. A β-glucuronide linker can be easily cleaved by the abundant lysosomal enzyme β-glucuronidase, facilitating the easy and selective release of the active drug. In other embodiments, the linker is not cleavable.

[0225] In some embodiments, the linker component comprises an amino acid unit. In one such embodiment, the amino acid unit allows for protease cleavage of the linker, thereby facilitating the release of the drug from the immunoconjugate upon exposure to intracellular proteases, such as lysosomal enzymes. 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). These dipeptide linkers exhibit good stability in serum, but can be recognized and rapidly hydrolyzed by certain lysosomal proteases, such as cathepsin B, after internalization. Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly).

[0226] The amino acid units can include naturally occurring amino acid residues and minor amino acids, as well as non-naturally occurring 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.

[0227] In some embodiments, the linker is cleaved by a protease, a matrix metalloproteinase, a serine protease, or a combination thereof. In some embodiments, the linker is cleaved by a reducing agent. In some embodiments, the linker is cleaved by an oxidizing agent or oxidative stress.

[0228] In some embodiments, the linker is cleaved by MMPs. The hydrolytic activity of matrix metalloproteinases (MMPs) is involved in the invasive migration of metastatic tumor cells. In some embodiments, the linker comprises the amino acid sequence PLG-C(Me)-AG, PLGLAG, which is cleaved by the metalloproteinase enzymes MMP-2, MMP-9, or MMP-7 (MMPs involved in cancer and inflammation).

[0229] In some embodiments, the linker is cleaved by proteolytic enzymes or reducing environments found near cancerous cells, where such environments or enzymes are not typically found near normal cells.

[0230] In some embodiments, the linker is cleaved by a serine protease, including, but not limited to, thrombin and cathepsin. In some embodiments, the linker is cleaved by cathepsin K, cathepsin S, cathepsin D, cathepsin E, cathepsin W, cathepsin F, cathepsin A, cathepsin C, cathepsin H, cathepsin Z, or any combination thereof. In some embodiments, the linker is cleaved by cathepsin K and / or cathepsin S.

[0231] In some embodiments, the linker is cleaved in a necrotic environment. Necrosis often results in the release of enzymes or other cellular contents that can be used to induce cleavage of the linker. In some embodiments, cleavage of the linker occurs by necrotic enzymes (e.g., by calpain).

[0232] In some embodiments, the linker comprises one or more disulfide bonds.

[0233] Alternatively, the linker may be a non-peptidyl linker. Typical examples of these types of linkers are based on linear or branched hydrocarbons of various lengths or polyethylene glycol. They may also incorporate other groups that confer solubility, rigidity, or isoelectric point, such as aromatic or non-aromatic rings, halogens, ketones, aldehydes, esters, sulfonyl, or phosphate groups.

[0234] In some embodiments, the linker component is "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 conjugate moiety upon enzymatic (e.g., proteolytic) cleavage. 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 a glycine-glycine spacer unit by a tumor cell-associated protease results in the release of the glycine-glycine-drug moiety from the remainder of the immunoconjugate. In one such embodiment, the glycine-glycine-drug moiety is subsequently subjected to a separate hydrolysis step in the tumor cell, thus cleaving the glycine-glycine spacer unit from the chelator moiety.

[0235] The "self-immolative" spacer unit allows for release of the drug moiety without a separate hydrolysis step. In certain embodiments, the spacer unit of the linker comprises a p-aminobenzyl unit. In one such embodiment, p-aminobenzyl alcohol is attached to the amino acid unit via an amide bond, and a carbamate, methylcarbamate, or carbonate is made between the benzyl alcohol and the cytotoxic agent (see, e.g., Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-103). In one embodiment, the spacer unit is p-aminobenzyloxycarbonyl (PAB). In certain embodiments, the phenylene portion of the p-aminobenzyl unit is substituted with Qm, where Q is halogen, nitro, cyano, -OH, -OR. 5 , -CO2H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C1-C6 alkyl, where the substituted C1-C6 alkyl is -OH, -CO2H, -NHR 5 , -C(=O)NHR 5 , and -NHC(=O)R 5and m is an integer ranging from 0 to 4 (see, for example, Yam B. Poudel, et al., ACS Medicinal Chemistry Letters 2020 11(11), 2190-2194). Further examples of self-immolative spacer units include, but are not limited to, aromatic compounds electronically similar to p-aminobenzyl alcohol (see, for example, US2005 / 0256030A1), 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 a-position of glycine (Kingsbury et al., J. Med. Chem., 1984, 27:1447) is also an example of a self-immolative spacer useful in ADCs.

[0236] In some embodiments, the immunoconjugate comprises a linker, such as a dendritic linker, for covalent attachment of more than one drug moiety to an antibody via, for example, a branched, multifunctional 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, many drug moieties can be attached via the dendritic linker.

[0237] In some embodiments, the chelating moiety (R 1 ) or its radionuclide complex, the linker connecting the tumor targeting moiety to 1 -LL c -, and L is -L 1 -L 2 -L 3 -L 4 -L 5 - and X 1 Ha-NR a -, -NR a S(=O)2-, -NR a S(=O)NR a or one or more independently selected natural or unnatural amino acids, any free amine of the amino acid optionally being substituted with R 5 or -C(=O)R 5 and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R; R a are each independently selected from hydrogen and C1-C4 alkyl; L 1 is unsubstituted or substituted C1-C 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, unsubstituted or substituted C-C20 Alkenylene, unsubstituted or substituted C-C 20 Alkynylene, C4-C 20 Polyethylene glycol, -(X 3 CH2CH2) t -, unsubstituted or substituted cycloalkylene, unsubstituted or substituted heterocycloalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; X 3 are each independently O and NR 4 is selected from each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; L 2 is absent or unsubstituted or substituted C1-C 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, -C(=O)-(unsubstituted or substituted C-C 10 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 alkylene)-, -C(=O)-(CH2CH2X 3 ) m -(CH2) P -, -C(=O)NR 4 -(CH2CH2X 3 ) m -(CH2) p -, -NR 4 C(=O)-(CH2CH2X 3 ) m -(CH2) p - or -(CH2CH2X 3 ) m -(CH2) p - and R 4 are each independently selected from hydrogen and C1-C6 alkyl; each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each p is independently 0, 1, or 2; L 3 is absent or is one or more independently selected groups selected from natural or unnatural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates, and any free amine (—NH) of an amino acid is optionally substituted with R 5 or -C(=O)R 5 and any free carboxylic acid of any amino acid is optionally substituted with -C(=O)NH-R5, and the substituted benzyl is independently substituted with halogen, -OH, -OR 5 , -CO2H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C1-C6 alkyl, wherein the substituted C1-C6 alkyl is selected from -OH, -CO2H, -NHR 5 , -C(=O)NHR 5 , and -NHC(=O)R 5 is replaced by L 4 is absent or unsubstituted or substituted C1-C 10 Alkylene, -C(=O)-(unsubstituted or substituted C-C 10 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 alkylene)-, -C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2X 3 ) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -(CH2CH2X 3 ) n -(CH2) q- or -(X 3 CH2CH2) n - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each q is independently 0, 1, or 2; L 5 does not exist or -C(=O)-(CH2) n -, -C(=O)NR 4 -(CH2) n -, -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2X 3 ) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -(CH2CH2X 3 ) n -(CH2) q -, -C(=O)-(X 3 CH2CH2) n - or -(X 3 CH2CH2) n - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each q is independently 0, 1, or 2; R a are each independently selected from hydrogen and C1-C4 alkyl; R b are each independently selected from hydrogen and C1-C4 alkyl; Heteroalkylene is a heteroalkylene group in which one carbon atom is -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NH)-, or -S(=O)(=NR 5 )-, -NR 5 -, -P(=O)OR b -, -NRa C(=O)-, -C(=O)NR a -, -OC(=O)NR a -, -NR a C(=O)NR a -, -NR a C(=N-CN)NR a -, -NR a C(=NR 5 )NR a -, or -NR a alkylene substituted with C(=O)O-; -L 1 -, -L 2 -, -L 3 -, -L 4 -, and -L 5 If one of - is replaced, -L 1 -, -L 2 -, -L 3 -, -L 4 -, and -L 5 - is halogen, -OH, -OR 5 , -CO2H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C1-C6 alkyl, wherein the substituted C1-C6 alkyl is selected from -OH, -CO2H, -NHR 5 , -C(=O)NHR 5 , and -NHC(=O)R 5 is replaced by R 5 are independently C1-C 10 Alkyl, C4-C 30 selected from polyethylene glycol, unsubstituted or substituted arylene, and unsubstituted or substituted heteroarylene; L c -C(=O)-, -, NHC(=S)-, -X-phenyl-NHC(=S)-, -S-, -C(=O)CH2-,

[0238] [ka] and X is absent, -O-, -S-, -S(=O)-, -S(=O)2-, or -NR a -, -C(=O)-, -NR a C(=O)-, -C(=O)NR a -, -C(=O)O-, -OC(=O)-, -OC(=O)NR a -, -NR a C(=O)NR a -, -NR a C(=S)NR a -, -NR a C(=O)O-, R a are each independently selected from hydrogen and C1-C4 alkyl.

[0239] In some embodiments, L c is —C(═O)—. In some embodiments, L c is -NHC(=S)-. In some embodiments, L c is -X-phenyl-NHC(=S)-. In some embodiments, X is absent or is -NHC(=S)NH-. In some embodiments, L c is -NHC(=S)NH-. In some embodiments, L c -S-, -C(=O)CH2-,

[0240] [ka] In some embodiments, L c is —C(═O)CH—. In some embodiments, L c teeth

[0241] [ka] is.

[0242] In some embodiments, L 1 is unsubstituted or substituted C1-C 20 Alkylene, unsubstituted or substituted C1-C20 Heteroalkylene, C4-C 20 It is polyethylene glycol, unsubstituted or substituted C3-C8 cycloalkylene, unsubstituted or substituted monocyclic C3-C8 heterocycloalkylene, unsubstituted or substituted phenylene, or unsubstituted or substituted monocyclic heteroarylene.

[0243] In some embodiments, L 1 represents unsubstituted or substituted C1-C6 alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 It is polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene.

[0244] In some embodiments, X 1 are one or more independently selected natural or unnatural amino acids, and any free amine of an amino acid is optionally substituted with R 5 or -C(=O)R 5 and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R5; and L 1 is -NR 4 CH2CH2-(OCH2CH2) t - and t is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0245] In some embodiments, L 2 does not exist or -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 Alkylene)-, -C(=O)-(CH2CH2O) m -(CH2) P -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) P -, -NR 4 C(=O)-(CH2CH2O) n -(CH2)P - or -(CH2CH2O) n -(CH2) P -, each m is independently 1, 2, 3, 4, 5, or 6, and each p is independently 1 or 2.

[0246] In some embodiments, L 3 is absent or is one or more independently selected groups selected from natural or unnatural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates, and any free amine (—NH) of an amino acid is optionally replaced by R 5 or -C(=O)R 5 and any free carboxylic acid of any amino acid is optionally substituted with -C(=O)NH-R5.

[0247] In some embodiments, L 3 is absent or is one or more independently selected groups selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, tyrosine, valine, and amino (unsubstituted or substituted benzyl) carbamate, and any free amine of an amino acid may optionally be replaced by R 5 or -C(=O)(R 5 ), and any free carboxylic acid of the amino acid is optionally substituted with —C(═O)NH(R 5 ) is replaced.

[0248] In some embodiments, L 3is lysine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, serine, tyrosine, valine, citrulline, methionine-valine-lysine, glycine-phenylalanine-glycine-glycine, tyrosine-arginine-valine, arginine-valine, valine-citrulline-(para-aminobenzylcarbamate), or combinations thereof, and any free amine (—NH) of an amino acid is optionally replaced by —C(═O) (unsubstituted or substituted C1-C 20 alkylene) or -C(=O)-C4-C 20 substituted with polyethylene glycol, and any free carboxylic acid (—COH) of any amino acid is optionally substituted with —C(═O)NH—(2,4,6-trimethyl-3-bromophenyl), —C(═O)NH—(unsubstituted or substituted C-C 20 alkylene), or -C(=O)NH-(C4-C 20 Polyethylene glycol).

[0249] In some embodiments, L 3 teeth,

[0250] [ka]

[0251] [ka] is.

[0252] In some embodiments, L 4 is absent, -C(=O)-(unsubstituted or substituted C1-C6 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C6 alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C6 alkylene)-, -C(=O)-(CH2CH2O) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2O) n-(CH2) q -, -NR 4 C(=O)-(CH2CH2O) n -(CH2) q - or -(CH2CH2O) n -(CH2) q -, wherein each n is independently 1, 2, 3, 4, 5, or 6, and each q is independently 1 or 2.

[0253] In some embodiments, L 5 is -C(=O)-(CH2) n -, -C(=O)NR 4 -(CH2) n -, -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2O) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2O) n -(CH2) q -, -(CH2CH2O) n -(CH2) q -, -C(=O)-(OCH2CH2) n - or -(OCH2CH2) n -, and each p is independently 0, 1, or 2.

[0254] In some embodiments, L 5 is -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2O) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) q -, or -NR 4 C(=O)-(CH2CH2O) n -(CH2) q - and each q is independently 1 or 2.

[0255] In some embodiments, L 1 represents unsubstituted or substituted C1-C6 alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L 2 does not exist or -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 Alkylene)-, -C(=O)-(CH2CH2O) m -(CH2) P -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) P -, -NR 4 C(=O)-(CH2CH2O) n -(CH2) P - or -(CH2CH2O) n -(CH2) P -, each m is independently 1, 2, 3, 4, 5, or 6, each p is independently 1 or 2, and L 4 is absent, -C(=O)-(unsubstituted or substituted C1-C6 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C6 alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C6 alkylene)-, -C(=O)-(CH2CH2O) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2O) n -(CH2) q - or -(CH2CH2O) n -(CH2) q - and L 5is -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2O) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) q -, or -NR 4 C(=O)-(CH2CH2O) n -(CH2) q -, wherein each n is independently 1, 2, 3, 4, 5, or 6, and each q is independently 1 or 2.

[0256] In some embodiments, L 1 represents unsubstituted or substituted C1-C6 alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene;

[0257] L 2 does not exist or -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 Alkylene)-, -C(=O)-(CH2CH2O) m -(CH2) P -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) P -, -NR 4 C(=O)-(CH2CH2O) n -(CH2) P - or -(CH2CH2O) n -(CH2) P -, each m is independently 1, 2, 3, 4, 5, or 6, each p is independently 1 or 2, and L 4 does not exist, and L 5 is -NR 4 C(=O)-(CH2)n -, -C(=O)-(CH2CH2O) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) q -, or -NR 4 C(=O)-(CH2CH2O) n -(CH2) q -, wherein each n is independently 1, 2, 3, 4, 5, or 6, and each q is independently 1 or 2.

[0258] In some embodiments, X 1 is alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, leucine, lysine, phenylalanine, serine, tyrosine, valine, or a combination thereof, and any free amine of an amino acid is optionally R 5 or -C(=O)(R 5 ), and any free carboxylic acid of the amino acid is optionally substituted with —C(═O)NH(R 5 ) and R 5 are each independently unsubstituted or substituted C1-C 10 Alkylene, C4-C 20 polyethylene glycol, or unsubstituted or substituted phenyl, wherein the substituted phenyl is substituted with 1, 2, 3, 4, or 5 groups independently selected from F, Cl, Br, I, —CH3, and CF3; L 1 is -NR 4 CH2CH2-(OCH2CH2) t where t is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and L 2 does not exist or -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 Alkylene)-, -C(=O)-(CH2CH2O) m -(CH2) P -, -C(=O)NR4 -(CH2CH2O) n -(CH2) P -, -NR 4 C(=O)-(CH2CH2O) n -(CH2) P - or -(CH2CH2O) n -(CH2) P - and

[0259] Each m is independently 1, 2, 3, 4, 5, or 6; each p is independently 1 or 2; L 3 does not exist, and L 4 does not exist, and L 5 is -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2O) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) q -, or -NR 4 C(=O)-(CH2CH2O) n -(CH2) q -, wherein each n is independently 1, 2, 3, 4, 5, or 6, and each q is independently 1 or 2.

[0260] In some embodiments, L 1 represents unsubstituted or substituted C1-C6 alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L 2 does not exist or -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 Alkylene)-, -C(=O)-(CH2CH2O) m -(CH2) P -, -C(=O)NR 4-(CH2CH2O) n -(CH2) P -, -NR 4 C(=O)-(CH2CH2O) n -(CH2) P - or -(CH2CH2O) n -(CH2) P -, each m is independently 1, 2, 3, 4, 5, or 6, each p is independently 1 or 2, and L 3 are one or more independently selected groups selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, tyrosine, valine, and amino (unsubstituted or substituted benzyl) carbamate, and any free amine of an amino acid may optionally be R 5 or -C(=O)(R 5 ), and any free carboxylic acid of the amino acid is optionally substituted with —C(═O)NH(R 5 ) and R 5 are each independently unsubstituted or substituted C1-C 10 Alkylene, C4-C 20 polyethylene glycol, or unsubstituted or substituted phenyl, wherein the substituted phenyl is substituted with 1, 2, 3, 4, or 5 groups independently selected from F, Cl, Br, I, —CH3, and CF3; L 4 does not exist, and L 5 is -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2O) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) q -, or -NR 4 C(=O)-(CH2CH2O) n -(CH2) q -, wherein each n is independently 1, 2, 3, 4, 5, or 6, and each q is independently 1 or 2.

[0261] In some embodiments, heteroalkylene is an alkyl group in which one carbon atom is —S(═O)(═NH)—, —S(═O)(═NR 5 )-, -P(=O)OH-, -NHC(=N-CN)NH-, or -NHC(=NR 5 ) alkylene substituted with NH-.

[0262] In some embodiments, —CH CH C(═O)X 1 -L- is

[0263] [ka]

[0264] [ka]

[0265] [ka]

[0266] [ka] is.

[0267] Conjugate moiety (R 2 ) Furthermore, the conjugate moiety (R 2 ) is used to facilitate binding to the tumor-targeting moiety. In some embodiments, R 2 is the tumor-targeting moiety R 3 In some embodiments, R 2 is the tumor-targeting moiety R 3 This is a moiety that can react with the amine (-NH2) in the side chain of the lysine residue.

[0268] In some embodiments, R 2is the tumor-targeting moiety R 3 and includes a tetrafluorophenyl ester, a pentafluorophenyl ester, a dinitrophenyl ester, a succinimide ester, a sulfosuccinimide ester, or an isothiocyanate.

[0269] In some embodiments, R 2 is the tumor-targeting moiety R 3 is a moiety that can react with an amine (-NH2) of

[0270] [ka] Including, X is absent, -O-, -S-, -S(=O)-, -S(=O)2-, or -NR a -, -C(=O)-, -NR a C(=O)-, -C(=O)NR a -, -C(=O)O-, -OC(=O)-, -OC(=O)NR a -, -NR a C(=O)NR a -, -NR a C(=S)NR a -, -NR a C(=O)O-, R a are each independently selected from hydrogen and C1-C4 alkyl.

[0271] In some embodiments, R 2 is the tumor-targeting moiety R 3 is a moiety that can react with an amine (-NH2) of

[0272] [ka] and X is absent or -NR a C(=S)NR a - and R aare each independently selected from hydrogen and C1-C4 alkyl. In some embodiments, X is absent or is -NHC(=S)NH-.

[0273] In some embodiments, R 2 is the tumor-targeting moiety R 3 is a moiety that can react with an amine (-NH2) of

[0274] [ka] and X is absent or -NHC(=S)NH-.

[0275] In some embodiments, R 2 is the tumor-targeting moiety R 3 is a moiety that can react with an amine (-NH2) of

[0276] [ka] Includes.

[0277] In some embodiments, R 2 is the tumor-targeting moiety R 3 is a moiety that can react with an amine (-NH2) of

[0278] [ka] and X is absent or -NHC(=S)NH-.

[0279] Alternatively / additionally, an isothiocyanate linker containing a lysine residue may be used in the immunoconjugates of the invention, for example, p-SCN-Bn-DOTA.

[0280] In some embodiments, R 2 is the tumor-targeting moiety R 3is a moiety capable of reacting with the thiol (-SH) in the side chain of a cysteine ​​residue in the compound of formula (I), and includes a maleimide group, a haloacetamide group, a haloacetyl group, a haloacetate group, a pyridinylthio group, a vinylcarbonyl group, an aziridinyl group, a disulfide group, an acetylene group, a hydroxysuccinimide group, or a thiol group.

[0281] In some embodiments, R 2 is the tumor-targeting moiety R 3 is a moiety that can react with a thiol (-SH) in

[0282] [ka] and m is 0, 1, 2, 3, 4, or 5.

[0283] In some embodiments, R 2 is the tumor-targeting moiety R 3 is a moiety that can react with a thiol (-SH) in

[0284] [ka] Includes.

[0285] In some embodiments, the linker can be conjugated to the antibody via a cysteine ​​bridging functionality, 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-bridge stretcher element is shown below (where the wavy line indicates the site of covalent attachment to the immunoconjugate):

[0286] [ka]

[0287] For further details regarding linkers and their use in the compounds described herein, see Wu, AM; Senter, PDNat. Biotechnol. 2005, 23(9): 1137-1146; Beck, A.; et al. Discov. Med. 2010, 10(53): 329-339; Nolting, B.; et al. Methods. Mol. Biol. 2013, 1045: 71-100; Jain, N.; et al. Pharm. Res. 2015, 32: 3526-3540; McCombs, JR; Owen, SCAAPS J. 2015, 17(2): 339-351; Jun Lu, et al., Int J Mol Sci. 2016 Apr; 17(4): 561. Each of these documents is incorporated herein by reference for its disclosure of the above linkers.

[0288] Exemplary Immunoconjugates In some embodiments, the immunoconjugate comprises:

[0289] [ka] A compound of formula (I) or a pharmaceutically acceptable salt thereof: During the ceremony, R 1 is a chelating moiety or a radionuclide complex thereof; R 2 is the tumor-targeting moiety R 3 is a moiety capable of reacting with an amine (-NH2) or thiol (-SH) of X 1 Ha-NR a -, -NR a S(=O)2-, -NR a S(=O)NR a or one or more independently selected natural or unnatural amino acids, any free amine of the amino acid optionally being substituted with R 5 or -C(=O)R5 and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R; R a are each independently selected from hydrogen and C1-C4 alkyl; L is -L 1 -L 2 -L 3 -L 4 -L 5 is a linker which is L 1 is unsubstituted or substituted C1-C 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, unsubstituted or substituted C-C 20 Alkenylene, unsubstituted or substituted C-C 20 Alkynylene, C4-C 20 Polyethylene glycol, -(X 3 CH2CH2) t -, unsubstituted or substituted cycloalkylene, unsubstituted or substituted heterocycloalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; X 3 are each independently O and NR 4 is selected from each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; L 2 is absent or unsubstituted or substituted C1-C 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, -C(=O)-(unsubstituted or substituted C-C 10 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 alkylene)-, -C(=O)-(CH2CH2X 3 ) m -(CH2) P -, -C(=O)NR 4-(CH2CH2X 3 ) m -(CH2) p -, -NR 4 C(=O)-(CH2CH2X 3 ) m -(CH2) p - or -(CH2CH2X 3 ) m -(CH2) p - and R 4 are each independently selected from hydrogen and C1-C6 alkyl; each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each p is independently 0, 1, or 2; L 3 is absent or is one or more independently selected groups selected from natural or unnatural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates, and any free amine (—NH) of an amino acid is optionally substituted with R 5 or -C(=O)R 5 and any free carboxylic acid of any amino acid is optionally substituted with -C(=O)NH-R5, and the substituted benzyl is independently substituted with halogen, -OH, -OR 5 , -CO2H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C1-C6 alkyl, wherein the substituted C1-C6 alkyl is selected from -OH, -CO2H, -NHR 5 , -C(=O)NHR 5 , and -NHC(=O)R 5 is replaced by L 4 is absent or unsubstituted or substituted C1-C 10 Alkylene, -C(=O)-(unsubstituted or substituted C-C 10 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR4 C(=O)-(unsubstituted or substituted C1-C 10 alkylene)-, -C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2X 3 ) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -(CH2CH2X 3 ) n -(CH2) q - or -(X 3 CH2CH2) n - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each q is independently 0, 1, or 2; L 5 does not exist or -C(=O)-(CH2) n -, -C(=O)NR 4 -(CH2) n -, -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2X 3 ) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -(CH2CH2X 3 ) n -(CH2) q -, -C(=O)-(X 3 CH2CH2) n - or -(X 3 CH2CH2) n - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each q is independently 0, 1, or 2; R a are each independently selected from hydrogen and C1-C4 alkyl; R b are each independently selected from hydrogen and C1-C4 alkyl; Heteroalkylene is a heteroalkylene group in which one carbon atom is -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NH)-, or -S(=O)(=NR 5 )-, -NR 5 -, -P(=O)OR b -, -NR a C(=O)-, -C(=O)NR a -, -OC(=O)NR a -, -NR a C(=O)NR a -, -NR a C(=N-CN)NR a -, -NR a C(=NR 5 )NR a -, or -NR a alkylene substituted with C(=O)O-; -L 1 -, -L 2 -, -L 3 -, -L 4 -, and -L 5 If one of - is replaced, -L 1 -, -L 2 -, -L 3 -, -L 4 -, and -L 5 - is halogen, -OH, -OR 5 , -CO2H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C1-C6 alkyl, wherein the substituted C1-C6 alkyl is selected from -OH, -CO2H, -NHR 5 , -C(=O)NHR 5 , and -NHC(=O)R5 is replaced by R 5 are independently C1-C 10 Alkyl, C4-C 30 polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; However, L 3 If there is no R, then there is at least one R 5 exists, or X 1 is -N(C1-C4 alkyl), -NR a S(=O)2-, -NR a S(=O)NR a - or one or more independently selected natural or unnatural amino acids.

[0290] In some embodiments, the immunoconjugate has Formula (II), Formula (III), or Formula (IV):

[0291] [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, R 1 is a chelating moiety or a radionuclide complex thereof; -NH-R 3 is a tumor-targeting moiety, X is absent, -O-, -S-, -S(=O)-, -S(=O)2-, or -NR a -, -C(=O)-, -NR a C(=O)-, -C(=O)NR a -, -C(=O)O-, -OC(=O)-, -OC(=O)NR a -, -NR a C(=O)NR a -, -NR a C(=S)NR a -, -NR a C(=O)O-, R aare each independently selected from hydrogen and C1-C4 alkyl; X 1 Ha-NR a -, -NR a S(=O)2-, -NR a S(=O)NR a or one or more independently selected natural or unnatural amino acids, any free amine of the amino acid optionally being substituted with R 5 or -C(=O)R 5 and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R; R a are each independently selected from hydrogen and C1-C4 alkyl; L is -L 1 -L 2 -L 3 -L 4 -L 5 is a linker which is L 1 is unsubstituted or substituted C1-C 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, unsubstituted or substituted C-C 20 Alkenylene, unsubstituted or substituted C-C 20 Alkynylene, C4-C 20 Polyethylene glycol, -(X 3 CH2CH2) t -, unsubstituted or substituted cycloalkylene, unsubstituted or substituted heterocycloalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; X 3 are each independently O and NR 4 is selected from each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; L 2 is absent or unsubstituted or substituted C1-C 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, -C(=O)-(unsubstituted or substituted C-C10 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 alkylene)-, -C(=O)-(CH2CH2X 3 ) m -(CH2) P -, -C(=O)NR 4 -(CH2CH2X 3 ) m -(CH2) p -, -NR 4 C(=O)-(CH2CH2X 3 ) m -(CH2) p - or -(CH2CH2X 3 ) m -(CH2) p - and R 4 are each independently selected from hydrogen and C1-C6 alkyl; each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each p is independently 0, 1, or 2; L 3 is absent or is one or more independently selected groups selected from natural or unnatural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates, and any free amine (—NH) of an amino acid is optionally substituted with R 5 or -C(=O)R 5 and any free carboxylic acid of any amino acid is optionally substituted with -C(=O)NH-R5, and the substituted benzyl is independently substituted with halogen, -OH, -OR 5 , -CO2H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C1-C6 alkyl, wherein the substituted C1-C6 alkyl is selected from -OH, -CO2H, -NHR 5 , -C(=O)NHR5 , and -NHC(=O)R 5 is replaced by L 4 is absent or unsubstituted or substituted C1-C 10 Alkylene, -C(=O)-(unsubstituted or substituted C-C 10 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 alkylene)-, -C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2X 3 ) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -(CH2CH2X 3 ) n -(CH2) q - or -(X 3 CH2CH2) n - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each q is independently 0, 1, or 2; L 5 does not exist or -C(=O)-(CH2) n -, -C(=O)NR 4 -(CH2) n -, -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2X 3 ) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2X3 ) n -(CH2) q -, -(CH2CH2X 3 ) n -(CH2) q -, -C(=O)-(X 3 CH2CH2) n - or -(X 3 CH2CH2) n - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each q is independently 0, 1, or 2; R a are each independently selected from hydrogen and C1-C4 alkyl; R b are each independently selected from hydrogen and C1-C4 alkyl; Heteroalkylene is a heteroalkylene group in which one carbon atom is -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NH)-, or -S(=O)(=NR 5 )-, -NR 5 -, -P(=O)OR b -, -NR a C(=O)-, -C(=O)NR a -, -OC(=O)NR a -, -NR a C(=O)NR a -, -NR a C(=N-CN)NR a -, -NR a C(=NR 5 )NR a -, or -NR a alkylene substituted with C(=O)O-; -L 1 -, -L 2 -, -L 3 -, -L 4 -, and -L 5 If one of - is replaced, -L 1 -, -L 2 -, -L 3 -, -L 4 -, and -L 5 - is halogen, -OH, -OR5 , -CO2H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C1-C6 alkyl, wherein the substituted C1-C6 alkyl is selected from -OH, -CO2H, -NHR 5 , -C(=O)NHR 5 , and -NHC(=O)R 5 is replaced by R 5 are independently C1-C 10 Alkyl, C4-C 30 polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; However, L 3 If there is no R, then there is at least one R 5 exists, or X 1 is -N(C1-C4 alkyl), -NR a S(=O)2-, -NR a S(=O)NR a - or one or more independently selected natural or unnatural amino acids.

[0292] In some embodiments, the immunoconjugate has formula (II):

[0293] [ka] or a pharmaceutically acceptable salt thereof.

[0294] In some embodiments, the immunoconjugate has formula (III):

[0295] [ka] or a pharmaceutically acceptable salt thereof.

[0296] In some embodiments, the immunoconjugate has formula (IV):

[0297] [ka] or a pharmaceutically acceptable salt thereof.

[0298] In some embodiments, R 2 is the tumor-targeting moiety R 3 This is the moiety that can react with the amine (-NH2) of lysine.

[0299] In some embodiments, the compound of formula (II) has the formula (IIa):

[0300] [ka] or a pharmaceutically acceptable salt thereof, where -NHCH2CH2CH2CH2- is the tumor targeting moiety R 3 is the side chain of the lysine residue.

[0301] In some embodiments, the compound of formula (III) has the formula (IIIa):

[0302] [ka] or a pharmaceutically acceptable salt thereof, where -NHCH2CH2CH2CH2- is the tumor targeting moiety R 3 is the side chain of the lysine residue.

[0303] In some embodiments, R 2 is the tumor-targeting moiety R 3 It is a moiety that can react with the thiol (-SH) of cysteine.

[0304] In some embodiments, the compound of formula (IV) has the formula (IVa):

[0305] [ka] or a pharmaceutically acceptable salt thereof, where -NHCH2CH2CH2CH2- is the tumor targeting moiety R 3 is the side chain of the lysine residue.

[0306] In some embodiments, the immunoconjugate has Formula (V), Formula (VI), Formula (VII), or Formula (VIII):

[0307] [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, R 1 is a chelating moiety or a radionuclide complex thereof; -SR 3 is a tumor-targeting moiety, X 1 Ha-NR a -, -NR a S(=O)2-, -NR a S(=O)NR a or one or more independently selected natural or unnatural amino acids, any free amine of the amino acid optionally being substituted with R 5 or -C(=O)R 5 and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R; R a are each independently selected from hydrogen and C1-C4 alkyl; L is -L 1 -L 2 -L 3 -L 4 -L 5 is a linker which is L 1 is unsubstituted or substituted C1-C 10 Alkylene, unsubstituted or substituted C1-C 10Heteroalkylene, unsubstituted or substituted C-C 20 Alkenylene, unsubstituted or substituted C-C 20 Alkynylene, C4-C 20 Polyethylene glycol, -(X 3 CH2CH2) t -, unsubstituted or substituted cycloalkylene, unsubstituted or substituted heterocycloalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; X 3 are each independently O and NR 4 is selected from each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; L 2 is absent or unsubstituted or substituted C1-C 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, -C(=O)-(unsubstituted or substituted C-C 10 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 alkylene)-, -C(=O)-(CH2CH2X 3 ) m -(CH2) P -, -C(=O)NR 4 -(CH2CH2X 3 ) m -(CH2) p -, -NR 4 C(=O)-(CH2CH2X 3 ) m -(CH2) p - or -(CH2CH2X 3 ) m -(CH2) p - and R 4 are each independently selected from hydrogen and C1-C6 alkyl; each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each p is independently 0, 1, or 2; L 3 is absent or is one or more independently selected groups selected from natural or unnatural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates, and any free amine (—NH) of an amino acid is optionally substituted with R 5 or -C(=O)R 5 and any free carboxylic acid of any amino acid is optionally substituted with -C(=O)NH-R5, and the substituted benzyl is independently substituted with halogen, -OH, -OR 5 , -CO2H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C1-C6 alkyl, wherein the substituted C1-C6 alkyl is selected from -OH, -CO2H, -NHR 5 , -C(=O)NHR 5 , and -NHC(=O)R 5 is replaced by L 4 is absent or unsubstituted or substituted C1-C 10 Alkylene, -C(=O)-(unsubstituted or substituted C-C 10 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 alkylene)-, -C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2X 3 ) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -(CH2CH2X 3 ) n -(CH2)q - or -(X 3 CH2CH2) n - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each q is independently 0, 1, or 2; L 5 does not exist or -C(=O)-(CH2) n -, -C(=O)NR 4 -(CH2) n -, -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2X 3 ) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -(CH2CH2X 3 ) n -(CH2) q -, -C(=O)-(X 3 CH2CH2) n - or -(X 3 CH2CH2) n - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each q is independently 0, 1, or 2; R a are each independently selected from hydrogen and C1-C4 alkyl; R b are each independently selected from hydrogen and C1-C4 alkyl; Heteroalkylene is a heteroalkylene group in which one carbon atom is -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NH)-, or -S(=O)(=NR 5 )-, -NR 5 -, -P(=O)OR b-, -NR a C(=O)-, -C(=O)NR a -, -OC(=O)NR a -, -NR a C(=O)NR a -, -NR a C(=N-CN)NR a -, -NR a C(=NR 5 )NR a -, or -NR a alkylene substituted with C(=O)O-; -L 1 -, -L 2 -, -L 3 -, -L 4 -, and -L 5 If one of - is replaced, -L 1 -, -L 2 -, -L 3 -, -L 4 -, and -L 5 - is halogen, -OH, -OR 5 , -CO2H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C1-C6 alkyl, wherein the substituted C1-C6 alkyl is selected from -OH, -CO2H, -NHR 5 , -C(=O)NHR 5 , and -NHC(=O)R 5 is replaced by R 5 are independently C1-C 10 Alkyl, C4-C 30 polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; However, L 3 If there is no R, then there is at least one R 5 exists, or X 1 is -N(C1-C4 alkyl), -NR a S(=O)2-, -NR a S(=O)NR a- or one or more independently selected natural or unnatural amino acids.

[0308] In some embodiments, the immunoconjugate has formula (V):

[0309] [ka] or a pharmaceutically acceptable salt thereof.

[0310] In some embodiments, the immunoconjugate has formula (VI):

[0311] [ka] or a pharmaceutically acceptable salt thereof.

[0312] In some embodiments, the immunoconjugate has formula (VII):

[0313] [ka] or a pharmaceutically acceptable salt thereof.

[0314] In some embodiments, the immunoconjugate has formula (VIII):

[0315] [ka] or a pharmaceutically acceptable salt thereof.

[0316] In some embodiments, the compound of formula (V) has the formula (Va):

[0317] [ka] or a pharmaceutically acceptable salt thereof, where -SCH2- is a tumor-targeting moiety R 3 The thiol (-SH) is the side chain of the cysteine ​​residue in

[0318] In some embodiments, the compound of formula (VI) has formula (VIa):

[0319] [ka] or a pharmaceutically acceptable salt thereof, where -SCH2- is a tumor-targeting moiety R 3 The thiol (-SH) is the side chain of the cysteine ​​residue in

[0320] In some embodiments, the compound of formula (VI) has formula (VIa):

[0321] [ka] or a pharmaceutically acceptable salt thereof, where -SCH2- is a tumor-targeting moiety R 3 The thiol (-SH) is the side chain of the cysteine ​​residue in

[0322] In some embodiments, the compound of formula (VII) has the formula (VIIa)

[0323] [ka] or a pharmaceutically acceptable salt thereof, where -SCH2- is a tumor-targeting moiety R 3 The thiol (-SH) is the side chain of the cysteine ​​residue in

[0324] In some embodiments, the compound of formula (VIII) has the formula (VIIIa)

[0325] [ka] or a pharmaceutically acceptable salt thereof, where -SCH2- is a tumor-targeting moiety R 3 The thiol (-SH) is the side chain of the cysteine ​​residue in

[0326] In some embodiments, R 1 is a chelating moiety selected from the list consisting of DOTA, DO3A, DOTAGA, DOTAGA anhydride, Py4Pa, Py4Pa-NCS, Crown, Macropa, Macropa-NCS, HEHA, CHXoctapa, Bispa, and Noneunpa, or radionuclide complexes thereof.

[0327] In some embodiments, R 1 is a chelating moiety selected from the list consisting of DOTMA, DOTPA, DO3AM-acetic acid, DOTP, DOTMP, DOTA-4AMP, CB-TE2A, NOTA, NOTP, TETPA, TETA, PEPA, H4Octapa, H2Dedpa, DO2P, EDTA, DTPA-BMA, 3,2,3-LI(HOPO), 3,2-HOPO, Neunpa, Neunpa-NCS, Octapa, PyPa, porphyrin, and deferoxamine, or a radionuclide complex thereof.

[0328] In some embodiments, R 1 teeth, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A), α,α',α'',α''''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTMA), 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid (DOTPA), 2,2',2''-(10-(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, 6,6'-(((pyridine-2,6-diylbis(methylene))bis((carboxymethyl)azanediyl))-bis(methylene))dipicolinic acid (H4pypa), 6,6',6'',6''''-(((pyridine-2,6-diylbis(methylene))bis(azanetriyl))tetrakis(methylene))-tetrapicolinic acid (H4py4pa), 3,6,9,12-tetrakis(carboxymethyl)-3,6,9,12-tetraazatetradecanedioic acid (TTHA), or a radionuclide complex thereof.

[0329] In some embodiments, R 1 teeth,

[0330] [ka] or a radionuclide complex thereof.

[0331] In some embodiments, R 1 teeth,

[0332] [ka] or a radionuclide complex thereof.

[0333] In some embodiments, R 1 teeth,

[0334] [ka] or a radionuclide complex thereof.

[0335] In some embodiments, R 2 is the tumor-targeting moiety R 3 and includes a tetrafluorophenyl ester, a pentafluorophenyl ester, a dinitrophenyl ester, a succinimide ester, a sulfosuccinimide ester, or an isothiocyanate.

[0336] In some embodiments, R 2 is the tumor-targeting moiety R 3 is a moiety that can react with an amine (-NH2) of

[0337] [ka] and X is absent, -O-, -S-, -S(=O)-, -S(=O)2-, or -NR a -, -C(=O)-, -NR a C(=O)-, -C(=O)NR a -, -C(=O)O-, -OC(=O)-, -OC(=O)NR a -, -NR a C(=O)NR a -, -NR a C(=S)NR a -, or -NR a C(=O)O-, and R a are each independently selected from hydrogen and C1-C4 alkyl.

[0338] In some embodiments, X is absent, —O—, —S—, —S(═O)—, —S(═O) 2 —, or —NR a -, -C(=O)-, -NR a C(=O)- or -C(=O)NR a In some embodiments, X is -O-, -S-, -S(=O)-, -S(=O)2-, or -NR aIn some embodiments, X is -O-. In some embodiments, X is -S-. In some embodiments, X is -NR a C(=O)NR a -, -NR a C(=S)NR a -, or -NR a In some embodiments, X is -OC(=O)NR a -or-NR a In some embodiments, X is -NR a C(=O)NR a In some embodiments, X is -NR a C(=S)NR a In some embodiments, X is absent.

[0339] In some embodiments, R 2 is the tumor-targeting moiety R 3 and includes a maleimide group, a haloacetamide group, a haloacetyl group, a haloacetate group, a pyridinylthio group, a vinylcarbonyl group, an aziridinyl group, a disulfide group, an acetylene group, a hydroxysuccinimide group, or a thiol group.

[0340] In some embodiments, the tumor-targeting moiety R 3 is a polypeptide that contains an antigen-binding region and an immunoglobulin heavy chain constant region, and the molecular weight of the polypeptide is 60 to 110 kDa.

[0341] In some embodiments, R 2 is the tumor-targeting moiety R 3 is a moiety that can react with a thiol (-SH) in

[0342] [ka] and m is 0, 1, 2, 3, 4, or 5.

[0343] In some embodiments, X 1 is -NR a -, -NR a S(═O)₂—, or one or more independently selected natural or unnatural amino acids, any free amine of the amino acid optionally being substituted with R 5 or -C(=O)R 5 and any free carboxylic acid of any amino acid is optionally substituted with -C(=O)NH-R5. In some embodiments, X 1 is -NH-, -N(CH3)-, -N(CH2CH3)-, -NHS(=O)2-, -N(CH3)S(=O)2-, or -N(CH2CH3)S(=O)2-. In some embodiments, X 1 is -NH-, -N(CH3)-, or -N(CH2CH3)-. In some embodiments, X 1 is —NHS(═O)—, —N(CH)S(═O)—, or —N(CHCH)S(═O)—. In some embodiments, X 1 is -NH-. In some embodiments, X 1 is —N(CH)—. In some embodiments, X 1 is —N(CH2CH3)—. In some embodiments, X 1 is -NHS(=O)2-. In some embodiments, X 1 is —N(CH 3 )S(═O) 2 —. In some embodiments, X 1 is -N(CH2CH3)S(=O)2-.

[0344] In some embodiments, X 1 are one or more independently selected natural or unnatural amino acids, and any free amine of an amino acid is optionally substituted with R 5 or -C(=O)R 5 and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R5; and L 1 is -NR 4 CH2CH2-(OCH2CH2)t - and t is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0345] In some embodiments, X 1 is lysine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, serine, tyrosine, valine, citrulline, methionine-valine-lysine, glycine-phenylalanine-glycine-glycine, tyrosine-arginine-valine, arginine-valine, or combinations thereof, and any free amine (—NH2) of an amino acid is optionally replaced by R 5 or -C(=O)R 5 and any free carboxylic acid of any amino acid is optionally substituted with -C(=O)NH-R5.

[0346] In some embodiments, L 1 represents unsubstituted or substituted C1-C6 alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L 2 does not exist or -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 Alkylene)-, -C(=O)-(CH2CH2O) m -(CH2) P -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) P -, -NR 4 C(=O)-(CH2CH2O) n -(CH2) P - or -(CH2CH2O) n -(CH2) P -, each m is independently 1, 2, 3, 4, 5, or 6, each p is independently 1 or 2, and L 4is absent, -C(=O)-(unsubstituted or substituted C1-C6 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C6 alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C6 alkylene)-, -C(=O)-(CH2CH2O) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2O) n -(CH2) q - or -(CH2CH2O) n -(CH2) q - and L 5 is -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2O) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) q -, or -NR 4 C(=O)-(CH2CH2O) n -(CH2) q -, wherein each n is independently 1, 2, 3, 4, 5, or 6, and each q is independently 1 or 2.

[0347] In some embodiments, L 1 represents unsubstituted or substituted C1-C6 alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L 2 does not exist or -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10Alkylene)-, -C(=O)-(CH2CH2O) m -(CH2) P -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) P -, -NR 4 C(=O)-(CH2CH2O) n -(CH2) P - or -(CH2CH2O) n -(CH2) P -, each m is independently 1, 2, 3, 4, 5, or 6, each p is independently 1 or 2, and L 4 does not exist, and L 5 is -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2O) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) q -, or -NR 4 C(=O)-(CH2CH2O) n -(CH2) q -, wherein each n is independently 1, 2, 3, 4, 5, or 6, and each q is independently 1 or 2.

[0348] In some embodiments, X 1 is alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, leucine, lysine, phenylalanine, serine, tyrosine, valine, or a combination thereof, and any free amine of an amino acid is optionally R 5 or -C(=O)(R 5 ), and any free carboxylic acid of the amino acid is optionally substituted with —C(═O)NH(R 5 ) and R 5 are each independently unsubstituted or substituted C1-C 10 Alkylene, C4-C 20polyethylene glycol, or unsubstituted or substituted phenyl, wherein the substituted phenyl is substituted with 1, 2, 3, 4, or 5 groups independently selected from F, Cl, Br, I, —CH3, and CF3; L 1 is -NR 4 CH2CH2-(OCH2CH2) t where t is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and L 2 does not exist or -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 Alkylene)-, -C(=O)-(CH2CH2O) m -(CH2) P -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) P -, -NR 4 C(=O)-(CH2CH2O) n -(CH2) P - or -(CH2CH2O) n -(CH2) P -, each m is independently 1, 2, 3, 4, 5, or 6, each p is independently 1 or 2, and L 3 does not exist, and L 4 does not exist, and L 5 is -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2O) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) q -, or -NR 4 C(=O)-(CH2CH2O) n -(CH2) q -, wherein each n is independently 1, 2, 3, 4, 5, or 6, and each q is independently 1 or 2.

[0349] In some embodiments, L1 represents unsubstituted or substituted C1-C6 alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L 2 does not exist or -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 Alkylene)-, -C(=O)-(CH2CH2O) m -(CH2) P -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) P -, -NR 4 C(=O)-(CH2CH2O) n -(CH2) P - or -(CH2CH2O) n -(CH2) P -, each m is independently 1, 2, 3, 4, 5, or 6, each p is independently 1 or 2, and L 3 is alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, tyrosine, valine, or a combination thereof, and any free amine of an amino acid is optionally R 5 or -C(=O)(R 5 ), and any free carboxylic acid of the amino acid is optionally substituted with —C(═O)NH(R 5 ) and R 5 are each independently unsubstituted or substituted C1-C 10 Alkylene, C4-C 20 polyethylene glycol, or unsubstituted or substituted phenyl, wherein the substituted phenyl is substituted with 1, 2, 3, 4, or 5 groups independently selected from F, Cl, Br, I, —CH3, and CF3; L 4 does not exist, and L5 is -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2O) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) q -, or -NR 4 C(=O)-(CH2CH2O) n -(CH2) q -, wherein each n is independently 1, 2, 3, 4, 5, or 6, and each q is independently 1 or 2.

[0350] In some embodiments, L 1 is unsubstituted or substituted C1-C 20 Alkylene, unsubstituted or substituted C1-C 20 Heteroalkylene, C4-C 20 In some embodiments, L is selected from the group consisting of polyethylene glycol, unsubstituted or substituted C3-C8 cycloalkylene, unsubstituted or substituted monocyclic C3-C8 heterocycloalkylene, unsubstituted or substituted phenylene, and unsubstituted or substituted monocyclic heteroarylene. 1 represents unsubstituted or substituted C1-C6 alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 In some embodiments, L is polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene. 1 does not exist.

[0351] In some embodiments, L 2 does not exist or -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 Alkylene)-, -C(=O)-(CH2CH2O) m -(CH2) P -, -C(=O)NR 4-(CH2CH2O) n -(CH2) P -, -NR 4 C(=O)-(CH2CH2O) n -(CH2) P - or -(CH2CH2O) n -(CH2) P -, each m is independently 1, 2, 3, 4, 5, or 6, and each p is independently 1 or 2. In some embodiments, L 2 does not exist.

[0352] In some embodiments, L 3 is absent, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, tyrosine, valine, or a combination thereof, and any free amine of an amino acid is optionally R 5 or -C(=O)(R 5 ), and any free carboxylic acid of the amino acid is optionally substituted with —C(═O)NH(R 5 ) is replaced.

[0353] In some embodiments, L 3 is lysine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, serine, tyrosine, valine, citrulline, methionine-valine-lysine, glycine-phenylalanine-glycine-glycine, tyrosine-arginine-valine, arginine-valine, valine-citrulline-(para-aminobenzylcarbamate), or combinations thereof, and any free amine (—NH) of an amino acid is optionally replaced by —C(═O) (unsubstituted or substituted C1-C 20 alkylene) or -C(=O)-C4-C 20 substituted with polyethylene glycol, and any free carboxylic acid (—COH) of any amino acid is optionally substituted with —C(═O)NH—(2,4,6-trimethyl-3-bromophenyl), —C(═O)NH—(unsubstituted or substituted C-C 20alkylene), or -C(=O)NH-(C4-C 20 In some embodiments, L is substituted with methyl methyl ether. 3 does not exist.

[0354] In some embodiments, L 4 is absent or unsubstituted or substituted C1-C 10 Alkylene, -C(=O)-(unsubstituted or substituted C-C 10 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C 10 (alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C 10 alkylene)-, -C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2X 3 ) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -(CH2CH2X 3 ) n -(CH2) q - or -(X 3 CH2CH2) n -, each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and each q is independently 0, 1, or 2. In some embodiments, L 4 is absent, -C(=O)-(unsubstituted or substituted C1-C6 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C1-C6 alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C6 alkylene)-, -C(=O)-(CH2CH2O) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2O) n-(CH2) q -, -NR 4 C(=O)-(CH2CH2O) n -(CH2) q - or -(CH2CH2O) n -(CH2) q -, each n is independently 1, 2, 3, 4, 5, or 6, and each q is independently 1 or 2. In some embodiments, L 4 does not exist.

[0355] In some embodiments, L 5 does not exist or -C(=O)-(CH2) n -, -C(=O)NR 4 -(CH2) n -, -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2X 3 ) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2X 3 ) n -(CH2) q -, -(CH2CH2X 3 ) n -(CH2) q -, -C(=O)-(X 3 CH2CH2) n - or -(X 3 CH2CH2) n -, each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and each q is independently 0, 1, or 2. In some embodiments, L 5 is -C(=O)-(CH2) n -, -C(=O)NR 4 -(CH2) n -, -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2O)n -(CH2) q -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) q -, -NR 4 C(=O)-(CH2CH2O) n -(CH2) q -, -(CH2CH2O) n -(CH2) q -, -C(=O)-(OCH2CH2) n - or -(OCH2CH2) n -, and each p is independently 0, 1, or 2. In some embodiments, L 5 is -NR 4 C(=O)-(CH2) n -, -C(=O)-(CH2CH2O) n -(CH2) q -, -C(=O)NR 4 -(CH2CH2O) n -(CH2) q -, or -NR 4 C(=O)-(CH2CH2O) n -(CH2) q - and each q is independently 1 or 2. In some embodiments, L 5 does not exist.

[0356] In some embodiments, heteroalkylene is an alkyl group in which one carbon atom is —S(═O)(═NH)—, —S(═O)(═NR 5 )-, -P(=O)OH-, -NHC(=N-CN)NH-, or -NHC(=NR 5 In some embodiments, heteroalkylene is an alkylene substituted with -S(=O)(=NH)- or -S(=O)(=NR 5 In some embodiments, the heteroalkylene is an alkylene substituted at one carbon atom with -P(=O)OH-. In some embodiments, the heteroalkylene is an alkylene substituted at one carbon atom with -NHC(=N-CN)NH- or -NHC(=NR 5) alkylene substituted with NH-.

[0357] In some embodiments, X 3 are each independently O. In some embodiments, X 3 are each independently, NR 4 is.

[0358] In some embodiments, R 4 Each is hydrogen. In some embodiments, R 4 are each C1-C6 alkyl.

[0359] In some embodiments, R 5 are independently C1-C 10 Alkyl, C4-C 30 In some embodiments, R is selected from polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene. 5 are independently C1-C 10 Alkyl or C4-C 30 In some embodiments, R 5 are each independently selected from unsubstituted or substituted arylene, or unsubstituted or substituted heteroarylene.

[0360] In some embodiments, R a are each independently selected from hydrogen and C1-C4 alkyl. a are each independently hydrogen. a are each independently C1-C4 alkyl.

[0361] In some embodiments, R b are each independently selected from hydrogen and C1-C4 alkyl. b are each independently hydrogen. bare each independently C1-C4 alkyl.

[0362] Exemplary compounds for use in preparing the immunoconjugates described herein include those shown in Table A.

[0363] [Table 2-1]

[0364] [Table 2-2]

[0365] [Table 2-3]

[0366] [Table 2-4]

[0367] [Table 2-5]

[0368] [Table 2-6]

[0369] [Table 2-7]

[0370] Radioimmunoconjugates In one embodiment, the present invention provides an immunoconjugate. In one embodiment, the immunoconjugate, when so labeled, linked, or loaded with an alpha emitter, is capable of delivering an alpha emitter in vivo. In one embodiment, the immunoconjugate, when so labeled, linked, or loaded, is further capable of delivering other radioisotopes (beta and / or gamma 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) can be delivered in vivo.

[0371] The immunoconjugates of the present disclosure may be loaded with a radioisotope for therapeutic or diagnostic effect. In certain embodiments, the chelator may further comprise a radioisotope. In certain embodiments, the radioisotope is an alpha emitter. In certain embodiments, the radioisotope is: 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi, and 213 In certain embodiments, the radioisotope is an alpha emitter selected from the list consisting of Bi. 225 In certain embodiments, the radioisotope is a β-emitter. In certain embodiments, the radioisotope is 177 Lu, 90 Y, 67 Cu, and 153 It is a beta emitter selected from Sm.

[0372] Also described herein are methods of making a radioimmunoconjugate, comprising loading or conjugating an immunoconjugate of the present disclosure to a radioisotope. In certain embodiments, the radioisotope is an alpha emitter. In certain embodiments, the radioisotope is: 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi, and 213 In certain embodiments, the radioisotope is an alpha emitter selected from the list consisting of 225-Ac, 225-Bi, 225-Ac, 225-Ac-I, 225-Bi ... 177 Lu, 90 Y, 67 Cu, and 153 It is a beta emitter selected from Sm.

[0373] In one aspect, the present invention provides a radioimmunoconjugate comprising an immunoconjugate of the present invention and an alpha-emitting radioisotope. In one embodiment, the alpha-emitting radioisotope of the radioimmunoconjugate is 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi, and 213 In one embodiment, the alpha-emitting radioisotope of the radioimmunoconjugate is selected from the group comprising: 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi, and 213 In one embodiment, the alpha-emitting radioisotope of the radioimmunoconjugate is selected from the group consisting of 225 In one embodiment, the alpha-emitting radioisotope of the radioimmunoconjugate is Ac. 223 In one embodiment, the alpha-emitting radioisotope of the radioimmunoconjugate is 224In one embodiment, the alpha-emitting radioisotope of the radioimmunoconjugate is 227 In one embodiment, the alpha-emitting radioisotope of the radioimmunoconjugate is 212 In one embodiment, the alpha-emitting radioisotope of the radioimmunoconjugate is Pb. 212 In one embodiment, the alpha-emitting radioisotope of the radioimmunoconjugate is Bi. 213 I'm Bi.

[0374] In some embodiments, the immunoconjugates of the invention are combined with a radioisotope to provide radioimmunoconjugates of the 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 In some embodiments, the radioisotope is, for example, Pd. 225 Ac, 223-Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi, and 213 In some embodiments, the radioisotope is an alpha emitter, such as Bi. 177 Lu, 90 Y, 67 Cu, 153In some embodiments, the radioisotope is a beta particle emitter, such as 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, and contains, both an alpha emitter and a beta emitter, depending on the alpha and beta emitter. In some embodiments, the radioisotope is, for example, 68 Ga, 64 Cu, 89 Zr, 111 In, 134 Ce are selected for use in radioactive imaging.

[0375] 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.

[0376] In some embodiments, the radioimmunoconjugate of the present invention comprises: 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 It contains a radioisotope selected from the group including Pd.

[0377] In some embodiments, the radioimmunoconjugate of the present invention comprises: 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.

[0378] In some embodiments, the radioisotope is 225 Ac, 223 Ra, 224 Ra, 227 Th,212 Pb, 212 It is an alpha particle emitting radioisotope containing Bi, or +Bi.

[0379] In some embodiments, the radioisotope is 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi, and 213 Bi is an alpha particle emitting radioisotope selected from the group consisting of:

[0380] Further embodiments of immunoconjugates, antigen-binding regions, and heavy chain variable regions are described below.

[0381] In some embodiments, the immunoconjugate comprises a dimerization domain or motif. In some further embodiments, the dimerization domain or motif is in the variant constant region, linker, or hinge region.

[0382] 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 assembled multimeric structures (see, e.g., Glockshuber R et al., Biochemistry 29:1362-7 (1990)). For example, the introduction of cysteine ​​residues at specific positions can be used to create disulfide-stabilized structures such as Cys-diabodies, scFv multimers, VHH multimers, VNAR multimers, and IgNAR multimers, such as by adding 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)).

[0383] Alternatively, two or more polypeptide chains can be linked using polypeptide domains that self-associate or multimerize with each other (see, e.g., U.S. Pat. No. 6,329,507). For example, the addition of a carboxy-terminal multimerization domain can be used to synthesize immunoglobulin domains, e.g., scFv, autonomous V H Domain, V H H, V NARand IgNARs. Examples of self-association domains known to those skilled in the art include immunoglobulin constant domains (e.g., knob-into-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 helix bundles (e.g., scFv-HLX), helix- These include turn-helix domains (e.g., scFv-dHlx), coiled-coil structures containing leucine zippers and cartilage oligomeric matrix proteins (e.g., scZIP), cAMP-dependent protein kinase (PKA) dimerization and docking domains (DDDs) (also called "dock-and-lock" or "DNLs") combined with A-kinase anchor protein (AKAP) anchor domains (ADs), streptavidin, verotoxin B multimerization domains, the tetramerization region from p53, and the 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);Chang C et al.,Clin Cancer Res 13:5586-91s(2007);Liu M et al.,Biochem J 406:237-46(2007);Zhang J et al.,Protein 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)).

[0384] Those skilled in the art can engineer the multimeric immunoconjugates of the present invention using various scFv-based polypeptide interactions known in the art, such as scFv-based dimeric complexes, trimeric complexes, tetrameric complexes, etc. For example, the length of the linker in the scFv can affect the spontaneous assembly of non-covalently based multimeric multivalent structures. Generally, linkers of 12 amino acids or less, including the absence of any linker, favor intermolecular domain swapping over intrachain domain pairing, thereby promoting the multimerization of scFv-containing polypeptides or proteins into high molecular weight species (see, for example, Dolezal O et al., Protein Eng 16:47-56 (2003)). However, scFvs with no 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 7:1027-33(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.

[0385] 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 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, substitution of hinge, linker, and / or chelator moieties, and 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 used to enhance the final construct to achieve desired characteristics, e.g., a particular binding affinity level for antigen binding, K D The final construct can be tailored to have a particular level of K and / or a particular level of Koff.

[0386] 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 (for example, 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 antibodies or to reduce the overall size of the immunoconjugate of the present invention.

[0387] In some embodiments, variants of the immunoconjugates of the present invention are made larger by the introduction of 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 conjugation 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 is 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.

[0388] Nucleic acids encoding the immunoconjugates of the invention can be modified to create chimeric or fusion immunoconjugate polypeptides, for example, by substituting human heavy and light chain constant domains (CH and CO sequences of the homologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison, et al., Proc Natl Acad Sci USA 81:6851 (1984)) or by fusing the immunoglobulin coding sequence with all or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide). The non-immunoglobulin polypeptide sequence can replace the constant domains of the immunoconjugate or 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.

[0389] Variations 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. Variations can be the substitution, deletion, or insertion 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 variation is by substituting at least one amino acid with any other amino acid in one or more of the domains of the immunoconjugate. 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. Allowable 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.

[0390] In certain embodiments, conservative substitutions of interest are shown in Table C, including under the heading of preferred substitutions. If such substitutions result in a change in biological activity, either designated as exemplary substitutions in Table C, or more substantial changes, such as those further described below with respect to amino acid classes, are introduced and the products screened.

[0391] [Table 3]

[0392] Substantial modifications 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 helix 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 grouped 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, gln, his, lys, arg, (5) Residues that affect chain orientation: gly, pro, and (6) Aromatic: trp, tyr, phe It can be divided into:

[0393] Non-conservative substitutions involve exchanging a member of one of these classes for another. Such substituted residues may also be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.

[0394] Variations 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 techniques can be performed on cloned DNA to produce DNA molecules encoding the immunoconjugate variants of the present invention.

[0395] In some embodiments, immunoconjugate variants having 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 may be introduced into the immunoconjugate of interest, and the products may be screened for desired activities, 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 may be introduced into the immunoconjugate of interest, and the products may be screened for activities, such as reduced or eliminated ADCC, CDC, target inhibition, and / or ADCP.

[0396] 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 variant selected for further study has a modification (e.g., an improvement) in a particular biological property (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and / or substantially retains a particular biological property of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be readily generated 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).

[0397] Modifications (e.g., substitutions) can be made in HVRs, for example, to improve immunoconjugate affinity. Such modifications can be made in HVR "hot spots," 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 is tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in 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 second library is then generated. The 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 multiple HVR residues (e.g., 4-6 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-L, in particular, are randomized. 3 are often targeted.

[0398] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such modifications do not significantly reduce the ability of the immunoconjugate to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not significantly reduce binding affinity may be made in HVRs. Such modifications may be outside the HVR "hot spots" or SDRs. In some embodiments of the variant VH and VL sequences provided above, each HVR is either unmodified or contains no more than one, two, or three amino acid substitutions.

[0399] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of multiple target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and substituted 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.

[0400] Alternatively / 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. Variants can be screened to determine whether they contain the desired properties.

[0401] In some embodiments, the immunoconjugates of the invention comprise antibody constructs comprising humanized immunoglobulin domains (used herein as antigen-binding regions).

[0402] 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 comprise a human immunoglobulin (recipient antibody) 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 instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may further comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being those of a human immunoglobulin consensus sequence. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321:522-5 (1986); Riechmann et al., Nature, 332:323-9 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-6 (1992)).

[0403] Methods for humanizing non-human antibodies are known in the art. Generally, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are typically obtained from an "import" variable domain. Humanization can essentially be performed according to the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Such "humanized" antibodies are thus chimeric antibodies (U.S. Patent No. 4,816,567) in which substantially less than an intact human variable domain is 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.

[0404] According to another method, antigen binding can be restored during antibody humanization through the selection of restored hypervariable regions (see, e.g., U.S. Application No. 11 / 061,841, filed February 18, 2005). The method involves incorporating non-human hypervariable regions onto an acceptor framework and further introducing one or more amino acid substitutions in one or more hypervariable regions without modifying the acceptor framework sequence. Alternatively, the introduction of one or more amino acid substitutions can be accompanied by modifications in the acceptor framework sequence.

[0405] Any cysteine ​​residue not involved in maintaining the proper conformation of the immunoconjugates of the invention may generally be substituted with serine to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bond(s) may be added to immunoconjugates of the invention to improve their stability, particularly where the antibody is an antibody fragment such as an Fv or VHH fragment.

[0406] 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, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies can be generated, for example, as described in US Pat. No. 7,521,541.

[0407] 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.

[0408] In some embodiments, the immunoconjugates provided herein are modified to increase or decrease the extent to which the immunoconjugate is glycosylated and / or to change the glycosylation pattern. For purposes herein, "modification of 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 that are not present in the native sequence immunoconjugate of the invention. In addition, 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.

[0409] 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. When X is any amino acid other than proline, the tripeptide sequences asparagine-X-serine and asparagine-X-threonine 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.

[0410] Addition or deletion of glycosylation sites to an immunoconjugate can be conveniently achieved by modifying the amino acid sequence so that one or more glycosylation sites are created or removed. Addition of glycosylation sites to an immunoconjugate of the present invention is conveniently achieved by modifying the amino acid sequence to include one or more of the above tripeptide sequences (for N-linked glycosylation sites). Modifications can also be made (in the case of O-linked glycosylation sites) by adding or substituting one or more serine or threonine residues to the sequence of the original immunoconjugate of the present invention. Immunoconjugates of the present invention can optionally be modified through changes at the DNA level, particularly by mutating the DNA encoding the immunoconjugate of the present invention at preselected bases to generate codons that translate into the desired amino acids.

[0411] If the immunoconjugate comprises an Fc region, the carbohydrate attached thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched, 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 invention can be performed to generate immunoconjugate variants with specific improved properties.

[0412] 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 Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0413] 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, in Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of various endoglycosidases and exoglycosidases, as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).

[0414] 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 WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (Eu numbering of Fc region residues) in the Fc region. However, due to minor sequence variability in antibodies, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function (see, eg, US2003 / 0157108; US2004 / 0093621). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; 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); US 2003 / 0157108; WO 2004 / 056312, Adams et al., especially Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); WO2003 / 085107).

[0415] Immunoconjugate variants are also provided that have bisected oligosaccharides, such as biantennary oligosaccharides attached to the Fc region of the antibody, 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; and US2005 / 0123546. Immunoconjugate variants are also provided that have 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.

[0416] 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 the N- or C-terminal residues of the immunoconjugates. 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 such as 3,3'-dithiobis(succinimidyl propionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane, and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.

[0417] Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, 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.

[0418] In some embodiments, immunoconjugates as provided herein can be further modified to include additional nonprotein moieties known and readily available in the art. Moieties suitable for derivatization of immunoconjugates include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, 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, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerin), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages because it is stable in water. The polymers can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the immunoconjugate can vary, and when more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the immunoconjugate to be improved, whether the immunoconjugate derivative will be used in therapy under defined conditions, etc.

[0419] PEG-derivatized immunoconjugates of the present invention can contain more than one -CH2CHO- containing linker, which can be used to alter the biodistribution and pharmacokinetics of the immunoconjugate. PEG can be prepared in polymeric form or as individual oligomers. Bifunctionalized versions of these polymers can link the immunoconjugate with 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.

[0420] Methods for producing immunoconjugates of the present invention The present invention provides compositions comprising any of the above embodiments or one or more of the 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.

[0421] 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.

[0422] 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 producing immunoconjugates of the invention, the methods 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 methods further comprise radiolabeling the radioisotope delivery platform with a suitable isotope, e.g., an α- or β-particle emitter.

[0423] Generation and Identification of Antigen-Binding Domains, Immunoconjugates, and Nucleic Acids The antigen-binding domain useful as 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, which can then be 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 (for example, Skerra et al., Curr. Opinion in Immunol., 5:256-262(1993) and Pluckthun, Immunol Revs.130:151-188(1992)).

[0424] In some embodiments, antigen-binding domains of immunoconjugates of the present invention, or fragments thereof, are isolated by screening phage libraries containing phage displaying various fragments of antibody variable regions (Fv, scFv, or VHH) fused to phage coat proteins. Such phage libraries are 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 are then eluted from the antigen and can be further enriched by additional cycles of antigen adsorption / elution.

[0425] 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 mouse and human antibodies, respectively, 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., Nuc 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).

[0426] Repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR), randomly recombined in phage libraries, and then 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 to 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 and achieve rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

[0427] 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, 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 a display library. The selection of antibodies with slow dissociation kinetics (and strong binding affinity) can be facilitated by long washes and monovalent phage display, as described in Bass et al., Proteins, 8:309-314 (1990) and WO1992 / 09690, and by using low coating density antigens, as described in Marks et al., Biotechnol., 10:779-783 (1992).

[0428] Techniques for screening cDNA libraries are well known in the art. Libraries can be screened using probes (such as oligonucleotides of at least about 20-80 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 present invention is to use PCR (Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)).

[0429] DNA encoding the immunoconjugates of the present invention can be obtained from a cDNA library prepared from tissues believed to have and express detectable levels of the mRNA immunoconjugates of the present invention. Thus, human immunoconjugates of the present invention can be easily obtained from a cDNA library prepared from human tissue. Immunoconjugates of the genes encoding the present invention can also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis). For some embodiments, the desired polynucleotide sequence encoding an antibody can be isolated and sequenced from antibody-producing cells, such as hybridoma cells.

[0430] The 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 full-length sequence can be determined using methods known in the art and described herein. Any of the antibody CDRs or heavy chain variable fragments of the present invention can be obtained by designing an appropriate antigen screening procedure to select a phage clone of interest, and then 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).

[0431] Production of immunoconjugates; host cells and expression vectors of the invention The following description primarily relates to the production of antibody constructs of the present invention by culturing cells transformed or transfected with vectors containing immunoconjugates of nucleic acids encoding the invention. It is, of course, contemplated that alternative methods well known in the art may be used to prepare antibody constructs of the present invention. For example, the appropriate amino acid sequence or portions thereof may be generated by direct peptide synthesis using solid-phase techniques (e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J., Am. Chem. Soc., 85:2149-54 (1963)). In vitro protein synthesis 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 immunoconjugates of the present invention.

[0432] 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 (e.g., transformed with this vector). In some other embodiments, the host cell comprises (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryote, 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 provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0433] 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 versions of the antibody construct.

[0434] 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. In general, 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.

[0435] Suitable host cells for cloning or expressing nucleic acids and vectors encoding immunoconjugates include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237; US 5,789,199; US 5,840,523, and Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli. After expression, the immunoconjugate can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0436] 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 (e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004); Li et al., Nat. Biotech. 24:210-215 (2006)).

[0437] The host cell suitable for the expression of glycosylated immunoconjugate can also be derived from multicellular organisms (for example, invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells.Many baculovirus strains have been identified that are suitable for use with insect cells, particularly for transfection of Spodoptera frugiperda cells.Plant cell cultures can also be used as hosts (see, for example, US5,959,177; US6,040,498; US6,420,548; US7,125,978; and US6,417,429).

[0438] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney line (293 or 293 cells as described in Graham et al., J Gen Viral. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as 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 hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep 02), mouse mammary tumor (MMT 060562), e.g., Mather et al., Annals TRI cells, MRC5 cells, and FS4 cells, as described in NYAcad. Sci. 383:44-68 (1982). 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).

[0439] 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 in 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. Bact., 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).

[0440] prokaryotic host cells Suitable prokaryotes include, but are not limited to, archaebacteria and eubacteria, e.g., gram-negative or gram-positive organisms, e.g., Enterobacteriaceae, such as E. coli. Various E. coli strains are publicly available, such as K12 strains MM294 (ATCC 31,446); X1776 (ATCC 31,537); W3110 (ATCC 27,325), and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae, e.g., Escherichia coli, e.g., E. coli, Enterobacteriaceae, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillus species, e.g., B. subtilis and B. licheniformis (e.g., B. licheniformis 41P, disclosed in DD 266,710, published April 12, 1989), Pseudomonas species, e.g., Pseudomonas aeruginosa, Rhizobium, Vitreoscilla, Paracoccus, and Streptomyces. These examples are illustrative and not limiting. E. coli strain W3110 is a common host strain for recombinant DNA product fermentation 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 Accession No. 27,325) can be modified to introduce genetic mutations into genes encoding proteins endogenous to the host, examples of which include E. coli W3110 strain 1A2 with the complete tonA genotype, E. coli W3110 strain 9E4 with the complete tonA ptr3 genotype, E. coli W3110 strain 27C7 (ATCC 55,244) with the complete tonA ptr3 phoA E15(argF-lac)169degP ompT kanr genotype, and E. coli W3110 strain 27C7 with the complete tonA ptr3 phoA E15(argF-lac)169degP ompT rbs7 ilvG genotype. 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 W3110ΔfhuA(ΔtonA)ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE)degP41 kanR (U.S. Pat. No. 5,639,635), and E. coli strains with mutant periplasmic proteases disclosed in U.S. Pat. No. 4,946,783, issued August 7, 1990. Other strains and their derivatives, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli λ1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608), 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.

[0441] Full-length antibodies, antibody fragments, and antibody fusion proteins can be produced in bacteria, especially when 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, for example, similar to the process for purifying antibodies expressed in CHO cells.

[0442] eukaryotic host cell In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for immunoconjugation of vectors encoding 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 Kluyveromyces lactis (K. lactis) (MW98-8C, CBS683, CBS4574; Louvencourt et al. al., J. Bacteriol., 154(2):737-742(1983)), Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces wickeramii (ATCC 24,178), Kluyveromyces waltii (ATCC 56,500), Kluyveromyces drosophilarum (ATCC 36,906; Van den Berg et al. al., Bio / Technology, 8:135 (1990)), Kluyveromyces thermotolerans (K. thermotolerans and Kluyveromyces marxianus), Yarrowia (EP402,226), Pichia pastoris (EP183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278 (1988)), Candida, Trichoderma reesia (EP244,234), Neurospora crassa (Case et al., Proc Natl Acad Sci USA 76:5259-5263 (1979)), Schwanniomyces, e.g., Schwanniomyces occidentalis (EP 394,538 published October 31, 1990), as well as filamentous fungi, e.g., Moldova, Penicillium, and 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); Tilburn et al., Gene, 26:205-221 (1983); Yelton et al. al., Proc Natl Acad Sci USA 81:1470-1474 (1984)) and Aspergillus niger (A. niger) (Kelly and Hynes, EMBO J., 4:475-479 (1985)). Methylotrophic yeasts are suitable herein and include, but are not limited to, yeasts capable of growing on methanol selected from the genera Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeast can be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

[0443] Suitable host cells for expression of the 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, as well as plant cells such as cotton, corn, potato, soybean, petunia, tomato, and tobacco cell cultures. Many baculovirus strains and variants have been identified, as well as corresponding permissive 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, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses may be used as viruses in accordance with the present invention, particularly for transfection of Spodoptera frugiperda cells.

[0444] 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 CCL 10), 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 CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL1442), human lung cells (W138, ATCC CCL 75), 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 hepatocellular carcinoma line (Hep G2).

[0445] Host cells are transformed with the above-described expression or cloning vectors for the production of the immunoconjugates of the present invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0446] 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.

[0447] Vectors can be, for example, in the form of plasmids, cosmids, virus particles, or phage.Appropriate nucleic acid sequences can be inserted into vectors by various procedures.Generally, DNA is inserted into an appropriate restriction endonuclease site using techniques known in the art.Vector components generally include, but are not limited to, one or more of the following: signal sequence, origin of replication, one or more marker genes, enhancer element, promoter, and transcription termination sequence.Construction of suitable vectors containing one or more of these components uses standard ligation techniques known to those skilled in the art.

[0448] Immunoconjugates of the present invention can be produced recombinantly not only directly but also as fusion polypeptides with heterologous polypeptides, which may be signal sequences or other polypeptides with specific cleavage sites 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 of the present invention-encoding DNA inserted into the vector. The signal sequence may be, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion, the signal sequence may be, for example, the yeast invertase leader, the alpha-factor leader (including the Saccharomyces and Kluyveromyces α-factor leaders, the latter of which is described in U.S. Pat. No. 5,010,182), 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 secretion of proteins, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.

[0449] Cultivation of host cells to generate radioisotope delivery platform The host cells used to produce the immunoconjugates of the present invention may be cultured in a variety of media and culture conditions.

[0450] 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. An example of a suitable medium is Luria Broth (LB) in addition to necessary nutritional supplements. In some embodiments, the medium further 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 for the growth of cells expressing an ampicillin resistance gene.

[0451] Any necessary nutritional supplements other than sources of carbon, nitrogen, and inorganic phosphate may also be included at appropriate concentrations introduced alone or in mixture with another nutritional 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.

[0452] Prokaryotic host cells are cultured at an appropriate temperature. For example, for growth of E. coli, 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 in the range of about 5 to about 9, depending primarily on the host organism. For E. coli, the pH is preferably about 6.8 to about 7.4, more preferably about 7.0.

[0453] When an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activating the promoter.In one aspect of the present invention, the PhoA promoter is used to control the transcription of the polypeptide.Therefore, the transformed host cell is cultured in a 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.

[0454] 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.

[0455] In one embodiment of the present invention, immunoconjugates are produced in large quantities by a fermentation process. A variety of large-scale fed-batch fermentation procedures are available for the production of recombinant proteins. Large-scale fermentations have a volume of at least 1000 liters, preferably between about 1,000 and 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, which can range from about 1 liter to about 100 liters.

[0456] 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 approximately 180-220, 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 typically induced for approximately 12-50 hours, although longer or shorter induction times may be used.

[0457] 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 that overexpresses chaperone proteins such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (peptidyl prolyl cis, trans-isomerase 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;USPatent No.6,083,715;USPatent No.6,027,888;Bothmann and Pluckthun(2000) J.Biol.Chem.275:17100-5;Ramm and Pluckthun(2000) J.Biol.Chem.275:17106-13;Arie et al.(2001) Mol.Microbiol.39:199-210.

[0458] 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).

[0459] 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.

[0460] 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. In addition, media such as those described in 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 No. Any of the media described in Re. 30,985 can be used as a culture medium for the host cells. Any of these media can be supplemented, as needed, with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., 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 nutritional supplements can also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, and the like, will be those previously used with the host cell selected for expression and will be apparent to those skilled in the art.

[0461] Purification of immunoglobulin-derived structures of the invention The immunoconjugate of the present invention can be recovered from culture medium or host cell lysate.If it is membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton-X100) or by enzymatic cleavage.The cells used to express the immunoconjugate of the present invention can be disrupted by various physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysis agents.

[0462] 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 using ion exchange column fractionation, ethanol precipitation, reverse-phase HPLC, chromatography on silica or on a cation exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75, a protein A Sepharose column to remove contaminants such as IgG, and a metal chelate column that binds to an epitope-tagged form of the immunoconjugate 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 steps selected will depend, for example, on the nature of the production process used and the particular immunoconjugate of the present invention being produced.

[0463] When using recombinant techniques, 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. Cell debris can be removed by centrifugation. If the immunoconjugate is secreted into the culture medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter (e.g., an Amicon or Millipore Pellicon ultrafiltration unit). A protease inhibitor such as PMSF may also be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0464] Immunoconjugate compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the immunoconjugate. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). While agarose is the most commonly used matrix for affinity ligand binding, other matrices are 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, silica chromatography, chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns) with heparin SEPHAROSE™ chromatography, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available, depending on the immunoconjugate to be recovered.

[0465] After any preliminary purification steps, the mixture containing the immunoconjugate of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5-4.5, generally with a low salt concentration (e.g., about 0-0.25 M salt).

[0466] Immunoconjugates (including antibody drug conjugates (ADCs)) In a further aspect of the invention, an immunoconjugate of the invention as described in any of the above embodiments or herein is conjugated to a heterologous moiety or agent, including, for example, as described below, and any additional exogenous material as described herein.

[0467] 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.

[0468] 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 generation of the radioconjugates of the present invention.

[0469] Immunoconjugates or conjugates of antibody constructs 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 (e.g., dimethyl adipimidate H), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 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).

[0470] The immunoconjugates or ADCs herein expressly contemplate 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 sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., available from Pierce Biotechnology, Inc., Rockford, Illinois, USA).

[0471] 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 reference herein to only immunoconjugates or antibody constructs), and such preparative methods are also encompassed by the present invention.

[0472] 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.

[0473] Bifunctional chelators, such as DOTA, DTPA, and related analogs, are suitable for coordinating metal ions, such as α- and β-emitting radionuclides. For example, these chelating molecules can be linked to targeting molecules by forming a new amide bond between an amine (e.g., a functional group of a lysine residue) on the antibody construct and a carboxylate on DOTA / DTPA. In the case of peptide synthesis, characterization and purification of the linker addition can be part of the overall synthesis of the antibody platform or immunoconjugate for radioisotope conjugation.

[0474] In some embodiments, the method of producing the immunoconjugate comprises a click chemistry process as described by Poty, S et al., Chem Commun (Camb) 54:2599 (2018).

[0475] In some embodiments, peptides can be biosynthesized or synthesized by chemical amino acid synthesis using appropriate amino acid precursors, for example, containing 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). Other methods are described in detail in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

[0476] Characterization of the Immunoconjugates of the Invention Immunoconjugates of the present invention can be identified, screened, or characterized for their physicochemical / 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 physicochemical / 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.

[0477] antigen binding Immunoconjugates of the 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 immunoconjugates of the 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.

[0478] In one embodiment, the K D is measured using a radiolabeled antigen ELISA performed with the immunoconjugate. D is measured using a surface plasmon resonance assay with a BIACORE®-2000 or BIACORE®-3000 instrument (BIACORE, Inc., Piscataway, NJ), for example, using an immobilized antigen CM5 chip at 25°C and 10 response units.

[0479] 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)).

[0480] 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).

[0481] 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.

[0482] 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.

[0483] The immunoconjugates or radioimmunoconjugates may be formulated in any form suitable for delivery to target cells / tissues. Pharmaceutical formulations of the immunoconjugates of the present invention are prepared by mixing such immunoconjugates having the desired purity with one or more pharmaceutically acceptable carriers, diluents, and / or excipients (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or aqueous solution. Pharmaceutically acceptable carriers, diluents, and excipients are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, sterile water, buffers, such as phosphate, citric acid, 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) polypeptides, proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, e.g., polyethylene glycol (PEG).

[0484] Pharmaceutical formulations to be used for in vivo administration are generally sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0485] 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.

[0486] Pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0487] The formulations herein may further 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.

[0488] The active ingredient can also be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, prepared by coacervation techniques or interfacial polymerization, respectively, in colloidal drug delivery systems (e.g., 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).

[0489] In some embodiments, the immunoconjugate may be formulated as an immunoliposome. A "liposome" is a vesicle composed of various types of lipids, phospholipids, and / or surfactants that is useful for delivering drugs to mammals. The components of a liposome are usually arranged in a bilayer formation, similar to the lipid arrangement of biolog...

Claims

1. Formula (I) 【Chemical 1】 or a pharmaceutically acceptable salt thereof, During the ceremony, R １ is a chelating moiety or a radionuclide complex thereof; R ２ is the tumor-targeting moiety R ３ Amine (-NH ２ ) or thiol (—SH), X １ Ha-NR ａ -, -NR ａ S (= O) ２ -, -NR ａ S (= O) ２ NR ａ -, or one or more independently selected natural or unnatural amino acids, any free amine of the amino acid optionally being substituted with R ５ or -C(=O)R ５ and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R ５ is replaced by R ａ are each independently hydrogen and C １ -C ４ alkyl, L is -L １ -L ２ -L ３ -L ４ -L ５ -, L １ is unsubstituted or substituted C １ -C １０ Alkylene, unsubstituted or substituted C １ -C １０ Heteroalkylene, unsubstituted or substituted C ２ -C ２０ Alkenylene, unsubstituted or substituted C ２ -C ２０ Alkynylene, C ４ -C ２０ Polyethylene glycol, -(X ３ CH ２ CH ２ ) ｔ -, unsubstituted or substituted cycloalkylene, unsubstituted or substituted heterocycloalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; X ３ are each independently O and NR ４ is selected from each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; L ２ is absent or is unsubstituted or substituted C １ -C １０ Alkylene, unsubstituted or substituted C １ -C １０ heteroalkylene, —C(═O)—(unsubstituted or substituted C １ -C １０ alkylene)-, -C(=O)NR ４ -(unsubstituted or substituted C １ -C １０ alkylene)-, -NR ４ C(=O)-(unsubstituted or substituted C １ -C １０ alkylene)-, -C(=O)-(CH ２ CH ２ X ３ ) ｍ - (CH ２ ) Ｐ -, -C(=O)NR ４ - (CH ２ CH ２ X ３ ) ｍ - (CH ２ ) ｐ -, -NR ４ C(=O)-(CH ２ CH ２ X ３ ) ｍ - (CH ２ ) ｐ - or -(CH ２ CH ２ X ３ ) ｍ - (CH ２ ) ｐ - and R ４ are each independently hydrogen and C １ -C ６ alkyl, each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; Each p is independently 0, 1, or 2; L ３ is absent or is one or more independently selected groups selected from natural or unnatural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates, and does not cleave any free amine (—NH ２ ) is also optional, R ５ or -C(=O)R ５ and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R ５ The substituted benzyl is substituted with halogen, —OH, —OR ５ , -CO ２ H, -NHR ５ , -C(=O)NHR ５ , -NHC(=O)R ５ , and substituted C １ -C ６ alkyl, and the substituted C １ -C ６ Alkyl is —OH, —CO ２ H, -NHR ５ , -C(=O)NHR ５ and —NHC(═O)R ５ is replaced by L ４ is absent or is unsubstituted or substituted C １ -C １０ alkylene, —C(═O)—(unsubstituted or substituted C １ -C １０ alkylene)-, -C(=O)NR ４ -(unsubstituted or substituted C １ -C １０ alkylene)-, -NR ４ C(=O)-(unsubstituted or substituted C １ -C １０ alkylene)-, -C(=O)-(CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -C(=O)NR ４ - (CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -NR ４ C(=O)-(CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -(CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ - or - (X ３ CH ２ CH ２ ) ｎ - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; Each q is independently 0, 1, or 2; L ５ does not exist or -C(=O)-(CH ２ ) ｎ -, -C(=O)NR ４ - (CH ２ ) ｎ -, -NR ４ C(=O)-(CH ２ ) ｎ -, -C(=O)-(CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -C(=O)NR ４ - (CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -NR ４ C(=O)-(CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -(CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -C(=O)-(X ３ CH ２ CH ２ ) ｎ - or - (X ３ CH ２ CH ２ ) ｎ - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; Each q is independently 0, 1, or 2; R ａ are each independently hydrogen and C １ -C ４ alkyl, R ｂ are each independently hydrogen and C １ -C ４ alkyl, Heteroalkylene is a group in which one carbon atom is —O—, —S—, —S(═O)—, or —S(═O) ２ -, -S(=O)(=NH)-, -S(=O)(=NR ５ ) -, -NR ５ -, -P(=O)OR ｂ -, -NR ａ C(=O)-, -C(=O)NR ａ -, -OC(=O)NR ａ -, -NR ａ C(=O)NR ａ -, -NR ａ C (=N-CN)NR ａ -, -NR ａ C(=N-R ５ ) NR ａ - or -NR ａ alkylene substituted with C(═O)O—; -L １ -, -L ２ -, -L ３ -, -L ４ -, and -L ５ -L １ -, -L ２ -, -L ３ -, -L ４ -, and -L ５ - is a halogen, -OH, -OR ５ , -CO ２ H, -NHR ５ , -C(=O)NHR ５ , -NHC(=O)R ５ , and substituted C １ -C ６ alkyl, wherein the substituted C １ -C ６ Alkyl is —OH, —CO ２ H, -NHR ５ , -C(=O)NHR ５ and —NHC(═O)R ５ is replaced by R ５ are each independently １ -C １０ Alkyl, C ４ -C ３０ polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; However, L ３ If no R is present, then at least one R ５ exists, or X １ HA-N(C １ -C ４ alkyl), -NR ａ C(=O)-, -NR ａ S (= O) ２ - or one or more independently selected natural or unnatural amino acids, or a pharmaceutically acceptable salt thereof.

2. R ２ is the tumor-targeting moiety R ３ Amine (-NH ２ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the moiety is a tetrafluorophenyl ester, a pentafluorophenyl ester, a dinitrophenyl ester, a succinimide ester, a sulfosuccinimide ester, or an isothiocyanate.

3. R ２ is the tumor-targeting moiety R ３ Amine (-NH ２ ) is a moiety that can react with 【Chemistry 2】 Including, X is absent, -O-, -S-, -S(=O)-, -S(=O) ２ -, -NR ａ -, -C(=O)-, -NR ａ C(=O)-, -C(=O)NR ａ -, -C(=O)O-, -OC(=O)-, -OC(=O)NR ａ -, -NR ａ C(=O)NR ａ -, -NR ａ C(=S)NR ａ -, -NR ａ C(═O)O—, R ａ are each independently hydrogen and C １ -C ４ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein:

4. Formula (II), Formula (III), or Formula (IV) 【Chemistry 3】 or a pharmaceutically acceptable salt thereof, During the ceremony, R １ is a chelating moiety or a radionuclide complex thereof; -NH-R ３ is a tumor-targeting moiety, X is absent, -O-, -S-, -S(=O)-, -S(=O) ２ -, -NR ａ -, -C(=O)-, -NR ａ C(=O)-, -C(=O)NR ａ -, -C(=O)O-, -OC(=O)-, -OC(=O)NR ａ -, -NR ａ C(=O)NR ａ -, -NR ａ C(=S)NR ａ -, -NR ａ C(═O)O—, R ａ are each independently hydrogen and C １ -C ４ alkyl, X １ Ha-NR ａ -, -NR ａ S (= O) ２ -, -NR ａ S (= O) ２ NR ａ -, or one or more independently selected natural or unnatural amino acids, any free amine of the amino acid optionally being substituted with R ５ or -C(=O)R ５ and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R ５ is replaced by R ａ are each independently hydrogen and C １ -C ４ alkyl, L is -L １ -L ２ -L ３ -L ４ -L ５ -, L １ is unsubstituted or substituted C １ -C １０ Alkylene, unsubstituted or substituted C １ -C １０ Heteroalkylene, unsubstituted or substituted C ２ -C ２０ Alkenylene, unsubstituted or substituted C ２ -C ２０ Alkynylene, C ４ -C ２０ Polyethylene glycol, -(X ３ CH ２ CH ２ ) ｔ -, unsubstituted or substituted cycloalkylene, unsubstituted or substituted heterocycloalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; X ３ are each independently O and NR ４ is selected from each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; L ２ is absent or is unsubstituted or substituted C １ -C １０ Alkylene, unsubstituted or substituted C １ -C １０ heteroalkylene, —C(═O)—(unsubstituted or substituted C １ -C １０ alkylene)-, -C(=O)NR ４ -(unsubstituted or substituted C １ -C １０ alkylene)-, -NR ４ C(=O)-(unsubstituted or substituted C １ -C １０ alkylene)-, -C(=O)-(CH ２ CH ２ X ３ ) ｍ - (CH ２ ) Ｐ -, -C(=O)NR ４ - (CH ２ CH ２ X ３ ) ｍ - (CH ２ ) ｐ -, -NR ４ C(=O)-(CH ２ CH ２ X ３ ) ｍ - (CH ２ ) ｐ - or -(CH ２ CH ２ X ３ ) ｍ - (CH ２ ) ｐ - and R ４ are each independently hydrogen and C １ -C ６ alkyl, each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; Each p is independently 0, 1, or 2; L ３ is absent or is one or more independently selected groups selected from natural or unnatural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates, and does not cleave any free amine (—NH ２ ) is also optional, R ５ or -C(=O)R ５ and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R ５ The substituted benzyl is substituted with halogen, —OH, —OR ５ , -CO ２ H, -NHR ５ , -C(=O)NHR ５ , -NHC(=O)R ５ , and substituted C １ -C ６ alkyl, and the substituted C １ -C ６ Alkyl is —OH, —CO ２ H, -NHR ５ , -C(=O)NHR ５ and —NHC(═O)R ５ is replaced by L ４ is absent or is unsubstituted or substituted C １ -C １０ alkylene, —C(═O)—(unsubstituted or substituted C １ -C １０ alkylene)-, -C(=O)NR ４ -(unsubstituted or substituted C １ -C １０ alkylene)-, -NR ４ C(=O)-(unsubstituted or substituted C １ -C １０ alkylene)-, -C(=O)-(CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -C(=O)NR ４ - (CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -NR ４ C(=O)-(CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -(CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ - or - (X ３ CH ２ CH ２ ) ｎ - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; Each q is independently 0, 1, or 2; L ５ does not exist or -C(=O)-(CH ２ ) ｎ -, -C(=O)NR ４ - (CH ２ ) ｎ -, -NR ４ C(=O)-(CH ２ ) ｎ -, -C(=O)-(CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -C(=O)NR ４ - (CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -NR ４ C(=O)-(CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -(CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -C(=O)-(X ３ CH ２ CH ２ ) ｎ - or - (X ３ CH ２ CH ２ ) ｎ - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; Each q is independently 0, 1, or 2; R ａ are each independently hydrogen and C １ -C ４ alkyl, R ｂ are each independently hydrogen and C １ -C ４ alkyl, Heteroalkylene is a group in which one carbon atom is —O—, —S—, —S(═O)—, or —S(═O) ２ -, -S(=O)(=NH)-, -S(=O)(=NR ５ ) -, -NR ５ -, -P(=O)OR ｂ -, -NR ａ C(=O)-, -C(=O)NR ａ -, -OC(=O)NR ａ -, -NR ａ C(=O)NR ａ -, -NR ａ C (=N-CN)NR ａ -, -NR ａ C(=N-R ５ ) NR ａ - or -NR ａ alkylene substituted with C(═O)O—; -L １ -, -L ２ -, -L ３ -, -L ４ -, and -L ５ -L １ -, -L ２ -, -L ３ -, -L ４ -, and -L ５ - is a halogen, -OH, -OR ５ , -CO ２ H, -NHR ５ , -C(=O)NHR ５ , -NHC(=O)R ５ , and substituted C １ -C ６ alkyl, wherein the substituted C １ -C ６ Alkyl is —OH, —CO ２ H, -NHR ５ , -C(=O)NHR ５ and —NHC(═O)R ５ is replaced by R ５ are each independently １ -C １０ Alkyl, C ４ -C ３０ polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; However, L ３ If no R is present, then at least one R ５ exists, or X １ HA-N(C １ -C ４ alkyl), -NR ａ C(=O)-, NR ａ S (= O) ２ - or one or more independently selected natural or unnatural amino acids, or a pharmaceutically acceptable salt thereof.

5. R ２ is the tumor-targeting moiety R ３ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the moiety is a maleimide group, a haloacetamide group, a haloacetyl group, a haloacetate group, a pyridinylthio group, a vinylcarbonyl group, an aziridinyl group, a disulfide group, an acetylene group, a hydroxysuccinimide group, or a thiol group.

6. R ２ is the tumor-targeting moiety R ３ is a moiety capable of reacting with a thiol (—SH) of 【Chemistry 4】 Including, 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein m is 0, 1, 2, 3, 4, or 5.

7. Formula (V), Formula (VI), or Formula (VII) 【Chemistry 5】 or a pharmaceutically acceptable salt thereof, During the ceremony, R １ is a chelating moiety or a radionuclide complex thereof; -S-R ３ is a tumor-targeting moiety, X １ Ha-NR ａ -, -NR ａ S (= O) ２ -, -NR ａ S (= O) ２ NR ａ -, or one or more independently selected natural or unnatural amino acids, any free amine of the amino acid optionally being substituted with R ５ or -C(=O)R ５ and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R ５ is replaced by R ａ are each independently hydrogen and C １ -C ４ alkyl, L is -L １ -L ２ -L ３ -L ４ -L ５ -, L １ is unsubstituted or substituted C １ -C １０ Alkylene, unsubstituted or substituted C １ -C １０ Heteroalkylene, unsubstituted or substituted C ２ -C ２０ Alkenylene, unsubstituted or substituted C ２ -C ２０ Alkynylene, C ４ -C ２０ Polyethylene glycol, -(X ３ CH ２ CH ２ ) ｔ -, unsubstituted or substituted cycloalkylene, unsubstituted or substituted heterocycloalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; X ３ are each independently O and NR ４ is selected from each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; L ２ is absent or is unsubstituted or substituted C １ -C １０ Alkylene, unsubstituted or substituted C １ -C １０ heteroalkylene, —C(═O)—(unsubstituted or substituted C １ -C １０ alkylene)-, -C(=O)NR ４ -(unsubstituted or substituted C １ -C １０ alkylene)-, -NR ４ C(=O)-(unsubstituted or substituted C １ -C １０ alkylene)-, -C(=O)-(CH ２ CH ２ X ３ ) ｍ - (CH ２ ) Ｐ -, -C(=O)NR ４ - (CH ２ CH ２ X ３ ) ｍ - (CH ２ ) ｐ -, -NR ４ C(=O)-(CH ２ CH ２ X ３ ) ｍ - (CH ２ ) ｐ - or -(CH ２ CH ２ X ３ ) ｍ - (CH ２ ) ｐ - and R ４ are each independently hydrogen and C １ -C ６ alkyl, each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; Each p is independently 0, 1, or 2; L ３ is absent or is one or more independently selected groups selected from natural or unnatural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates, and does not cleave any free amine (—NH ２ ) is also optional, R ５ or -C(=O)R ５ and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R ５ The substituted benzyl is substituted with halogen, —OH, —OR ５ , -CO ２ H, -NHR ５ , -C(=O)NHR ５ , -NHC(=O)R ５ , and substituted C １ -C ６ alkyl, and the substituted C １ -C ６ Alkyl is —OH, —CO ２ H, -NHR ５ , -C(=O)NHR ５ and —NHC(═O)R ５ is replaced by L ４ is absent or is unsubstituted or substituted C １ -C １０ alkylene, —C(═O)—(unsubstituted or substituted C １ -C １０ alkylene)-, -C(=O)NR ４ -(unsubstituted or substituted C １ -C １０ alkylene)-, -NR ４ C(=O)-(unsubstituted or substituted C １ -C １０ alkylene)-, -C(=O)-(CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -C(=O)NR ４ - (CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -NR ４ C(=O)-(CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -(CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ - or - (X ３ CH ２ CH ２ ) ｎ - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; Each q is independently 0, 1, or 2; L ５ does not exist or -C(=O)-(CH ２ ) ｎ -, -C(=O)NR ４ - (CH ２ ) ｎ -, -NR ４ C(=O)-(CH ２ ) ｎ -, -C(=O)-(CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -C(=O)NR ４ - (CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -NR ４ C(=O)-(CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -(CH ２ CH ２ X ３ ) ｎ - (CH ２ ) ｑ -, -C(=O)-(X ３ CH ２ CH ２ ) ｎ - or - (X ３ CH ２ CH ２ ) ｎ - and each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; Each q is independently 0, 1, or 2; R ａ are each independently hydrogen and C １ -C ４ alkyl, R ｂ are each independently hydrogen and C １ -C ４ alkyl, Heteroalkylene is a group in which one carbon atom is —O—, —S—, —S(═O)—, or —S(═O) ２ -, -S(=O)(=NH)-, -S(=O)(=NR ５ ) -, -NR ５ -, -P(=O)OR ｂ -, -NR ａ C(=O)-, -C(=O)NR ａ -, -OC(=O)NR ａ -, -NR ａ C(=O)NR ａ -, -NR ａ C (=N-CN)NR ａ -, -NR ａ C(=N-R ５ ) NR ａ - or -NR ａ alkylene substituted with C(═O)O—; -L １ -, -L ２ -, -L ３ -, -L ４ -, and -L ５ -L １ -, -L ２ -, -L ３ -, -L ４ -, and -L ５ - is a halogen, -OH, -OR ５ , -CO ２ H, -NHR ５ , -C(=O)NHR ５ , -NHC(=O)R ５ , and substituted C １ -C ６ alkyl, wherein the substituted C １ -C ６ Alkyl is —OH, —CO ２ H, -NHR ５ , -C(=O)NHR ５ and —NHC(═O)R ５ is replaced by R ５ are each independently １ -C １０ Alkyl, C ４ -C ３０ polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; However, L ３ If no R is present, then at least one R ５ exists, or X １ HA-N(C １ -C ４ alkyl), -NR ａ C(=O)-, NR ａ S (= O) ２ - or one or more independently selected natural or unnatural amino acids, or a pharmaceutically acceptable salt thereof.

8. the tumor-targeting moiety R ３ is a polypeptide comprising an antigen-binding region and an immunoglobulin heavy chain constant region, and the molecular weight of the polypeptide is 60 to 110 kDa, or a pharmaceutically acceptable salt thereof.

9. 9. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein the antigen-binding region comprises an scFv polypeptide or a VHH polypeptide.

10. 10. The compound of claim 8 or 9, or a pharmaceutically acceptable salt thereof, wherein the immunoglobulin heavy chain constant region comprises an immunoglobulin CH2 domain, an immunoglobulin CH3 domain, or an immunoglobulin CH2 and CH3 domain.

11. 11. The compound of any one of claims 8 to 10, or a pharmaceutically acceptable salt thereof, wherein the immunoglobulin heavy chain constant region is of the IgA, IgG1, IgG2, IgG3, or IgG4 isotype.

12. 12. The compound of any one of claims 8 to 11, or a pharmaceutically acceptable salt thereof, wherein the antigen-binding region is humanized, the immunoglobulin heavy chain constant region is a human immunoglobulin heavy chain constant region, or both.

13. 13. The compound of any one of claims 8 to 12, or a pharmaceutically acceptable salt thereof, wherein the immunoglobulin heavy chain constant region comprises a modification 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 the neonatal Fc receptor (FcRn), or wherein the immunoglobulin heavy chain constant region comprises a modification 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 the neonatal Fc receptor (FcRn).

14. 14. The compound of any one of claims 8 to 13, or a pharmaceutically acceptable salt thereof, wherein the immunoglobulin heavy chain constant region comprises a modification to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region, or the immunoglobulin heavy chain constant region comprises a modification to one or more amino acid residues that alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn), or both.

15. 15. The compound according to claim 13 or 14, or a pharmaceutically acceptable salt thereof, wherein the modification to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region is a modification that reduces complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or a combination thereof.

16. R ２ is the tumor-targeting moiety R ３ The amine of the side chain of the lysine residue (-NH ２ 16. The compound of any one of claims 8 to 15, or a pharmaceutically acceptable salt thereof, wherein R is a moiety capable of reacting with R, ...

17. The compound of formula (II) has the formula (IIa) 【Chemistry 6】 or a pharmaceutically acceptable salt thereof, In the formula, -NHCH ２ CH ２ CH ２ CH ２ - is the tumor targeting moiety R ３ 16. The compound according to any one of claims 4 or 8 to 15, or a pharmaceutically acceptable salt thereof, wherein:

18. The compound of formula (III) has the formula (IIIa) 【Chemistry 7】 or a pharmaceutically acceptable salt thereof, In the formula, -NHCH ２ CH ２ CH ２ CH ２ - is the tumor targeting moiety R ３ 16. The compound according to any one of claims 4 or 8 to 15, or a pharmaceutically acceptable salt thereof, wherein:

19. The compound of formula (IV) may be represented by the formula (IVa) 【Chemistry 8】 or a pharmaceutically acceptable salt thereof, In the formula, -NHCH ２ CH ２ CH ２ CH ２ - is the tumor targeting moiety R ３ 16. The compound according to any one of claims 4 or 8 to 15, or a pharmaceutically acceptable salt thereof, wherein:

20. R ２ is the tumor-targeting moiety R ３ 17. The compound according to any one of claims 8 to 16, or a pharmaceutically acceptable salt thereof, wherein the moiety is capable of reacting with a thiol (-SH) in the side chain of a cysteine ​​residue of

21. The compound of formula (V) has the formula (Va) 【Chemistry 9】 or a pharmaceutically acceptable salt thereof, In the formula, -SCH ２ - is a tumor-targeting moiety R ３ The compound according to any one of claims 7 to 15, or a pharmaceutically acceptable salt thereof, wherein the thiol (-SH) is in the side chain of the cysteine ​​residue of

22. The compound of formula (VI) has the formula (VIa) 【Chemistry 10】 or a pharmaceutically acceptable salt thereof, In the formula, -SCH ２ - is a tumor-targeting moiety R ３ The compound according to any one of claims 7 to 15, or a pharmaceutically acceptable salt thereof, wherein the thiol (-SH) is in the side chain of the cysteine ​​residue of

23. The compound of formula (VII) can be represented by the formula (VIIa) 【Chemistry 11】 or a pharmaceutically acceptable salt thereof, In the formula, -SCH ２ - is a tumor-targeting moiety R ３ The compound according to any one of claims 7 to 15, or a pharmaceutically acceptable salt thereof, wherein the thiol (-SH) is in the side chain of the cysteine ​​residue of

24. The compound of formula (VIII) can be prepared by the reaction of formula (VIIIa) 【Chemistry 12】 or a pharmaceutically acceptable salt thereof, In the formula, -SCH ２ - is a tumor-targeting moiety R ３ The compound according to any one of claims 7 to 15, or a pharmaceutically acceptable salt thereof, wherein the thiol (-SH) is in the side chain of the cysteine ​​residue of

25. R １ 25. The compound of any one of claims 1 to 24, or a pharmaceutically acceptable salt thereof, wherein is a chelating moiety selected from the list consisting of DOTA, DO3A, DO3Apic, DOTAGA, DOTAGA anhydride, Py4Pa, Py4Pa-NCS, Crown, Macropa, Macropa-NCS, HEHA, CHXoctapa, Bispa, and Noneunpa, or a radionuclide complex thereof.

26. R １ 25. The compound of any one of claims 1 to 24, or a pharmaceutically acceptable salt thereof, wherein is a chelating moiety selected from the list consisting of DOTMA, DOTPA, DO3AM-acetate, DO3Apic, DOTP, DOTMP, DOTA-4AMP, CB-TE2A, NOTA, NOTP, TETPA, TETA, PEPA, H4Octapa, H2Dedpa, DO2P, EDTA, DTPA-BMA, 3,2,3-LI(HOPO), 3,2-HOPO, Neunpa, Neunpa-NCS, Octapa, PyPa, porphyrin, and deferoxamine, or a radionuclide complex thereof.

27. R １ teeth, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A), α,α',α'',α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTMA), 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid (DOTPA), 2,2',2''-(10-(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, 6,6'-(((pyridine-2,6-diylbis(methylene))bis((carboxymethyl)azanediyl))-bis(methylene))dipicolinic acid (H ４ pypa), 6,6',6'',6'''-(((pyridine-2,6-diylbis(methylene))bis(azanetriyl))tetrakis(methylene))-tetrapicolinic acid (H ４ py4pa), 10-((6-carboxypyridin-2-yl)methyl)-1,4,7,10-tetra-azacyclododecane-1,4,7-triacetic acid (DO3Apic), 3,6,9,12-tetrakis(carboxymethyl)-3,6,9,12-tetraazatetradecanedioic acid (TTHA), 25. The compound of any one of claims 1 to 24, or a pharmaceutically acceptable salt thereof, wherein the chelating moiety is selected from the group consisting of: or a radionuclide complex thereof.

28. R １ teeth, 【Chemistry 13】 25. The compound of any one of claims 1 to 24, or a pharmaceutically acceptable salt thereof, wherein the chelating moiety is selected from the group consisting of: or a radionuclide complex thereof.

29. R ２ teeth, 【Chemistry 14】 25. The compound of any one of claims 1 to 24, or a pharmaceutically acceptable salt thereof, which is a chelating moiety which is a radionuclide complex thereof.

30. X １ is -NR ａ -, -NR ａ S (= O) ２ -, or one or more independently selected natural or unnatural amino acids, any free amine of the amino acid optionally being substituted with R ５ or -C(=O)R ５ and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R ５ 30. The compound of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof, substituted with:

31. X １ is -NH-, -N(CH ３ ) -, -N(CH ２ CH ３ )-,-NHS(=O) ２ -, -N(CH ３ ) S(=O) ２ -, or -N(CH ２ CH ３ ) S(=O) ２ 30. The compound of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof, wherein:

32. X １ is lysine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, serine, tyrosine, valine, citrulline, methionine-valine-lysine, glycine-phenylalanine-glycine-glycine, tyrosine-arginine-valine, arginine-valine, or combinations thereof, and does not include any free amine (—NH ２ ) is also optional, R ５ or -C(=O)R ５ and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R ５ 30. The compound of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof, substituted with:

33. L １ is unsubstituted or substituted C １ -C ２０ Alkylene, unsubstituted or substituted C １ -C ２０ Heteroalkylene, C ４ -C ２０ Polyethylene glycol, unsubstituted or substituted C ３ -C ８ Cycloalkylene, unsubstituted or substituted monocyclic C ３ -C ８ The compound of any one of claims 1 to 32, which is heterocycloalkylene, unsubstituted or substituted phenylene, or unsubstituted or substituted monocyclic heteroarylene, or a pharmaceutically acceptable salt thereof.

34. L １ is unsubstituted or substituted C １ -C ６ Alkylene, unsubstituted or substituted C １ -C １０ Heteroalkylene, C ４ -C ２０ 33. The compound of any one of claims 1 to 32, which is polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene, or a pharmaceutically acceptable salt thereof.

35. X １ are one or more independently selected natural or unnatural amino acids, and any free amine of an amino acid is optionally substituted with R ５ or -C(=O)R ５ and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R ５ is replaced by L １ is -NR ４ CH ２ CH ２ -(OCH ２ CH ２ ) ｔ - and 30. The compound of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof, wherein t is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

36. L ２ does not exist or -C(=O)NR ４ -(unsubstituted or substituted C １ -C １０ alkylene)-, -NR ４ C(=O)-(unsubstituted or substituted C １ -C １０ alkylene)-, -C(=O)-(CH ２ CH ２ O) ｍ - (CH ２ ) Ｐ -, -C(=O)NR ４ - (CH ２ CH ２ O) ｎ - (CH ２ ) Ｐ -, -NR ４ C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) Ｐ - or -(CH ２ CH ２ O) ｎ - (CH ２ ) Ｐ - and m is independently 1, 2, 3, 4, 5, or 6; 35. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt thereof, wherein each p is independently 1 or 2.

37. L ３ is absent or is one or more independently selected groups selected from natural or unnatural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates, and does not cleave any free amine (—NH ２ ) is also optional, R ５ or -C(=O)R ５ and any free carboxylic acid of any amino acid is optionally substituted with —C(═O)NH—R ５ is replaced by L ３ is absent or is one or more independently selected groups selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamate, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, tyrosine, valine, and amino (unsubstituted or substituted benzyl) carbamate, and any free amine of an amino acid may optionally be R ５ or -C(=O)(R ５ ), and any free carboxylic acid of the amino acid is optionally substituted with —C(═O)NH(R ５ 37. The compound of any one of claims 1 to 36, or a pharmaceutically acceptable salt thereof, substituted with:

38. L ３ is lysine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, serine, tyrosine, valine, citrulline, methionine-valine-lysine, glycine-phenylalanine-glycine-glycine, tyrosine-arginine-valine, arginine-valine, valine-citrulline-(para-aminobenzylcarbamate), or combinations thereof; Any free amine (-NH ２ ) is also optionally —C(═O) (unsubstituted or substituted C １ -C ２０ alkylene) or —C(═O)—C ４ -C ２０ substituted with polyethylene glycol, Any free carboxylic acid (—CO ２ H) also optionally includes —C(═O)NH—(2,4,6-trimethyl-3-bromophenyl), —C(═O)NH—(unsubstituted or substituted C １ -C ２０ alkylene), or —C(═O)NH—(C ４ -C ２０ 37. The compound of any one of claims 1 to 36, or a pharmaceutically acceptable salt thereof, substituted with methyl methyl ether (polyethylene glycol).

39. L ４ is absent or -C(=O)- (unsubstituted or substituted C １ -C ６ alkylene)-, -C(=O)NR ４ -(unsubstituted or substituted C １ -C ６ alkylene)-, -NR ４ C(=O)-(unsubstituted or substituted C １ -C ６ alkylene)-, -C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) ｑ -, -C(=O)NR ４ - (CH ２ CH ２ O) ｎ - (CH ２ ) ｑ -, -NR ４ C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) ｑ - or -(CH ２ CH ２ O) ｎ - (CH ２ ) ｑ - and n is independently 1, 2, 3, 4, 5, or 6; 39. The compound of any one of claims 1 to 38, or a pharmaceutically acceptable salt thereof, wherein each q is independently 1 or 2.

40. L ５ is -C(=O)-(CH ２ ) ｎ -, -C(=O)NR ４ -(CH ２ ) ｎ -, -NR ４ C(=O)-(CH ２ ) ｎ -, -C(=O)-(CH ２ CH ２ O) ｎ -(CH ２ ) ｑ -, -C(=O)NR ４ -(CH ２ CH ２ O) ｎ -(CH ２ ) ｑ -, -NR ４ C(=O)-(CH ２ CH ２ O) ｎ -(CH ２ ) ｑ -, -(CH<()000946>CH ２ O) ｎ -(CH ２ ) ｑ -, -C(=O)-(OCH ２ [[ID=()4]]CH ２ ) ｎ -, or -(OCH ２ CH ２ ) ｎ - and is 39. The compound of any one of claims 1 to 38, or a pharmaceutically acceptable salt thereof, wherein each p is independently 0, 1, or 2.

41. L ５ is -NR ４ C(=O)-(CH ２ ) ｎ -, -C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) ｑ -, -C(=O)NR ４ - (CH ２ CH ２ O) ｎ - (CH ２ ) ｑ - or -NR ４ C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) ｑ - and 39. The compound of any one of claims 1 to 38, or a pharmaceutically acceptable salt thereof, wherein each q is independently 1 or 2.

42. L １ is unsubstituted or substituted C １ -C ６ Alkylene, unsubstituted or substituted C １ -C １０ Heteroalkylene, C ４ -C ２０ polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L ２ does not exist or -C(=O)NR ４ -(unsubstituted or substituted C １ -C １０ alkylene)-, -NR ４ C(=O)-(unsubstituted or substituted C １ -C １０ alkylene)-, -C(=O)-(CH ２ CH ２ O) ｍ - (CH ２ ) Ｐ -, -C(=O)NR ４ - (CH ２ CH ２ O) ｎ - (CH ２ ) Ｐ -, -NR ４ C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) Ｐ - or -(CH ２ CH ２ O) ｎ - (CH ２ ) Ｐ - and m is independently 1, 2, 3, 4, 5, or 6; Each p is independently 1 or 2; L ４ is absent or -C(=O)- (unsubstituted or substituted C １ -C ６ alkylene)-, -C(=O)NR ４ -(unsubstituted or substituted C １ -C ６ alkylene)-, -NR ４ C(=O)-(unsubstituted or substituted C １ -C ６ alkylene)-, -C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) ｑ -, -C(=O)NR ４ - (CH ２ CH ２ O) ｎ - (CH ２ ) ｑ -, -NR ４ C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) ｑ - or -(CH ２ CH ２ O) ｎ - (CH ２ ) ｑ - and L ５ is -NR ４ C(=O)-(CH ２ ) ｎ -, -C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) ｑ -, -C(=O)NR ４ - (CH ２ CH ２ O) ｎ - (CH ２ ) ｑ - or -NR ４ C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) ｑ - and n is independently 1, 2, 3, 4, 5, or 6; 30. The compound of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof, wherein each q is independently 1 or 2.

43. L １ is unsubstituted or substituted C １ -C ６ Alkylene, unsubstituted or substituted C １ -C １０ Heteroalkylene, C ４ -C ２０ polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L ２ does not exist or -C(=O)NR ４ -(unsubstituted or substituted C １ -C １０ alkylene)-, -NR ４ C(=O)-(unsubstituted or substituted C １ -C １０ alkylene)-, -C(=O)-(CH ２ CH ２ O) ｍ - (CH ２ ) Ｐ -, -C(=O)NR ４ - (CH ２ CH ２ O) ｎ - (CH ２ ) Ｐ -, -NR ４ C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) Ｐ - or -(CH ２ CH ２ O) ｎ - (CH ２ ) Ｐ - and m is independently 1, 2, 3, 4, 5, or 6; Each p is independently 1 or 2; L ４ does not exist, L ５ is -NR ４ C(=O)-(CH ２ ) ｎ -, -C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) ｑ -, -C(=O)NR ４ - (CH ２ CH ２ O) ｎ - (CH ２ ) ｑ - or -NR ４ C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) ｑ - and n is independently 1, 2, 3, 4, 5, or 6; 30. The compound of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof, wherein each q is independently 1 or 2.

44. X １ is alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, leucine, lysine, phenylalanine, serine, tyrosine, valine, or a combination thereof, and any free amine of an amino acid is optionally substituted with R ５ or -C(=O)(R ５ ), and any free carboxylic acid of the amino acid is optionally substituted with —C(═O)NH(R ５ ) and R ５ are each independently unsubstituted or substituted C １ -C １０ Alkylene, C ４ -C ２０ polyethylene glycol, or unsubstituted or substituted phenyl, where substituted phenyl is F, Cl, Br, I, —CH ３ , and C.F. ３ and is substituted with 1, 2, 3, 4, or 5 groups independently selected from L １ is -NR ４ CH ２ CH ２ -(OCH ２ CH ２ ) ｔ - and t is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; L ２ does not exist or -C(=O)NR ４ -(unsubstituted or substituted C １ -C １０ alkylene)-, -NR ４ C(=O)-(unsubstituted or substituted C １ -C １０ alkylene)-, -C(=O)-(CH ２ CH ２ O) ｍ - (CH ２ ) Ｐ -, -C(=O)NR ４ - (CH ２ CH ２ O) ｎ - (CH ２ ) Ｐ -, -NR ４ C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) Ｐ - or -(CH ２ CH ２ O) ｎ - (CH ２ ) Ｐ - and m is independently 1, 2, 3, 4, 5, or 6; Each p is independently 1 or 2; L ３ does not exist, L ４ does not exist, L ５ is -NR ４ C(=O)-(CH ２ ) ｎ -, -C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) ｑ -, -C(=O)NR ４ - (CH ２ CH ２ O) ｎ - (CH ２ ) ｑ - or -NR ４ C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) ｑ - and n is independently 1, 2, 3, 4, 5, or 6; 30. The compound of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof, wherein each q is independently 1 or 2.

45. L １ is unsubstituted or substituted C １ -C ６ Alkylene, unsubstituted or substituted C １ -C １０ Heteroalkylene, C ４ -C ２０ polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L ２ does not exist or -C(=O)NR ４ -(unsubstituted or substituted C １ -C １０ alkylene)-, -NR ４ C(=O)-(unsubstituted or substituted C １ -C １０ alkylene)-, -C(=O)-(CH ２ CH ２ O) ｍ - (CH ２ ) Ｐ -, -C(=O)NR ４ - (CH ２ CH ２ O) ｎ - (CH ２ ) Ｐ -, -NR ４ C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) Ｐ - or -(CH ２ CH ２ O) ｎ - (CH ２ ) Ｐ - and m is independently 1, 2, 3, 4, 5, or 6; Each p is independently 1 or 2; L ３ are one or more independently selected groups selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, tyrosine, valine, and amino (unsubstituted or substituted benzyl) carbamate, and any free amine of an amino acid may optionally be R ５ or -C(=O)(R ５ ), and any free carboxylic acid of the amino acid is optionally substituted with —C(═O)NH(R ５ ) and R ５ are each independently unsubstituted or substituted C １ -C １０ Alkylene, C ４ -C ２０ polyethylene glycol, or unsubstituted or substituted phenyl, where substituted phenyl is F, Cl, Br, I, —CH ３ , and C.F. ３ and is substituted with 1, 2, 3, 4, or 5 groups independently selected from L ４ does not exist, L ５ is -NR ４ C(=O)-(CH ２ ) ｎ -, -C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) ｑ -, -C(=O)NR ４ - (CH ２ CH ２ O) ｎ - (CH ２ ) ｑ - or -NR ４ C(=O)-(CH ２ CH ２ O) ｎ - (CH ２ ) ｑ - and n is independently 1, 2, 3, 4, 5, or 6; 30. The compound of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof, wherein each q is independently 1 or 2.

46. Heteroalkylene is a group in which one carbon atom is —S(═O)(═NH)—, —S(═O)(═NR ５ )-, -P(=O)OH-, -NHC(=N-CN)NH-, or -NHC(=N-R ５ 46. ​​The compound of any one of claims 1 to 45, or a pharmaceutically acceptable salt thereof, wherein: R is 1 or 2; and R is 2 or 3; and R is 4 or 5; and R is 5 or 6; and R is 7 or 8.

47. -CH ２ CH ２ C(=O)X １ -L-は、 【Chemistry 15-1】 【Chemistry 15-2】 【Chemistry 15-3】 【Chemistry 15-4】 30. The compound of any one of claims 1 to 29, wherein:

48. 【Catalog 16】 teeth, 【Chemistry 17-1】 【Chemistry 17-2】 【Chemistry 17-3】 【Chemistry 17-4】 【Chemistry 17-5】 【Chemistry 17-6】 24. The compound of any one of claims 1 to 23, which is a radionuclide complex thereof, or a pharmaceutically acceptable salt thereof.

49. 49. The compound of any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof, wherein the radionuclide is a lanthanide or an actinide.

50. 49. The compound of any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof, wherein the radionuclide is actinium, bismuth, cesium, cobalt, copper, dysprosium, erbium, gold, indium, iridium, gallium, lead, lutetium, manganese, palladium, platinum, radium, rhenium, samarium, strontium, technetium, ytterbium, yttrium, or zirconium.

51. 49. The compound of any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof, wherein the radionuclide is a diagnostic or therapeutic radionuclide.

52. 49. The compound of any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof, wherein the radionuclide is an Auger electron-emitting radionuclide, an alpha-emitting radionuclide, a beta-emitting radionuclide, or a gamma-emitting radionuclide.

53. 49. The compound of any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof, wherein the radionuclide is an alpha-emitting radionuclide.

54. The radionuclide is indium-111 ( １１１ In), Gallium-67 ( ６７ Ga), Gallium-68 ( ６８ Ga), technetium-99m ( ９９ｍ Tc), or platinum-195m ( １９５ｍ 49. The compound of any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof, wherein the Auger electron emitting radionuclide is Benzylpyrrolidone (Pt).

55. The radionuclide is actinium-225 ( ２２５ Ac), Bismuth-213 ( ２１３ Bi), radium-223 ( ２２３ Ra), or lead-212 ( ２１２ 49. The compound of any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof, wherein the alpha-emitting radionuclide is

56. The radionuclide is yttrium-90 ( ９０ Y), lutetium-177 ( １７７ Lu), rhenium-186 ( １８６ Re), rhenium-188 ( １８８ Re), copper-64 ( ６４ Cu), copper-67 ( ６７ Cu), samarium-153 ( １５３ Sm), strontium-89 ( ８９ Sr), Gold-198 ( １９８ Au), erbium-169 ( １６９ Er), dysprosium-165 ( １６５ Dy), technetium-99m ( ９９ｍ Tc), zirconium-89 ( ８９ Zr), or manganese-52 ( ５２ 49. The compound of any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof, wherein the beta-emitting radionuclide is

57. The radionuclide is cobalt-60 ( ６０ Co), palladium-103 ( １０３ Pd), Cesium-137 ( １３７ Cs), ytterbium-169 ( １６９ Yb), iridium-192 ( １９２ Ir), or radium-226 ( ２２６ 49. The compound of any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof, wherein the gamma-emitting radionuclide is Ra).

58. 58. The compound of any one of claims 1 to 57, or a pharmaceutically acceptable salt thereof, wherein the radionuclide is suitable for positron emission tomography (PET) analysis, single photon emission tomography (SPECT), or magnetic resonance imaging (MRI).

59. The radionuclide is actinium-225 ( ２２５ 49. The compound of any one of claims 1 to 48, wherein R is R, ...

60. 60. A pharmaceutical composition comprising a compound according to any one of claims 1 to 59, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

61. 61. The pharmaceutical composition of claim 60, wherein the pharmaceutical composition is formulated for administration to a mammal by intravenous administration.

62. 60. A method of treating a tumor in a mammal, comprising administering to said mammal a compound of any one of claims 1 to 59, thereby treating said tumor.

63. 63. The method of claim 62, wherein the mammal is a human.

64. 64. The method of claim 62 or 63, wherein the tumor is a solid tumor.

65. 65. The method of any one of claims 62 to 64, wherein the tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer.

66. 66. The method of any one of claims 62-65, comprising administering to said mammal between 0.5 μCi and 30.0 μCi per kilogram.

67. 66. The method of any one of claims 62 to 65, comprising administering to the mammal 10 mCi to 75 mCi per square meter of body area.

68. The tumor is administered by tumor-targeting polypeptide R ３ The method of any one of claims 62 to 67, wherein the vector expresses an antigen specifically bound by the antigen-binding region of

69. 60. A method for targeting delivery of a radionuclide to a tumor in a mammal, comprising administering to a mammal having a tumor a compound according to any one of claims 1 to 59, or a pharmaceutically acceptable salt thereof, wherein the tumor is a tumor-targeting polypeptide R ３ and expressing an antigen specifically bound by the antigen-binding region of the antibody.

70. 70. The method of claim 69, wherein the compound of any one of claims 1 to 59, or a pharmaceutically acceptable salt thereof, comprises a diagnostic or therapeutic radionuclide.

71. 10. A method for identifying a tissue or organ in a mammal having a tumor, comprising the steps of: i) administering to said mammal a compound according to any one of claims 1 to 59, or a pharmaceutically acceptable salt thereof; and ii) performing positron emission tomography (PET) analysis, single photon emission computed tomography (SPECT), or magnetic resonance imaging (MRI), wherein the tumor cells are identified as having a tumor-targeting polypeptide R. ３ and expressing an antigen specifically bound by the antigen-binding region of the antibody.

72. 1. A method for in vivo imaging of a tissue or organ in a mammal having a tumor, comprising: i) administering to said mammal a compound according to any one of claims 1 to 59, or a pharmaceutically acceptable salt thereof; ii) performing positron emission tomography (PET) analysis, single photon emission computed tomography (SPECT), or magnetic resonance imaging (MRI); The tumor cells are infected with tumor-targeting polypeptide R ３ and expressing an antigen specifically bound by the antigen-binding region of the antibody.

73. 73. The method of claim 71 or 72, wherein the compound of any one of claims 1 to 59, or a pharmaceutically acceptable salt thereof, comprises a diagnostic radionuclide.

74. 74. The method of any one of claims 71 to 73, wherein step (ii) is initiated after step (i) has elapsed a sufficient time for interaction between the compound of any one of claims 1 to 59, or a pharmaceutically acceptable salt thereof, and an antigen expressed on a tumor cell.

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