VHH antibody DOTA conjugates

Abstract

Described herein are VHH antibody DOTA conjugates. In particular, described herein are immunoconjugates comprising: a) an antigen binding region; b) an immunoglobulin heavy chain constant region; and c) a radioisotope chelating agent; wherein the molecular weight of the immunoconjugate is between 60 kDa and 110 kDa. The immunoconjugates can be used to deliver alpha and beta emitters for the treatment of tumors or cancers.

Description

[0001] This application is a divisional application of Chinese patent application No. 202380074393.8, entitled "VHH Antibody DOTA Conjugate", filed on August 21, 2023 (the corresponding PCT application was filed on August 21, 2023, and has the application number PCT / US2023 / 072589).

[0002] Cross-references

[0003] This application claims the benefit of U.S. Application No. 63 / 373,189, filed August 22, 2022, which is incorporated herein by reference in its entirety. Background Technology

[0004] The keen specificity of antibodies (such as IgG) to their antigens makes them a primary targeting platform for therapeutics; however, the typical serum half-life of IgG is at least three weeks, which is less than that of radioactive isotopes (including alpha emission isotopes, such as...). 255 Ac (actinium-225), and β-emission isotopes, such as 177 Lu (lutetium-177) and 90 Delivery of Y (yttrium-90) is unfavorable, particularly due to prolonged exposure and chronic off-target toxicity. The emergence of engineered smaller antibody forms (e.g., monomeric scFv, heavy-chain-only antibodies, or single-domain antibody fragments) has provided keen specificity for full-size antibodies (e.g., IgG (approximately 150 kDa)) with smaller forms (e.g., 15 kDa to 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, and furthermore, plasma clearance of these small antibody forms by the renal system can lead to isotope accumulation in renal tissue and problematic off-target toxicity.

[0005] 225Ac is one of the most cytotoxic alpha-emitting radioisotopes, and a single decay event can effectively destroy cancer cells by inducing double-strand DNA breaks and subsequent cell death. The potency of alpha-emitting radioisotopes makes them attractive as cytotoxic agents, overcoming acquired resistance observed in response to other therapies. However, unfortunately, due to decay events occurring at different locations in the body, many challenges remain regarding systemic administration and achieving the desired dose determination in target and non-target tissues. A key aspect of the application of alpha-emitting radioisotopes as targeted therapeutic agents is the ability to modulate the distribution of daughter radioisotopes in the body to limit toxicity. This, in turn, relates to the timing of parent radionuclide production, the timing of therapeutic administration, the decay pathway and half-life of the daughter radionuclide, circulation time, and the biodistribution and pharmacokinetics of the delivery medium. Unfortunately, the emission of alpha particles typically also generates recoil energy large enough to detach the daughter nuclide from the chelator, potentially separating it from its target medium and leading to a subsequent redistribution of the 'free' daughter nuclide, which can cause various toxicities. See, for example, Robertson A et al., CurrRadiopharm 11:156 (2018). Therefore, by 225 Nephrotoxicity caused by Ac recoil progeny nuclides (e.g., 213-Bi) has so far been recognized as a leading cause of death and nephrotoxicity. 225 The main limitations of the therapeutic use of Ac (see, for example, Jaggi J et al., Cancer Res. 65:4888 (2005)).

[0006] Another perplexing issue regarding the use of antibodies and antibody fragments containing alpha-emitting radioisotopes in therapeutic agents is that interference with radioactive decay can particularly impair antibody components and targeting sequences, even prior to treatment. Radiodegradation of antibody fragments can occur before alpha-emitter-tagged antibody fragments can be administered to patients, reducing the amount targeted (see, for example, Larsen R, Bruland O, J Labelled Cmpd Radiopharm. 36:1009-18 (1995)), and at the higher specific activity required for therapeutic administration, immunoreactivity can rapidly decline with radiochemical quality. Salako et al., J Nucl Med. 39(4):667-670 (1998). For instance, the high ionization density released by alpha emitters at doses of 1,000 Gy or higher impairs the immunoreactivity of isotopically labeled Fab fragments via radiodegradation. Similarly, significant radiodegradation of antibodies labeled with alpha-emitting isotopes has been observed at doses exceeding 1,200 Gy (Zalutsky M et al., J Nucl Med. 42(10):1508-15(2001)). Therefore, identifying suitable targeted delivery vehicles for alpha-emitting radioisotopes is not straightforward.

[0007] In addition, there are other issues with targeted radioscopy delivery platforms (including alpha- and beta-emitted radioisotopes) that need to be optimized simultaneously when designing such platforms, such as immunogenicity, specificity, tissue penetration, stability, ease of manufacture, and acceptable therapeutic window. Summary of the Invention

[0008] This invention relates to immunoconjugates or radioimmunoconjugates, compositions comprising them, and methods of using such immunoconjugates and compositions. The immunoconjugates and compositions of this invention have multiple uses, for example, for delivering radioisotopes to kill target cells (e.g., cancer cells expressing a target antigen bound by the radioimmunoconjugate); for detecting and characterizing malignant cells in a subject (e.g., target antigen expression); and for diagnosing and treating a variety of diseases and conditions, such as cancer, tumors, and other growth abnormalities involving cells expressing antigens.

[0009] This invention addresses many of the inherent challenges in the targeted delivery of alpha particle emitters in vivo through the selection and specific combination of components from a dedicated delivery platform. The alpha particle-emitting radioisotope delivery platform of this invention provides a shorter half-life compared to conventional IgG, but a longer half-life compared to smaller monomeric antibody fragments. This half-life allows for reduced toxicity caused by alpha emitters while enabling antibody fragments to remain in vivo long enough to exert therapeutic activity. For example, the alpha particle-emitting radioisotope delivery platform of this disclosure exhibits enhanced tumor targeting and reduced accumulation in radiosensitive tissues such as bone marrow and kidney. Furthermore, and surprisingly, the alpha particle-emitting radioisotope delivery platform of this invention exhibits excellent tumor binding and labeling properties against tumors with varying antigen densities, which may limit the use of some immunoconjugates.

[0010] In one aspect, this document describes an immunoconjugate comprising: a) an antigen-binding region; b) an immunoglobulin heavy chain constant region; and c) a chelating agent; wherein the molecular weight of said immunoconjugate is between 60 kDa and 110 kDa. In some embodiments, the antigen-binding region comprises an scFv peptide or a VHH peptide. In some embodiments, the antigen-binding region comprises an scFv peptide. In some embodiments, the antigen-binding region comprises a VHH peptide. In some embodiments, the antigen-binding region is humanized.

[0011] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is described herein:

[0012]

[0013] in:

[0014] R 1 It is a chelated portion or a radionuclide complex thereof;

[0015] R 2 In order to be able to target the tumor-specific R 3 The portion of the reaction between amines (-NH2) or thiols (-SH);

[0016] X 1 For -NR a -、-NR a S(=O)2-、-NR a S(=O)2NR a - or one or more independently selected natural or non-natural amino acids, wherein any free amine of the amino acid is optionally independently controlled by R 5 or -C(=O)R 5 Substitution, and optionally, any free carboxylic acid of any amino acid is replaced by -C(=O)NH-R. 5replace;

[0017] Each R a Independently selected from hydrogen and C1-C4 alkyl groups;

[0018] L is the connector, which is -L. 1 -L 2 -L 3 -L 4 -L 5 -;

[0019] L 1 C1-C, whether unsubstituted or substituted 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, unsubstituted or substituted C2-C 20 alkenyl, unsubstituted or substituted C2-C 20 Ethyne group, C4-C 20 Polyethylene glycol, -(X 3 CH2CH2) t - Unsubstituted or substituted cycloalkylene, unsubstituted or substituted heteroalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene;

[0020] Each X 3 Independently selected from O and NR 4 ;

[0021] Each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0022] L 2 C1-C that is absent, unsubstituted, or substituted 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, -C(=O)- (unsubstituted or substituted C1-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 -;

[0023] Each R 4 Independently selected from hydrogen and C1-C6 alkyl groups;

[0024] Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0025] Each p is independently 0, 1, or 2;

[0026] L 3 The group is absent, or one or more independently selected from the group consisting of: natural or non-natural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates; wherein any free amine (-NH2) of the amino acid is optionally independently controlled by R. 5 or -C(=O)R 5 Substitution, wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 Substitution, wherein the substituted benzyl group is selected from halogen, -OH, -OR. 5 -CO2H, -NHR 5 -C(=O)NHR 5 -NHC(=O)R 5 The substituted C1-C6 alkyl groups are replaced by -OH, -CO2H, or -NHR groups. 5 -C(=O)NHR 5 and -NHC(=O)R 5 replace;

[0027] L 4 C1-C that is absent, unsubstituted, or substituted 10 Alkylene, -C(=O)- (unsubstituted or substituted C1-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 -;

[0028] Each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0029] Each q is independently 0, 1, or 2;

[0030] L 5 This does not exist; -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 -;

[0031] Each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0032] Each q is independently 0, 1, or 2;

[0033] Each R a Independently selected from hydrogen and C1-C4 alkyl groups;

[0034] Each R bIndependently selected from hydrogen and C1-C4 alkyl groups;

[0035] Wherein, the heteroalkylene group is an alkylene group in which one carbon atom is replaced by one of the following groups: -O-, -S-, -S(=O)-, -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(=NR 5 )NR a -or-NR a C(=O)O-;

[0036] Where -L 1 -、-L 2 -、-L 3 -、-L 4 -and-L 5 When any one of - is replaced, then -L 1 -、-L 2 -、-L 3 -、-L 4 -and-L 5 - Selected by 1, 2, 3 or 4 from halogen, -OH, -OR 5 -CO2H, -NHR 5 -C(=O)NHR 5 -NHC(=O)R 5 The substituted C1-C6 alkyl groups are replaced by -OH, -CO2H, or -NHR groups. 5 -C(=O)NHR 5 and -NHC(=O)R 5 replace;

[0037] Each R 5 Independently selected from C1-C 10 Alkyl, C4-C 30 Polyethylene glycol and unsubstituted or substituted arylene groups, unsubstituted or substituted heteroarylene groups;

[0038] The premise is that when L 3 If it does not exist, then there exists at least one R. 5 ;or

[0039] The premise is X 1 -N (C1-C4 alkyl), -NR a S(=O)2-、-NR a S(=O)2NR a -Or one or more independently selected natural or non-natural amino acids.

[0040] In some embodiments, the immunoconjugate is a compound of formula (II), formula (III) or formula (IV) or a pharmaceutically acceptable salt thereof:

[0041]

[0042] in:

[0043] R 1 It is a chelated portion or a radionuclide complex thereof;

[0044] -NH-R 3 For tumor targeting;

[0045] X represents non-existent, -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-;

[0046] Each R a Independently selected from hydrogen and C1-C4 alkyl groups;

[0047] X 1 For -NR a -、-NR a S(=O)2-、-NR a S(=O)2NR a - or one or more independently selected natural or non-natural amino acids, wherein any free amine of the amino acid is optionally independently controlled by R 5 or -C(=O)R 5 Substitution, and optionally, any free carboxylic acid of any amino acid is replaced by -C(=O)NH-R. 5 Replace; for each R a Independently selected from hydrogen and C1-C4 alkyl groups;

[0048] L is the connector, which is -L. 1 -L 2 -L 3 -L 4 -L 5 -;

[0049] L 1 C1-C, whether unsubstituted or substituted 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, unsubstituted or substituted C2-C 20 alkenyl, unsubstituted or substituted C2-C 20 Ethyne group, C4-C 20 Polyethylene glycol, -(X 3 CH2CH2) t - Unsubstituted or substituted cycloalkylene, unsubstituted or substituted heteroalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene;

[0050] Each X 3 Independently selected from O and NR 4 ;

[0051] Each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0052] L 2 C1-C that is absent, unsubstituted, or substituted 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, -C(=O)- (unsubstituted or substituted C1-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 -;

[0053] Each R 4 Independently selected from hydrogen and C1-C6 alkyl groups;

[0054] Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0055] Each p is independently 0, 1, or 2;

[0056] L 3 The group is absent, or one or more independently selected from the group consisting of: natural or non-natural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates; wherein any free amine (-NH2) of the amino acid is optionally independently controlled by R. 5 or -C(=O)R 5 Substitution, wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 Substitution, wherein the substituted benzyl group is selected from halogen, -OH, -OR. 5 -CO2H, -NHR 5 -C(=O)NHR 5 -NHC(=O)R 5 The substituted C1-C6 alkyl groups are replaced by -OH, -CO2H, or -NHR groups. 5 -C(=O)NHR 5 and -NHC(=O)R 5 replace;

[0057] L 4 C1-C that is absent, unsubstituted, or substituted 10 Alkylene, -C(=O)- (unsubstituted or substituted C1-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 -;

[0058] Each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0059] Each q is independently 0, 1, or 2;

[0060] L 5 This does not exist; -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 -;

[0061] Each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0062] Each q is independently 0, 1, or 2;

[0063] Each R a Independently selected from hydrogen and C1-C4 alkyl groups;

[0064] Each R b Independently selected from hydrogen and C1-C4 alkyl groups;

[0065] Wherein, the heteroalkylene group is an alkylene group in which one carbon atom is replaced by one of the following groups: -O-, -S-, -S(=O)-, -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(=NR 5 )NR a -or-NR a C(=O)O-;

[0066] Where -L 1 -、-L 2 -、-L 3 -、-L 4 -and-L 5 When any one of - is replaced, then -L 1 -、-L 2 -、-L 3 -、-L 4 -and-L 5 - Selected by 1, 2, 3 or 4 from halogen, -OH, -OR 5 -CO2H, -NHR 5 -C(=O)NHR 5 -NHC(=O)R 5 The substituted C1-C6 alkyl groups are replaced by -OH, -CO2H, or -NHR groups. 5 -C(=O)NHR 5 and -NHC(=O)R 5 replace;

[0067] Each R 5 Independently selected from C1-C 10 Alkyl, C4-C 30 Polyethylene glycol, and unsubstituted or substituted arylene groups, unsubstituted or substituted heteroarylene groups;

[0068] The premise is that when L 3 If it does not exist, then there exists at least one R. 5 ;or

[0069] The premise is X1 -N (C1-C4 alkyl), -NR a S(=O)2-、-NR a S(=O)2NR a -Or one or more independently selected natural or non-natural amino acids.

[0070] In some embodiments, the immunoconjugate is a compound of formula (II) or a pharmaceutically acceptable salt thereof:

[0071]

[0072] In some embodiments, the immunoconjugate is a compound of formula (III) or a pharmaceutically acceptable salt thereof:

[0073]

[0074] In some implementations, the immunoconjugate is a compound of formula (IV) or a pharmaceutically acceptable salt thereof:

[0075]

[0076] In some implementation schemes, R 2 It is able to target the tumor-specific R 3 The part of the amine (-NH2) reaction of lysine.

[0077] In some embodiments, the compound of formula (II) has the structure of formula (IIa) or a pharmaceutically acceptable salt thereof:

[0078]

[0079] in:

[0080] -NHCH2CH2CH2CH2- is the tumor-targeting fraction R. 3 The side chain of lysine residues.

[0081] In some embodiments, the compound of formula (III) has the structure of formula (IIIa) or a pharmaceutically acceptable salt thereof:

[0082]

[0083] in:

[0084] -NHCH2CH2CH2CH2- is the tumor-targeting fraction R. 3 The side chain of lysine residues.

[0085] In some implementation schemes, R 2 It is able to target the tumor-specific R 3The part of the thiol (-SH) reaction of cysteine.

[0086] In some embodiments, the compound of formula (IV) has the structure of formula (IVa) or a pharmaceutically acceptable salt thereof:

[0087]

[0088]

[0089] in:

[0090] -NHCH2CH2CH2CH2- is the tumor-targeting fraction R. 3 The side chain of lysine residues.

[0091] In some embodiments, the immunoconjugate is a compound of formula (V), (VI), (VII), or (VIII) or a pharmaceutically acceptable salt thereof:

[0092]

[0093] in:

[0094] R 1 It is a chelated portion or a radionuclide complex thereof;

[0095] -SR 3 For tumor targeting;

[0096] X 1 For -NR a -、-NR a S(=O)2-、-NR a S(=O)2NR a - or one or more independently selected natural or non-natural amino acids, wherein any free amine of the amino acid is optionally independently controlled by R 5 or -C(=O)R 5 Substitution, and optionally, any free carboxylic acid of any amino acid is replaced by -C(=O)NH-R. 5 replace;

[0097] Each R a Independently selected from hydrogen and C1-C4 alkyl groups;

[0098] L is the connector, which is -L. l -L 2 -L 3 -L 4 -L 5 -;

[0099] L 1 C1-C, whether unsubstituted or substituted 10Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, unsubstituted or substituted C2-C 20 alkenyl, unsubstituted or substituted C2-C 20 Ethyne group, C4-C 20 Polyethylene glycol, -(X 3 CH2CH2) t - Unsubstituted or substituted cycloalkylene, unsubstituted or substituted heteroalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene;

[0100] Each X 3 Independently selected from O and NR 4 ;

[0101] Each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0102] L 2 C1-C that is absent, unsubstituted, or substituted 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, -C(=O)- (unsubstituted or substituted C1-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 -;

[0103] Each R 4 Independently selected from hydrogen and C1-C6 alkyl groups;

[0104] Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0105] Each p is independently 0, 1, or 2;

[0106] L 3 The group is absent, or one or more independently selected from the group consisting of: natural or non-natural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates; wherein any free amine (-NH2) of the amino acid is optionally independently controlled by R. 5 or -C(=O)R 5 Substitution, wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 Substitution, wherein the substituted benzyl group is selected from halogen, -OH, -OR. 5 -CO2H, -NHR 5 -C(=O)NHR 5 -NHC(=O)R 5 The substituted C1-C6 alkyl groups are replaced by -OH, -CO2H, or -NHR groups. 5 -C(=O)NHR 5 and -NHC(=O)R 5 replace;

[0107] L 4 C1-C that is absent, unsubstituted, or substituted 10 Alkylene, -C(=O)- (unsubstituted or substituted C1-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 -;

[0108] Each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0109] Each q is independently 0, 1, or 2;

[0110] L 5 For non-existent, -C(=O)-(CH2)n-, -C(=O)NR 4 -(CH2) n -, -NR4C(=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 -;

[0111] Each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0112] Each q is independently 0, 1, or 2;

[0113] Each R a Independently selected from hydrogen and C1-C4 alkyl groups;

[0114] Each R b Independently selected from hydrogen and C1-C4 alkyl groups;

[0115] Wherein, the heteroalkylene group is an alkylene group in which one carbon atom is replaced by one of the following groups: -O-, -S-, -S(=O)-, -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 -、-NRa C(=O)NR a -、-NR a C(=N-CN)NR a -、-NR a C(=NR 5 )NR a -or-NR a C(=O)O-;

[0116] Where -L 1 -、-L 2 -、-L 3 -、-L 4 -and-L 5 When any one of - is replaced, then -L 1 -、-L 2 -、-L 3 -、-L 4 -and-L 5 - Selected by 1, 2, 3 or 4 from halogen, -OH, -OR 5 -CO2H, -NHR 5 -C(=O)NHR 5 -NHC(=O)R 5 The substituted C1-C6 alkyl groups are replaced by -OH, -CO2H, or -NHR groups. 5 -C(=O)NHR 5 and -NHC(=O)R 5 replace;

[0117] Each R 5 Independently selected from C1-C 10 Alkyl, C4-C 30 Polyethylene glycol, and unsubstituted or substituted arylene groups, unsubstituted or substituted heteroarylene groups;

[0118] The premise is that when L 3 If it does not exist, then there exists at least one R. 5 ;or

[0119] The premise is X 1 -N (C1-C4 alkyl), -NR a S(=O)2-、-NR a S(=O)2NR a -Or one or more independently selected natural or non-natural amino acids.

[0120] In some implementations, the immunoconjugate is a compound of formula (V) or a pharmaceutically acceptable salt thereof:

[0121]

[0122] In some implementations, the immunoconjugate is a compound of formula (VI) or a pharmaceutically acceptable salt thereof:

[0123]

[0124] In some implementations, the immunoconjugate is a compound of formula (VII) or a pharmaceutically acceptable salt thereof:

[0125]

[0126] In some implementations, the immunoconjugate is a compound of formula (VIII) or a pharmaceutically acceptable salt thereof:

[0127]

[0128] In some embodiments, the compound of formula (V) has the structure of formula (Va) or a pharmaceutically acceptable salt thereof:

[0129]

[0130] in:

[0131] -SCH2- is the tumor-targeting component R 3 Thiols (-SH) on the side chains of cysteine ​​residues.

[0132] In some embodiments, the compound of formula (VI) has the structure of formula (VIa) or a pharmaceutically acceptable salt thereof:

[0133]

[0134] in:

[0135] -SCH2- is the tumor-targeting component R 3 Thiols (-SH) on the side chains of cysteine ​​residues.

[0136] In some embodiments, the compound of formula (VI) has the structure of formula (VIa) or a pharmaceutically acceptable salt thereof:

[0137]

[0138] in:

[0139] -SCH2- is the tumor-targeting component R 3 Thiols (-SH) on the side chains of cysteine ​​residues.

[0140] In some embodiments, the compound of formula (VII) has the structure of formula (VIIa) or a pharmaceutically acceptable salt thereof:

[0141]

[0142] in:

[0143] -SCH2- is the tumor-targeting component R 3 Thiols (-SH) on the side chains of cysteine ​​residues.

[0144] In some embodiments, the compound of formula (VIII) has the structure of formula (VIIIa) or a pharmaceutically acceptable salt thereof:

[0145]

[0146] in:

[0147] -SCH2- is the tumor-targeting component R 3 Thiols (-SH) on the side chains of cysteine ​​residues.

[0148] In some implementations, the tumor-targeting component R 3 It is a polypeptide containing an antigen-binding region and an immunoglobulin heavy chain constant region, wherein the molecular weight of the polypeptide is between 60 kDa and 110 kDa.

[0149] 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 the CH2 domain, the CH3 domain, or both CH2 and CH3 domains of an immunoglobulin. 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, and 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 alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region or alter the binding of the immune conjugate to the neonatal Fc receptor (FcRn); or the immunoglobulin heavy chain constant region comprises alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region and alter the binding of the immune conjugate to the neonatal Fc receptor (FcRn). In some embodiments, the immunoglobulin heavy chain constant region includes alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region; or the immunoglobulin heavy chain constant region includes alterations to one or more amino acid residues that alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn); or both. In some embodiments, the alteration to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region is an alteration that reduces complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or a combination thereof.

[0150] In some embodiments, the antigen-binding region specifically binds to HER2 or DLL3. In some embodiments, the antigen-binding region specifically binds to HER2. In some embodiments, the antigen-binding region of the immunoconjugate comprises: a) heavy chain CDR1, which comprises the amino acid sequence shown in SEQ ID NO:21; b) heavy chain CDR2, which comprises the amino acid sequence shown in SEQ ID NO:22; and c) heavy chain CDR3, which comprises the amino acid sequence shown in SEQ ID NO:23, and binds to HER2. In some embodiments, the antigen-binding region of the immunoconjugate comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:20 and binding to HER2. In some embodiments, the antigen-binding region specifically binds to DLL3. In some embodiments, the antigen-binding region of the immunoconjugate comprises: a) heavy chain CDR1, which comprises the amino acid sequence shown in SEQ ID NO:31; b) heavy chain CDR2, which comprises the amino acid sequence shown in SEQ ID NO:32;and c) heavy chain CDR3, which comprises the amino acid sequence shown in SEQ ID NO:33 and binds DLL3. In some embodiments, the antigen-binding region of the immunoconjugate comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:30 and binding DLL3. In some embodiments, the immunoglobulin heavy chain constant region comprises the CH2 domain, the CH3 domain, or the CH2 and CH3 domains of an immunoglobulin. In some embodiments, the immunoglobulin heavy chain constant region comprises the CH2 and CH3 domains of an immunoglobulin. In some embodiments, the immunoglobulin heavy chain constant region is the human immunoglobulin heavy chain constant region. In some embodiments, the immunoglobulin heavy chain constant region is an IgA, IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the immunoglobulin heavy chain constant region is the IgG1 isotype. In some embodiments, the immunoglobulin heavy chain constant region is the IgG4 isotype. In some embodiments, the immunoglobulin heavy chain constant region includes alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region or alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In some embodiments, the immunoglobulin heavy chain constant region includes alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region and alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In some embodiments, the immunoglobulin heavy chain constant region includes alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region. In some embodiments, the immunoglobulin heavy chain constant region includes alterations to one or more amino acid residues that alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In some embodiments, the alteration to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region is an alteration that reduces complement-dependent cytotoxicity (CDC), antibody-dependent cell-cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or a combination thereof. In some implementations, the alteration of one or more amino acid residues that reduces the effector function of the constant region of the immunoglobulin heavy chain is selected from a list of the following components according to EU numbers: (a) 297A, 297Q, 297G, or 297D; (b) 279F, 279K, or 279L; (c) 228P; (d) 235A, 235E, 235G, 235Q, 235R, or 235S; (e) 237A, 237E, 237K, 237N, or 237R; (f) 234A, 234V, or 234F; (g) 233P; (h) 328A; (i) 327Q or 327T; (j) 329A, 329G, 329Y, or 329R;(k) 331S, (l) 236F or 236R; (m) 238A, 238E, 238G, 238H, 238I, 238V, 238W or 238Y; (n) 248A; (o) 254D, 254E, 254G, 254H, 254I, 254N, 254P, 254Q, 254T or 254V; (p) 255N; (q) 256H, 256K, 256R or 256V; (r) 264S; (s) 265H, 265K, 265S, 265Y or 265A; (t) 267G, 267H, 267I or 267K; (u) 268K, (v) 269N or 269Q; (w) 270 A) 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 4 24V; (pp)434I; (qq)438G; (rr)439E, 439H, or 439Q; (ss)440A, 440D, 440E, 440F, 440M, 440T, or 440V; (tt)K322A; (uu)L235E; (vv)L234A and L235A; (ww)L234A, L235A, and G237A; (xx)L234A, L235A, and P329G; (yy)L234F, L235E, and P331S; (zz)L234A, L235E, and G237A; (aaa)L234A, L235E, G237A, and P331S; (bbb)L234 A, 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 any combination of (ppp)(a)-(ppp). In some embodiments, alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region include L234A, L235E, G237A, A330S, and P331S according to EU numbers. In some embodiments, amino acid alterations to one or more amino acid residues that alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) shorten the serum half-life of the immunoconjugate. In some embodiments, alterations to one or more amino acid residues that alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are for amino acid residues selected from the list of the following compositions according to EU numbers: 251, 252, 253, 254, 255, 288, 309, 310, 312, 385, 386, 388, 400, 415, 433, 435, 436, 439, 447, and combinations thereof. In some embodiments, the alteration of one or more amino acid residues that changes the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is directed to amino acid residues selected from the list of the following compositions according to EU numbers: 253, 254, 310, 435, 436, and combinations thereof. In some embodiments, the alteration of one or more amino acid residues that changes the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is directed to amino acid residues selected from the list of the following compositions according to EU numbers: I253A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof. In some embodiments, the alteration of one or more amino acid residues that changes the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is directed to amino acid residues selected from the list of the following compositions according to EU numbers: I253A, S254A, H310A, H435Q, Y436A, and combinations thereof. In some embodiments, the alteration of one or more amino acid residues that changes the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is directed to amino acid residues selected from the list of the following compositions according to EU numbers: I253A, H310A, H435Q, and combinations thereof. In some embodiments, the serum half-life of the immunoconjugate is less than 15 days. In some embodiments, the serum half-life of the immunoconjugate is less than 10 days. In some embodiments, the serum half-life of the immunoconjugate is less than 120 hours. In some embodiments, the serum half-life of the immunoconjugate is less than 72 hours. In some embodiments, the antigen-binding region is coupled to the immunoglobulin heavy chain constant region via a linker amino acid sequence or a human IgG hinge region. In some implementations, the antigen-binding region is coupled to the immunoglobulin heavy chain constant region via the human IgG hinge region.

[0151] In some embodiments, the chelating agent is coupled to the antigen-binding region and / or the immunoglobulin heavy chain constant region at a ratio of 1:1 to 8:1. In some embodiments, the chelating agent is coupled to the antigen-binding region and / or the immunoglobulin heavy chain constant region at a ratio of 1:1 to 6:1. In some embodiments, the chelating agent is coupled to the antigen-binding region and / or the immunoglobulin heavy chain constant region at a ratio of 2:1 to 6:1. In some embodiments, the immunoconjugate further comprises a radioactive isotope. In some embodiments, the radioactive isotope is an alpha emitter. In some embodiments, the radioactive isotope is an alpha emitter selected from the list of the following compositions: 225 Ac、 223 Ra、 224 Ra、 227 Th、 212 Pb, 212 Bihe 213 Bi. In some implementations, the radioactive isotope is 225 Ac. In some embodiments, the radioactive isotope is a beta emitter. In some embodiments, the radioactive isotope is a beta emitter selected from: 177 Lu、 90 Y、 67 Cu and 153 Sm. In some embodiments, the molecular weight of the immunoconjugate is between 60 kDa and 100 kDa. In some embodiments, the molecular weight of the immunoconjugate is between 60 kDa and 90 kDa. In some embodiments, the molecular weight of the immunoconjugate is between 65 kDa and 90 kDa. In some embodiments, the molecular weight of the immunoconjugate is between 70 kDa and 90 kDa. In some embodiments, the immunoconjugate forms a dimer with another immunoconjugate. In some embodiments, the immunoconjugate further comprises a pharmaceutically acceptable excipient or carrier. In some embodiments, the immunoconjugate is formulated for intravenous administration.

[0152] This document also describes a method for preparing an immunoconjugate, the method comprising loading the immunoconjugate with a radioactive isotope. In some embodiments, the radioactive isotope is an alpha emitter. In some embodiments, the radioactive isotope is an alpha emitter selected from the list of the following: 225 Ac、 223 Ra、 224 Ra、 227 Th、 212 Pb, 212 Bihe 213 Bi. In some implementations, the radioactive isotope is 225 Ac. In some embodiments, the radioactive isotope is a beta emitter. In some embodiments, the radioactive isotope is a beta emitter selected from:177 Lu、 90 Y、 67 Cu and 153 Sm. In some implementations, the radioactive isotope is 177 Lu.

[0153] This document also describes a method for treating an individual's cancer or tumor, the method comprising administering an immune conjugate to the individual thereby treating the cancer or tumor. In some embodiments, the individual is a human individual. In some embodiments, the cancer or tumor is a solid cancer or tumor. In some embodiments, the cancer or tumor includes lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer. In some embodiments, the method further includes administering 0.5 μCi to 30.0 μCi per kilogram to the individual. In some embodiments, the cancer or tumor expresses an antigen specifically bound to the immune conjugate.

[0154] This document also describes an immune conjugate for a method of treating cancer or tumor in an individual. In some embodiments, the individual is a human individual. In some embodiments, the cancer or tumor is a solid cancer or tumor. In some embodiments, the cancer or tumor includes lung cancer, breast cancer, ovarian cancer, or neuroendocrine carcinoma. In some embodiments, the individual is administered 0.5 μCi to 30.0 μCi per kilogram. In some embodiments, the cancer or tumor expresses an antigen that the immune conjugate specifically binds to.

[0155] This document also describes a method for killing cancer cells in an individual, the method comprising administering an immune conjugate to the individual to kill the cancer cells. In some embodiments, the individual is a human individual. In some embodiments, the cancer cells include lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells. In some embodiments, the method comprises administering 0.1 μCi to 30.0 μCi per kilogram to the individual. In some embodiments, the method comprises administering 10 mCi to 75 mCi per square meter of body area to the individual. In some embodiments, the cancer cells express an antigen specifically bound to the immune conjugate.

[0156] This document also describes the use of the immune conjugate in a method for killing cancer cells in an individual. In some embodiments, the individual is a human individual. In some embodiments, the cancer cells include lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells. In some embodiments, the method includes administering 0.5 μCi to 30.0 μCi per kilogram to the individual. In some embodiments, the cancer cells express an antigen that the immune conjugate specifically binds to.

[0157] This document also describes a method for delivering a radioactive isotope to cancer cells or tumor cells in an individual, the method comprising administering an immunoconjugate to the individual thereby delivering the radioactive isotope to the cancer cells or tumor cells. In some embodiments, the individual is a human individual. In some embodiments, the cancer cells or tumor cells include lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells. In some embodiments, the method comprises administering 0.5 μCi to 30.0 μCi per kilogram to the individual. In some embodiments, the cancer cells or tumor cells express an antigen specifically bound to the immunoconjugate.

[0158] This document also describes immune conjugates for delivering radioactive isotopes to cancer cells or tumor cells in an individual. In some embodiments, the individual is a human individual. In some embodiments, the cancer cells or tumor cells include lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells. In some embodiments, the cancer cells or tumor cells express an antigen that the immune conjugate specifically binds to.

[0159] This document also describes a method for imaging tumors in an individual, the method comprising administering an immune conjugate to the individual. In some embodiments, the individual is a human individual. In some embodiments, the cancer or tumor includes lung cancer, breast cancer, ovarian cancer, or neuroendocrine carcinoma. In some embodiments, the tumor expresses an antigen that the immune conjugate specifically binds to.

[0160] This document also describes immune conjugates for methods of imaging tumors in an individual. In some embodiments, the individual is a human individual. In some embodiments, the cancer or tumor includes lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer. In some embodiments, the tumor expresses an antigen that the immune conjugate specifically binds to.

[0161] This document also describes a nucleic acid that encodes an immunoconjugate. In some embodiments, the expression vector comprises a nucleic acid. In some embodiments, the cell comprises a nucleic acid or an expression vector. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a CHO cell.

[0162] In some embodiments, the molecular size of the subject radioisotope delivery platform is sufficiently large (e.g., 60 kDa to 110 kDa) to significantly reduce off-target toxicity, particularly kidney injury (e.g., from alpha-emitted isotope cargo), and its size is sufficiently small to achieve increased tissue penetration compared to conventional IgG, maintain target specificity, and increase the probability of first decay events in the target tissue. This size also allows for preferential elimination by the liver relative to the kidneys, thus protecting the kidneys from radiotoxicity.

[0163] In some implementations, the subject radioisotope delivery platform can be used to safely and efficiently deliver alpha emitters in vivo in part due to the reduction of certain adverse effects caused by platforms with a half-life of more than 5 days and / or a molecular weight of less than 60 kDa.

[0164] In some implementations, the subject radioisotope delivery platform can be used to safely and efficiently target and deliver alpha emitters in vivo, partly because it exhibits a reduced loss of targeting capability due to radiodegradation compared to other possible delivery platforms.

[0165] In some implementations, the subject radioisotope delivery platform can be used for the safe and efficient targeted delivery of alpha emitters in vivo, partly due to increased stability at the temperatures required for certain radiolabeling processes (e.g., high-temperature chelation using certain chelating factors) compared to other possible delivery platforms using antibody fragments.

[0166] In one embodiment, the present invention provides an immunoconjugate for in vivo delivery of an alpha-emitting radioisotope. In one embodiment, the immunoconjugate is also capable of delivering other atoms in vivo. In one embodiment, the immunoconjugate is capable of delivering imaging metals (e.g., 111-In, 89-Zr, 64-Cu, 68-Ga, or 134-Ce) in vivo.

[0167] In one embodiment, the immunoconjugate comprises an antibody construct and a chelating agent, and its molecular weight is between 60 kDa and 110 kDa, preferably between 60 kDa and 100 kDa, preferably between 60 kDa and 90 kDa, preferably between 65 kDa and 90 kDa, and preferably between 70 kDa and 90 kDa. The chelating agent is capable of chelating an alpha-emitting radioisotope, thereby linking the antibody construct to the alpha-emitting radioisotope.

[0168] At least one of the variant constant regions of 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, which may be the same or different.

[0169] 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.

[0170] In one embodiment, the chelating agent comprises a radioisotope chelating component and a functional group that allows covalent bonding with an antigen-binding arm. In one embodiment, the functional group is directly connected to the radioisotope chelating component. In one embodiment, the chelating agent further comprises a connector between the functional group and the radioisotope chelating component.

[0171] In one embodiment, the radioisotope chelating component comprises DOTA or a DOTA derivative. In one embodiment, the radioisotope chelating component comprises DOTAGA. In one embodiment, the radioisotope chelating component comprises macropa or a macropa derivative. In one embodiment, the radioisotope chelating component comprises Py4Pa or a Py4Pa derivative.

[0172] In one embodiment, the present invention provides a pharmaceutical composition comprising the radioimmunoconjugate of the present invention and a pharmaceutically acceptable carrier.

[0173] In one embodiment, the present invention provides a method for delivering an alpha-emitting radioisotope to cancer cells in a patient, the method comprising administering to the patient a radioimmunoconjugate or pharmaceutical composition of the present invention. In one embodiment, the patient is a human patient.

[0174] In one embodiment, the present invention provides a method for inhibiting the growth of cancer cells, the method comprising contacting the cancer cells with a radioimmunoconjugate of the present invention. In one embodiment, the cancer cells are in a patient. In one embodiment, the method involves administering the pharmaceutical composition of the present invention to a patient. In one embodiment, the patient is a human patient.

[0175] In one embodiment, the present invention provides a method for killing cancer cells, the method comprising contacting the cancer cells with a radioimmunoconjugate of the present invention. In one embodiment, the cancer cells are in a patient. In one embodiment, the method involves administering the pharmaceutical composition of the present invention to a patient. In one embodiment, the patient is a human patient.

[0176] In one embodiment, the present invention provides a method for treating cancer in a patient in need, the method comprising administering to the patient a radioimmunoconjugate or pharmaceutical composition of the present invention. In one embodiment, the patient is a human patient.

[0177] In one embodiment, the present invention provides a targeted imaging complex comprising the immunoconjugate of the present invention and further comprising an imaging metal. In one aspect, the present invention provides a targeted imaging complex comprising an antibody construct of the immunoconjugate of the present invention and further comprising an imaging metal. In one embodiment, the imaging metal is a radioisotope. In one embodiment, the imaging metal is selected from the group consisting of: 111 In (Indium-111) 89 Zr (Zr-89) 64 Cu (Copper-64) 68 Ga (Ga-69) and 134 Ce(cesium-134). In one embodiment, the imaging metal is selected from: 111 In、 89 Zr、 64 Cu、 68 Ga and 134 Ce. In one implementation, the imaging metal is 111 In one embodiment, the imaging metal is covalently bound to an immunoconjugate or antibody construct. In one embodiment, the imaging metal is associated with a chelating agent of the immunoconjugate. In one embodiment, the present invention provides a method for determining the location of cancer cells in a patient, the method comprising administering the targeted imaging complex of the present invention to the patient. In one embodiment, the patient is a human patient.

[0178] In one embodiment, the present invention provides a kit for preparing the radiopharmaceutical of the present invention, the kit comprising the immunoconjugate of the present invention. In one embodiment, the present invention provides a kit comprising the radioimmunoconjugate of the present invention. In one embodiment, the present invention provides a kit for preparing a pharmaceutical composition of the present invention, the kit comprising the immunoconjugate of the present invention. In one embodiment, the present invention provides a kit for preparing a pharmaceutical composition of the present invention, the kit comprising the radioimmunoconjugate of the present invention. In one embodiment, the present invention provides a kit comprising the pharmaceutical composition of the present invention.

[0179] In some embodiments, the immunoconjugates or radioimmunoconjugates of the present invention comprise dimerizing domains or motifs. In some other embodiments, the dimerizing domains or motifs are located in hinge regions and / or variant-constant regions.

[0180] In some embodiments, the immunoconjugates or radioimmunoconjugates or pharmaceutical compositions of the present invention have a half-life of less than 96 hours in human serum. In some other embodiments, the half-life in human serum is less than 72 hours. In some other embodiments, the half-life is less than 48 hours, 36 hours, 24 hours, and / or 12 hours. In some embodiments, the half-life is between 4 and 8 hours, between 6 and 12 hours, between 8 and 16 hours, between 12 and 24 hours, or between 24 and 48 hours.

[0181] In one aspect, the present invention provides a radioimmunoconjugate comprising the immunoconjugate of the present invention and further comprising a β-particle emitter, such as, for example... 177 Lu、 90 Y、 67 Cu or 153 Sm. In one aspect, the present invention provides a pharmaceutical composition comprising such radioimmunoconjugates.

[0182] In one aspect, the present invention provides a radioimmunoconjugate comprising the immunoconjugate of the present invention and further comprising an alpha particle emitter and a beta and / or gamma particle emitter. In another aspect, the present invention provides a pharmaceutical composition comprising such a radioimmunoconjugate.

[0183] In some embodiments, the kit of the present invention includes, in addition to the immunoconjugate, radioimmunoconjugate or pharmaceutical composition of the present invention, a reagent or pharmaceutical device.

[0184] In some embodiments, the kit of the present invention is an immunoassay kit for the specific detection of antigens in biological samples, the kit comprising: (a) an immunoconjugate, radioimmunoconjugate, or targeted imaging complex and / or a combination thereof as described herein; and (b) instructions for the detection of the immunoconjugate, radioimmunoconjugate, or targeted imaging complex.

[0185] In another aspect, the present invention provides an isolated nucleic acid encoding an antigen-binding arm or a component thereof as provided herein. In one aspect, the present invention provides an isolated nucleic acid encoding an antigen-binding region of an immunoconjugate described herein. In one aspect, the present invention provides an isolated nucleic acid encoding a VHH polypeptide of an immunoconjugate described herein. In one aspect, the present invention provides an isolated nucleic acid encoding a hinge region of an immunoconjugate described herein. In one aspect, the present invention provides an isolated nucleic acid encoding a variant-constant region of an immunoconjugate described herein. In one aspect, the present invention provides an isolated nucleic acid encoding a VHH polypeptide of an immunoconjugate described herein and a hinge region of an immunoconjugate described herein. In one aspect, the present invention provides an isolated nucleic acid encoding a VHH polypeptide of an immunoconjugate described herein, a hinge region of an immunoconjugate described herein, and a variant-constant region of an immunoconjugate described herein.

[0186] In another aspect, the present invention provides a vector comprising the nucleic acid as provided herein. In some embodiments, the vector is an expression vector.

[0187] In another aspect, the present invention provides methods for using the immunoconjugates, radioimmunoconjugates, targeted imaging complexes, or pharmaceutical compositions of the present invention. In some embodiments, the present invention provides a method for treating a disease, symptom, or condition, the method comprising administering a pharmaceutically effective amount of the radioimmunoconjugates or pharmaceutical compositions of the present invention to a patient in need.

[0188] In some embodiments, the method of the present invention includes the step of administering any one of the radioimmunoconjugates or pharmaceutical compositions described herein to a subject in need. In some other embodiments, the method is used to inhibit the growth of cancer cells or tumors and / or kill cancer cells or tumors.

[0189] In some embodiments, the use of the immunoconjugates or radioimmunoconjugates described herein is provided for the manufacture of medicaments for treating a disease, condition, or illness (such as, for example, cancer).

[0190] In another aspect, the present invention provides a method for preparing the radioimmunoconjugate or pharmaceutical composition of the present invention, the method comprising radiolabeling the immunoconjugate with a suitable isotope (such as, for example, an alpha or beta particle emitter).

[0191] These and other features, aspects, and advantages of the invention will become more readily understood with reference to the following description and the appended claims. The foregoing elements of the invention may be combined individually or freely removed to form other embodiments of the invention, and no objection to such combinations or removals is stated below. Attached Figure Description

[0192] Figure 1A and 1B This demonstrates the combination of anti-HER2 and anti-DLL3 VHH-Fc constructs.

[0193] Figure 2A , 2B 2C showed that the anti-HER2 and anti-DLL3 VHH-Fc constructs bound to cells expressing HER2 and / or DLL3.

[0194] Figure 3A and 3B This demonstrates the internalization of the anti-HER2 and anti-DLL3 VHH-Fc constructs in cells expressing HER2 and DLL3.

[0195] Figure 4 Shows self-interaction data for the anti-HER2 and anti-DLL3 VHH-Fc constructs.

[0196] Figure 5 A schematic diagram showing the chemical synthesis of the linker molecule.

[0197] Figure 6 A schematic diagram showing the chemical synthesis of the linker molecule.

[0198] Figure 7A , 7B 7C shows the immunoreactivity scores of different VHH-Fc constructs.

[0199] Figure 8 Display 111 Imaging with In-labeled VHH-Fc and 225 Comparison of the biological distribution of Ac-labeled VHH-Fc.

[0200] Figure 9A , 9B 9C and 9D show the biodistribution of the labeled anti-HER2 VHH-Fc constructs over time.

[0201] Figure 10A , 10B The ratio of tumor to non-tumor tissue labeled with the anti-HER2 VHH-Fc construct and 10C.

[0202] Figure 11 The biodistribution of the labeled anti-HER2 VHH-Fc construct is shown.

[0203] Figure 12 Display 111 Systemic clearance of In-labeled VHH-Fc (H101) and VHH-Fc variants (H105, H107 and H108).

[0204] Figure 13The biological distribution of the labeled anti-DLL3 VHH-Fc construct over time is shown.

[0205] Figure 14 The biodistribution of the labeled anti-DLL3 VHH-Fc construct is shown.

[0206] Figure 15A and 15B show 225 Biodistribution of Ac-labeled anti-HER2 (15A) and anti-DLL3 (15B) VHH-Fc constructs.

[0207] Figure 16A , 16B and 16C display 225 Results of toxicity studies on Ac-tagged anti-HER2 VHH-Fc constructs.

[0208] Figure 17 The load is displayed. 177 Immunoreactivity scores of different anti-DDL3 VHH-Fc constructs of Lu.

[0209] Figure 18 This paper shows the chemical structures of some of the chelating agents described in this paper.

[0210] Figure 19A and Figure 19B Shown in young mice using 111 Imaging experiments were conducted to measure the whole-body clearance of radioactivity using different conjugates (see Example B-26). Detailed Implementation

[0211] The invention is described more fully below using illustrative and non-limiting embodiments. However, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth below. Rather, these embodiments are provided so that this disclosure is thorough and conveys the scope of the invention to those skilled in the art. Certain terms are defined below to facilitate understanding of the invention. Additional definitions can be found in the detailed description of the invention.

[0212] Specifically, in embodiments, the present invention addresses many of the inherent challenges in the targeted delivery of radioisotopes in vivo through the selection and specific assembly of specific immunoconjugates and radioimmunoconjugate components. The radiodelivery platform of the present invention provides a shorter half-life compared to conventional IgG, but a longer half-life compared to smaller monomeric antibody fragments. In some embodiments, the molecular size of the subject radioisotope delivery platform is sufficiently large (e.g., 60 kDa to 110 kDa) to significantly reduce off-target toxicity, particularly kidney injury (e.g., from α or β emitted isotope cargo), and its size is sufficiently small to achieve increased tissue penetration compared to conventional IgG, while maintaining target specificity and increasing the probability of first decay time in the target tissue. In some embodiments, the subject radioisotope delivery platform can be used for the safe and efficient targeted delivery of radioisotopes (such as α or β emitters) in vivo, partly due to the reduction of certain adverse effects caused by platforms with half-lives exceeding 5 days and / or molecular weights below 60 kDa. In some embodiments, the subject radioisotope delivery platform is used for the safe and efficient targeted delivery of radioisotopes (such as alpha or beta emitters) in vivo, partly due to a reduced loss of targeting ability caused by radiodegradation compared to other possible delivery platforms. In some embodiments, the subject radioisotope delivery platform is used for the safe and efficient targeted delivery of radioisotopes (such as alpha or beta emitters) in vivo, partly due to increased manufacturing stability at temperatures required for certain radiolabeling processes (e.g., high-temperature chelation using certain chelating factors) compared to other possible delivery platforms using antibody fragments.

[0213] Immunoconjugates

[0214] 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 are characterized by K... D ≤1μM, <100nM, <10nM, <1nM, <0.1nM, <0.01nM, or <0.001nM (e.g., 10 μM) -8 M or smaller, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M) has the affinity to bind to specific antigens and / or epitopes.

[0215] The immunoconjugates described herein can serve as platforms for the delivery of radioisotopes. This document provides radioisotope delivery platforms with relatively short half-lives (e.g., less than one or two weeks, but more than two to eight hours).

[0216] In one embodiment, the immunoconjugate of this 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 another embodiment, the immunoconjugate of this 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 molecular weight of the immunoconjugate is between 60 kDa and 110 kDa.

[0217] In one embodiment, the immunoconjugate of this 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 another embodiment, the immunoconjugate of this 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 molecular weight of the immunoconjugate is between 60 kDa and 110 kDa.

[0218] In one embodiment, the immunoconjugate of this disclosure comprises: a) a VHH antigen-binding region; b) an immunoglobulin Fc region, collectively referred to as aVHH-Fc; and c) a chelating moiety or a radionuclide complex thereof. In another embodiment, the immunoconjugate of this 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 molecular weight of the immunoconjugate is between 60 kDa and 110 kDa.

[0219] In one embodiment, the immunoconjugate of this 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 another embodiment, the immunoconjugate of this 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 molecular weight of the immunoconjugate is between 60 kDa and 110 kDa. In some embodiments, the variant immunoglobulin Fc region comprises one or more amino acid alterations that shorten the serum or plasma half-life of the immunoconjugate.

[0220] In some embodiments, the size of the radioisotope delivery platform is greater than about 60 kDa to avoid certain toxicities from alpha-emitted isotope cargo, such as, for example, off-target nephrotoxicity. In some embodiments, the size of the radioisotope delivery platform is less than about 110 kDa to improve tumor penetration. In some embodiments, the size of the radioisotope delivery platform is between 60 kDa and 110 kDa due to its dimer structure having two separate antigen-binding arms, each having a VHH polypeptide fused to a hinge region and 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 shorten the half-life and / or eliminate Fc effector function.

[0221] In one embodiment, the antibody construct of the immunoconjugate comprises two antigen-binding arms covalently linked to each other (e.g., via disulfide bonds between associated heavy chain constant regions or immunoglobulin hinge regions). Each of the antigen-binding arms independently comprises 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 connected to the hinge region of the arm, and the hinge region of the arm is covalently connected 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 within the antigen-binding arm, thereby connecting them.

[0222] In a preferred embodiment, at least one of the two antigen-binding regions of the immunoconjugate is composed of one or two heavy-chain only variable (VHH) polypeptides. In a preferred embodiment, at least one of the two antigen-binding regions is composed of one VHH polypeptide. In a preferred embodiment, each of the two antigen-binding regions of the immunoconjugate is composed of one VHH polypeptide, which may be the same or different.

[0223] In one embodiment, the antigen-binding region of the immunoconjugate binds to the same antigen. In one embodiment, the antigen-binding region of the immunoconjugate binds to different antigens. In one embodiment, the antigen-binding regions of the immunoconjugate are identical. 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.

[0224] In one embodiment, the antigen-binding region of one antigen-binding arm consists of two VHH peptides, and the antigen-binding region of the other antigen-binding arm does not contain a VHH peptide. In one embodiment, the two antigen-binding arms bind the same antigen. In one embodiment, the two antigen-binding arms bind different antigens. In one embodiment, the two VHH peptides are identical. In one embodiment, the two VHH peptides are different. In one embodiment, the immunoconjugate is bispecific.

[0225] In one embodiment, the antigen-binding region of one antigen-binding arm consists of one VHH polypeptide, and the antigen-binding region of another antigen-binding arm consists of two VHH polypeptides. In one embodiment, the two antigen-binding arms bind the same antigen. In one embodiment, the two antigen-binding arms bind different antigens. In one embodiment, all three VHH polypeptides are identical. In one embodiment, two of the three VHH polypeptides are identical and different from the third VHH polypeptide. In one embodiment, the three VHH polypeptides are different. In one embodiment, the immunoconjugate is bispecific.

[0226] In one embodiment, the antigen-binding region of each antigen-binding arm of the immunoconjugate consists of a VHH polypeptide. In one embodiment, the VHH polypeptide binds to the same antigen. In one embodiment, the VHH polypeptide binds to different antigens. In one embodiment, the VHH polypeptide is the same. In one embodiment, the VHH polypeptide is different. In one embodiment, the immunoconjugate is bispecific.

[0227] antigen-binding region

[0228] The antigen-binding region imparts specificity to the immunoconjugate and may suitably contain small antigen-binding peptides. Such small antigen-binding peptides offer advantages such as reducing the overall size of the immunoconjugate molecule to achieve tumor penetration and labeling. Small antigen-binding peptides may lack certain regions that are dispensable for binding, such as light chain constant regions, heavy chain constant regions, CH1 regions, or hinge regions. In some embodiments, the antigen-binding region may lack light chain variable regions. In some embodiments, the molecular weight of the small antigen-binding region may be between 10 kDa and 40 kDa.

[0229] In some implementations, the molecular weight of the small antigen-binding region is from about 10 kDa to about 40 kDa. In some embodiments, the molecular weight of the small antigen-binding region is 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, 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 molecular weight of the small antigen-binding region is 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 molecular weight of the small antigen-binding region is at least about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, or about 35 kDa. In some embodiments, the molecular weight of the small antigen-binding region is at most about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, or about 40 kDa.

[0230] The antigen-binding region may contain a VHH peptide, an scFv peptide, or a VNAR peptide. In some embodiments, the antigen-binding region contains a VHH peptide. In some embodiments, the antigen-binding region contains an scFv peptide. In some embodiments, the antigen-binding region contains a VNAR peptide. In some embodiments, the antigen-binding region is humanized.

[0231] The antigen region may contain specificity for an antigen selected by a technician to achieve a desired function, such as targeting a specific cancer, tumor, or cell type suitable for treatment with the said immunoconjugate or radioimmunoconjugate. As described herein, the antigen-binding region may be a fragment or form of antibody known in the art. Complete antibodies may be engineered to conform to the various small antigen-binding region forms described herein (e.g., scFv). The antigen-binding region may specifically bind to tumor antigens (e.g., antigens specifically expressed or enriched in cancer cells). In some implementations, tumor antigens include 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, and Glypican. 3. Trail receptor 2 (DR5), PD-L1, mesothelin, bombesin, EpCAM, DARPP, CSPG4, galactagogue-3, integrin αvβ1, integrin αvβ3, integrin αvβ5, integrin αvβ6, integrin α5β1, integrin α-3, integrin α-5, integrin β-6, Nectin-4, Wnt-activated inhibitory factor 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 some embodiments, the tumor antigen includes human epidermal growth factor receptor 2 (HER2), delta-like ligand 3 (DLL3), folate receptor α (FOLR1), or Wnt-activated inhibitory factor 1 (WAIF1). In some embodiments, the tumor antigen includes HER2. In some embodiments, the tumor antigen includes DLL3. In some embodiments, the tumor antigen includes FOLR1. In some embodiments, the tumor antigen includes WAIF1. In some embodiments, the tumor antigen includes TROP2. In some embodiments, the tumor antigen includes EGFR. In some embodiments, the tumor antigen includes PSA.In some embodiments, the tumor antigen includes MUC-1. In some embodiments, the tumor antigen includes CEA. In some embodiments, the tumor antigen includes NY-ESO-1.

[0232] In some embodiments, the antigen-binding region of the immunoconjugate comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:20 and binding to HER2.

[0233] In some embodiments, the antigen-binding region of the immunoconjugate comprises: a) CDR1, which comprises the amino acid sequence shown in SEQ ID NO:21; b) CDR2, which comprises the amino acid sequence shown in SEQ ID NO:22; and c) CDR3, which comprises the amino acid sequence shown in SEQ ID NO:23.

[0234] In some embodiments, the antigen-binding region of the immunoconjugate comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:30 and binding to DLL3.

[0235] In some embodiments, the antigen-binding region of the immunoconjugate comprises: a) CDR1, which comprises the amino acid sequence shown in SEQ ID NO:31; b) CDR2, which comprises the amino acid sequence shown in SEQ ID NO:32; and c) CDR3, which comprises the amino acid sequence shown in SEQ ID NO:33.

[0236] In some embodiments, the immunoconjugates of the present invention comprise synthetically engineered antibody derivatives, such as, for example, those comprising autonomous V H Proteins or polypeptides with structural domains (such as, for example, those derived from camels, mice, or humans); single-domain antibody domains (sdAbs); heavy-chain antibody domains derived from camels (V... H H fragment or V H (structural domain fragment); derived from camel family V H H fragment or V H Heavy chain antibody domain of a domain fragment; heavy chain antibody domain derived from cartilaginous fish; immunoglobulin neoantigen receptor (IgNAR); V NAR Fragments; single-chain variable (scFv) fragments; nanobodies; containing V H The "camelized" or "camelised" scaffold of the structural domain; composed of heavy chains and C H Fd fragments composed of 1 structural domain; single-chain Fv-C H3. Microantibodies; Fc antigen-binding domain (Fcab); scFv-Fc fusion compounds; polymerized scFv fragments (bi-antibody, tri-antibody, tetra-antibody); disulfide-bonded stabilized antibody variable (Fv) fragments (dsFv); composed of V L V H C L and C H 1. Disulfide-bonded antigen-binding (Fab) fragments composed of disulfide-bonded domains; scFv (sc-dsFv) containing disulfide-bonded heavy and light chains; bivalent nanobodies; bivalent microbodies; bivalent F(ab')2 fragments (Fab dimers); bispecific tandem V H H fragment; bispecific tandem scFv fragment; bispecific nanobody; bispecific microbody; and any gene manipulation pair that retains the complementary site and target antigen binding function mentioned above.

[0237] In some embodiments, the immunoconjugate is monovalent. In other embodiments, the immunoconjugate is multivalent, such as, for example, divalent. In some further embodiments, the immunoconjugate is divalent and dimer. In some still embodiments, the divalent immunoconjugate is homodimer.

[0238] In one aspect, the present invention provides an antibody construct (alone or in the context of the present invention’s immunoconjugates, radioimmunoconjugates, or targeted imaging complexes) comprising a VHH fragment containing a heavy chain variable region comprising three heavy chain CDRs derived from camels that bind antigens with specificity and high affinity.

[0239] In some embodiments, the antibody construct, immune conjugate, radioimmunoconjugate, or targeted imaging complex specifically binds to at least one extracellular portion of an antigen expressed on a cell surface. In some embodiments, the immune conjugate specifically binds to at least one extracellular portion of an antigen expressed by a target cell (such as, for example, a tumor cell).

[0240] In some embodiments, this disclosure provides immunoconjugates that specifically bind to antigens. In some embodiments, the immunoconjugate comprises an antibody construct containing a heavy chain variable region (HVR-H) comprising three CDRs: hCDR1, hCDR2, and hCDR3, such as, for example, those derived from camel antibodies or IgNARs. 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.

[0241] In some embodiments, the immunoconjugates of the present invention comprise an antibody construct containing an antigen-binding domain, which is an antibody fragment, including but not limited to, for example, Fv, Fab, Fab', scFv, HcAb fragment, VHH fragment, sdAb fragment, biantibody, or F(ab')2 fragment. In some other embodiments, the immunoconjugates of the present invention comprise polymers of two or more antibody fragments, such as, for example, homodimers or heterodimers comprising two antibody fragments, each antibody fragment capable of binding an antigen with specificity and high affinity, and each antibody fragment comprising a heavy chain variable region (HVR-H) containing three CDRs: hCDR1, hCDR2, and hCDR3.

[0242] Heavy chain constant region

[0243] The antigen-binding region of the immunoconjugates described herein may include an Fc or heavy chain constant region. Antigen-binding molecules can be coupled directly to the Fc or heavy chain constant region, either through a suitable linker or via an IgG hinge region. Including a heavy chain constant region or Fc region provides advantages such as allowing for optimization and modulation of serum half-life, the addition of sites for additional conjugating chelators or cytotoxic agents, and the use of standard procedures and methods to purify the immunoconjugates. The addition of a heavy chain constant region also increases the size, thereby shifting the catabolic metabolism and elimination of the immunoconjugate from the kidney to the liver. This can provide safety advantages, particularly for radioimmunoconjugates, as the kidney is more sensitive to radiation than the liver. Residues present in the heavy chain constant region responsible for binding to the neonatal Fc receptor (FcRn) can be altered to affect effector function or serum half-life. Binding to FcRn generally contributes to an increase in the half-life of molecules containing immunoglobulin Fc; therefore, reducing binding to FcRn can shorten the half-life of molecules containing Fc. Reduction in FcRn binding can impart advantages such as a shorter half-life of the immunoconjugate and thus reduced toxicity subsequently attributed to cytotoxic agents or radioisotopes. In some embodiments, the immunoglobulin constant region comprises or is composed of an Fc region. In some embodiments, the immunoglobulin heavy chain constant region comprises the CH2 domain, the CH3 domain, or both CH2 and CH3 domains of an immunoglobulin. In some embodiments, the immunoglobulin heavy chain constant region comprises both CH2 and CH3 domains of an immunoglobulin. For treatment or imaging in a human individual, the immunoglobulin heavy chain constant region can be human, thereby preventing or reducing the endogenous immune response to the immunoconjugate. In some embodiments, the immunoglobulin heavy chain constant region is a human immunoglobulin heavy chain constant region. In some embodiments, the immunoglobulin heavy chain constant region is an IgA, IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the immunoglobulin heavy chain constant region is an IgG1 isotype. In some embodiments, the immunoglobulin heavy chain constant region is an IgG4 isotype.

[0244] The immunoglobulin heavy chain constant region can be a variant constant region, comprising one or more amino acid residue alterations that confer additional utility and beneficial properties to the immunoconjugates described herein. In some embodiments, the immunoglobulin heavy chain constant region comprises alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region or alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In some embodiments, the immunoglobulin heavy chain constant region comprises alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region or reduce the binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In some embodiments, the immunoglobulin heavy chain constant region comprises alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region and reduce the binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In some embodiments, the immunoglobulin heavy chain constant region comprises alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region. In some embodiments, the immunoglobulin heavy chain constant region includes alterations to one or more amino acid residues that reduce the binding of the immunoconjugate to the neonatal Fc receptor (FcRn).

[0245] Alterations to the heavy chain constant region of immunoglobulin conjugates 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 some embodiments, alterations to one or more amino acid residues that reduce effector function of the immunoglobulin heavy chain constant region are alterations that reduce complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or combinations thereof. In some embodiments, alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region are selected from a list of the following compositions according to EU numbers: (a) 297A, 297Q, 297G, or 297D; (b) 279F, 279K, or 279L; (c) 228P; (d) 235A, 235E, 235G, 235Q, 235R, or 235S; (e) 237A, 237E, 237K, 237N, or 237R; (f) 234A, 234V, or 234F; (g) 233P; (h) 328A (i) 327Q or 327T; (j) 329A, 329G, 329Y or 329R; (k) 331S, (l) 236F or 236R; (m) 238A, 238E, 238G, 238H, 238I, 238V, 238W or 238Y; (n) 248A; (o) 254D, 254E, 254G, 254H, 254I, 254N, 254P, 254Q, 254T or 254V; (p) 255N; (q) 256H, 256K, 256R or 256V; (r) 264S; (s) 265H, 265K, 265S, 265Y, or 265A; (t) 267G, 267H, 267I, or 267K; (u) 268K, (v) 269N, or 269Q; (w) 270A, 270G, 270M, or 270N; (x) 271T, (y) 272N; (z) 292E, 292F, 292G, or 292I; (aa) 293S; (bb) 301W; (cc) 304E; (dd) 311E, 311G, or 311S; (ee) 316F; (ff) 328V; (gg) 330R (hh) 339E or 339L; (ii) 343I or 343V; (jj) 373A, 373G or 373S; (kk) 376E, 376W or 376Y; (ll) 380D; (mm) 382D or 382P; (nn) 385P; (oo) 424H, 424M or 424V; (pp) 434I; (qq) 438G; (rr) 439E, 439H or 439Q; (ss) 440A, 440D, 440E, 440F, 440M, 440T or 440V; (tt) K322A;(uu)L235E; (vv)L234A and L235A; (ww)L234A, L235A and G237A; (xx)L234A, L235A and P329G; (yy)L234F, L235E and P331S; (zz)L234A, L235E and G237A; (aaa)L234A, L235E, G237A and P331S; (bbb)L234A, L235A, G237A, P238S, H268A, A330S and P331S; (ccc)L234A, L235A and P329A; (ddd)G236R and L328R; (eee)G237A; (fff)F241A; (ggg)V264A; (hhh)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 any combination of (ppp)(a)-(ooo). In some implementations, alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region include those designated L234A, L235E, G237A, A330S, and P331S according to EU designations.

[0246] Alterations to the heavy chain constant region of an immunoconjugate can shorten its serum half-life. In some embodiments, alterations to the amino acid composition that change or reduce the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) shorten the serum half-life of the immunoconjugate. In some embodiments, alterations to the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) target amino acid residues selected from the list of the following compositions according to EU numbers: 251, 252, 253, 254, 255, 288, 309, 310, 312, 385, 386, 388, 400, 415, 433, 435, 436, 439, 447, and combinations thereof. In some embodiments, alterations to the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) target amino acid residues selected from the list of the following compositions according to EU numbers: 253, 254, 310, 435, 436, and combinations thereof. In some embodiments, the alteration or reduction of the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is directed against amino acid residues selected from the list of the following compositions according to EU numbers: I253A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof. In some embodiments, the alteration or reduction of the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is directed against amino acid residues selected from the list of the following compositions according to EU numbers: I253A, S254A, H310A, H435Q, Y436A, and combinations thereof. In some embodiments, the alteration or reduction of the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is directed against amino acid residues selected from the list of the following compositions according to EU numbers: I253A, H310A, H435Q, and combinations thereof. In some implementations, the alteration or reduction of the binding of the immune conjugate to the neonatal Fc receptor (FcRn) is directed against amino acid residues selected from the list consisting of H310A, H435Q, and combinations thereof (according to EU designations).

[0247] In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:1. In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence identical to that in SEQ ID NO:1. In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:1, wherein the heavy chain constant region comprises an I253A substitution (according to EU designation).

[0248] In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:2. In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence identical to that in SEQ ID NO:2. In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:2, wherein the heavy chain constant region comprises an S254A substitution (according to EU designation).

[0249] In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:3. In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence identical to that in SEQ ID NO:3. In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:3, wherein the heavy chain constant region comprises an H310A substitution (according to EU designation).

[0250] In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:4. In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence identical to that in SEQ ID NO:4. In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:4, wherein the heavy chain constant region comprises an H435Q substitution (according to EU designation).

[0251] In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:5. In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence identical to that in SEQ ID NO:5. In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:5, wherein the heavy chain constant region comprises a Y436A substitution (according to EU designation).

[0252] In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:6. In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence identical to that in SEQ ID NO:6. In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:6, wherein the heavy chain constant region comprises H310A / H435Q substitution (according to EU designation).

[0253] In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:7. In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence identical to that in SEQ ID NO:7. In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:7, wherein the heavy chain constant region comprises L234A, L235E, G237A, A330S, and P331S substitutions (according to EU designations).

[0254] In some embodiments, the heavy chain constant region of the immunoconjugate contains a sequence having at least 90%, 95%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:8. In some embodiments, the heavy chain constant region of the immunoconjugate contains the same sequence as SEQ ID NO:8, wherein the heavy chain constant region contains L234A, L235E, G237A, H310A, A330S, and P331S substitutions (according to EU designations).

[0255] In some embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:9. In some embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:9. In some embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:9, wherein the heavy chain constant region comprises L234A, L235E, G237A, H435Q, A330S, and P331S substitutions (according to EU designations).

[0256] In some embodiments, the heavy chain constant region of the immunoconjugate contains a sequence having at least 90%, 95%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:10. In some embodiments, the heavy chain constant region of the immunoconjugate contains the same sequence as in SEQ ID NO:10 (according to EU numbering).

[0257] In one embodiment, each of the two variant constant regions has at least one FcRn binding mutation. In another embodiment, each of the two variant constant regions has the same FcRn binding mutation. In yet another embodiment, each of the two variant constant regions has a different FcRn binding mutation.

[0258] In one embodiment, at least one of the variant constant regions of 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, which may be the same or different.

[0259] Alterations affecting FcRn binding can shorten the serum half-life of the immunoconjugate, thus allowing technicians to select a half-life suitable for a specific imaging or therapeutic target. In some embodiments, the serum half-life of the immunoconjugate is from about 12 hours to about 120 hours. In some embodiments, the serum half-life of the immunoconjugate is 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 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 some embodiments, the serum half-life of the immunoconjugate is 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 some embodiments, the serum half-life of the immunoconjugate is 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 some embodiments, the serum half-life of the immunoconjugate is at most about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours, or about 120 hours.

[0260] In some embodiments, the serum half-life of the immunoconjugate is about 1 day to about 10 days. In some embodiments, the serum half-life of the immunoconjugate is 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... Approximately 8 days, approximately 3 to approximately 9 days, approximately 3 to approximately 10 days, approximately 4 to approximately 5 days, approximately 4 to approximately 6 days, approximately 4 to approximately 7 days, approximately 4 to approximately 8 days, approximately 4 to approximately 9 days, approximately 4 to approximately 10 days, approximately 5 to approximately 6 days, approximately 5 to approximately 7 days, approximately 5 to approximately 8 days, approximately 5 to approximately 9 days, approximately 5 to approximately 10 days, approximately 6 to approximately 7 days, approximately 6 to approximately 8 days, approximately 6 to approximately 9 days, approximately 6 to approximately 10 days, approximately 7 to approximately 8 days, approximately 7 to approximately 9 days, approximately 7 to approximately 10 days, approximately 8 to approximately 9 days, approximately 8 to approximately 10 days, or approximately 9 to approximately 10 days. In some embodiments, the serum half-life of the immunoconjugate is approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, or approximately 10 days. In some embodiments, the serum half-life of the immunoconjugate is 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 some embodiments, the serum half-life of the immunoconjugate is 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.

[0261] In some embodiments, the molecular weight of the heavy chain constant region is about 10 kDa to about 25 kDa. In some embodiments, the molecular weight of the heavy chain constant region is about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, about 15 kDa to about 20 kDa, about 15 kDa to about 25 kDa, or about 20 kDa to about 25 kDa. In some embodiments, the molecular weight of the heavy chain constant region is about 10 kDa, about 15 kDa, about 20 kDa, or about 25 kDa. In some embodiments, the molecular weight of the heavy chain constant region is at least about 10 kDa, about 15 kDa, or about 20 kDa. In some embodiments, the molecular weight of the heavy chain constant region is at most about 15 kDa, about 20 kDa, or about 25 kDa.

[0262] In some embodiments, the immunoconjugates of the present invention comprise a linker or hinge region, which is a polypeptide that links an antigen-binding region to a heavy chain constant region or variant constant region of the present invention. Naturally occurring and synthetic hinge regions linking immunoglobulin components are well known in the art and can be used in the present invention. See, for example, US 8,067,548 and its references.

[0263] In one embodiment, the hinge regions of the immunoconjugates are identical. In another embodiment, the hinge regions of the immunoconjugates are different.

[0264] The antigen-binding region and the heavy chain constant region (with or without amino acid sequence alteration) can be linked by a suitable hinge or linker sequence. In some embodiments, the antigen-binding region is coupled to the immunoglobulin heavy chain constant region via a linker amino acid sequence or a human IgG hinge region. A suitable IgG hinge region comprises and includes an IgG1 or IgG4 hinge region. In some embodiments, the hinge region is an IgG1 hinge region. In some embodiments, the hinge region is an IgG1 hinge region with a C220S substitution (according to EU designation). Suitable hinge regions include those described below: 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, Vol. 14, No. 12, December 2001, pp. 1025-1033; Shu et al., “Secretion of a single-gene-encoded immunoglobulin from myeloma cells.” Proceedings of the National Academy of Sciences, Sep 1993, 90(17): 7995-7999; Davis et al., “Abatacept binds to the Fcreceptor CD64 but does not mediate complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity.” J Rheumatol., Nov 2007; 34(11): 2204-10. Suitable hinges may also include non-IgG-based peptide linkers. The linker amino acid sequence may primarily contain the following amino acid residues: Gly, Ser, Ala, or Thr. The length of the linker peptide should be sufficient to allow the two molecules to conform correctly relative to each other and thus retain the desired active manner of linking the two molecules. In one embodiment, the linker is about 1 to 50 amino acids long or about 1 to 30 amino acids long. In one embodiment, a linker of 1 to 20 amino acids in length can be used. Useful linkers include glycine-serine polymers (including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.For example, the adapter for linking antibody fragments or single-chain variable fragments may include AAEPKSS, AAEPKSSDKTHTCPPCP, GGGG, or GGGGDKTHTCPPCP. Alternatively, a variety of non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyethylene, or copolymers of polyethylene glycol and polypropylene glycol, may be used as adapters.

[0265] The overall size of the immunoconjugate can facilitate tissue penetration, stability, and / or clearance. In some embodiments, the molecular weight of the immunoconjugate is from about 60 kDa to about 120 kDa. In some embodiments, the molecular weight of the immunoconjugate is about 60 kDa to about 65 kDa, about 60 kDa to about 70 kDa, about 60 kDa to about 75 kDa, about 60 kDa to about 80 kDa, about 60 kDa to about 90 kDa, about 60 kDa to about 100 kDa, about 60 kDa to about 110 kDa, about 60 kDa to about 120 kDa, about 65 kDa to about 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. 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 100kDa, approximately 80kDa to approximately 110kDa, approximately 80kDa to approximately 120kDa, approximately 90kDa to approximately 100kDa, approximately 90kDa to approximately 110kDa, approximately 90kDa to approximately 120kDa, approximately 100kDa to approximately 110kDa, approximately 100kDa to approximately 120kDa, or approximately 110kDa to approximately 120kDa. In some embodiments, the molecular weight of the immunoconjugate is about 60 kDa, about 65 kDa, about 70 kDa, about 75 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 110 kDa, or about 120 kDa. In some embodiments, the molecular weight of the immunoconjugate is 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. In some embodiments, the molecular weight of the immunoconjugate is 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.

[0266] In some embodiments, the molecular weight of the immunoconjugate is greater than 60 kDa, 70 kDa, 75 kDa, 80 kDa, 82 kDa, 83 kDa, 85 kDa, 86 kDa, 87 kDa, 88 kDa, or 89 kDa. In some embodiments, the molecular weight of the immunoconjugate is less than 110 kDa, 100 kDa, 95 kDa, 93 kDa, 91 kDa, 90 kDa, 89 kDa, 88 kDa, 87 kDa, 86 kDa, 85 kDa, 84 kDa, 83 kDa, 82 kDa, 81 kDa, or 80 kDa. In some embodiments, the molecular weight of the immunoconjugate is greater than 60 kDa, 65 kDa, 70 kDa, 71 kDa, 72 kDa, 73 kDa, 74 kDa, 75 kDa, 76 kDa, 77 kDa, 78 kDa, or 79 kDa and less than 110 kDa, 100 kDa, 95 kDa, 93 kDa, 91 kDa, or 90 kDa.

[0267] The size of the immunoconjugates and / or heavy chain constant region variants described herein increases the safety or therapeutic index of the immunoconjugates included herein. This safety can be reflected in reduced radiation accumulation in radiosensitive primary tissues (such as the kidneys and bone marrow) and / or increased radiation accumulation in target tissues (i.e., tumors or cancerous tissues) or more radiotolerant organs (such as the liver).

[0268] In some embodiments, the immunoconjugates of this disclosure result in total radiation exposure per treatment, as measured in glycerols (Gy). In some embodiments, the kidney is exposed to 20 Gy or less per treatment. In some embodiments, the kidney is exposed to 19 Gy or less per treatment. In some embodiments, the kidney is exposed to 18 Gy or less per treatment. In some embodiments, the kidney is exposed to 17 Gy or less per treatment. In some embodiments, the kidney is exposed to 16 Gy or less per treatment. In some embodiments, the kidney is exposed to 15 Gy or less per treatment. In some embodiments, the kidney is exposed to 14 Gy or less per treatment. In some embodiments, the kidney is exposed to 13 Gy or less per treatment. In some embodiments, the kidney is exposed to 12 Gy or less per treatment. In some embodiments, the kidney is exposed to 11 Gy or less per treatment. In some embodiments, the kidney is exposed to 10 Gy or less per treatment. In some embodiments, the kidney is exposed to 9 Gy or less per treatment. In some implementations, the kidney is exposed to 8 Gy or less per treatment. In some implementations, the kidney is exposed to 5 Gy or less per treatment.

[0269] In some embodiments, the immunoconjugates of this disclosure result in total radiation exposure per treatment, as measured in glycerols (Gy). In some embodiments, bone marrow exposure is 4 Gy or less per treatment. In some embodiments, bone marrow exposure is 3 Gy or less per treatment. In some embodiments, bone marrow exposure is 2 Gy or less per treatment. In some embodiments, bone marrow exposure is 1.5 Gy or less per treatment. In some embodiments, bone marrow exposure is 1.0 Gy or less per treatment. In some embodiments, bone marrow exposure is 0.5 Gy or less per treatment.

[0270] In some embodiments, when measured as a percentage of the injected dose per gram, the immunoconjugates of this disclosure result in an increased radiation dose in the tumor compared to the kidney. In some embodiments, the ratio of the percentage of the injected dose per gram in the tumor to the percentage of the injected dose per gram in the kidney is greater than 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0271] In some embodiments, when measured as a percentage of the injected dose per gram, the immunoconjugates of this disclosure result in an increased radiation dose in the tumor compared to the blood. In some embodiments, the ratio of the percentage of the injected dose per gram in the tumor to the percentage of the injected dose per gram in the blood is greater than 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0272] In some embodiments, when measured as a percentage of the injected dose per gram, the immunoconjugates of this disclosure result in an increased radiation dose in the tumor compared to the bone marrow. In some embodiments, the ratio of the percentage of the injected dose per gram in the tumor to the percentage of the injected dose per gram in the bone marrow is greater than 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0273] In some embodiments, when measured per gram of injected dose, the immunoconjugates of this disclosure result in an increased radiation dose in the liver compared to the kidney. In some embodiments, the ratio of the percentage of injected dose per gram of tumor to the percentage of injected dose per gram of bone marrow is greater than 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0274] In some embodiments, the invention contemplates variants of the immunoconjugates of the invention comprising the Fc region, wherein the variant possesses some, but not all, effector functions, making it a desirable candidate for applications where the half-life of the immunoconjugate in vivo is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the immunoconjugate lacks FcγγR binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. The primary cells used to mediate ADCC, NK cells, express only FcγγRIII, while monocytes express FcγγRI, FcγγRII, and FcγRIII. Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991) summarizes FcR expression on hematopoietic cells. Non-limiting examples of in vitro assays for assessing ADCC activity of molecules of interest are described in US 5,500,362 (see, for example, Hellstrom, I. et al., Proc Natl Acad Sci USA 83:7059-7063 (1986)) and Hellstrom 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 assays may be used (see, for example, ACTI for flow cytometry). TM Non-radioactive cytotoxicity assay (Cell Technology, Inc., Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assays (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, such as in animal models, as disclosed in Clynes et al., ProcNatl Acad Sci USA 95:652-656 (1998). Clq binding assays can also be performed to confirm that the immunoconjugates do not bind Clq and therefore lack CDC activity (see, for example, Clq and C3c binding ELISAs in WO2006 / 029879 and WO 2005 / 100402). To assess complement activation, CDC assays can be performed (see, for example, 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)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0275] Immunoconjugates with reduced effector function include those with one or more substituted residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (US 6,737,056). Such Fc mutants include those with substitutions at two or more of the amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant (US 7,332,581) which substitutes residues 265 and 297 for alanine.

[0276] Immunoconjugates can have altered effector functions by incorporating the following modifications according to EU designations: L234A, L235E, G237A, A330S, and P331S, which reduce Fc receptor binding. See, for example, 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 July; 177(1):219-33.

[0277] Certain immunoconjugate variants that enhance or weaken binding to FcR are described (see, for example, US 6,737,056; WO 2004 / 056312; Shields et al., J. Biol. Chem. 9(2):6591-6604(2001)).

[0278] In some implementations, the alteration is performed in the Fc region, resulting in changes in Clq binding and / or complement-dependent cytotoxicity (CDC) (i.e., enhancement or reduction), as described, for example, in US6,194,551; WO 1999 / 051642; Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0279] US2005 / 0014934 describes antibodies with prolonged half-life and enhanced binding to the neonatal Fc receptor (FcRn) responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976); Kim et al., J. Immunol. 24:249 (1994)). These antibodies contain an Fc region having one or more substitutions that enhance the binding of the Fc region to the FcRn. Such Fc variants include those having substitutions at one or more of the Fc region residues 434 or 435, such as substitutions at Fc region residues N434A or R435A (US 7,371,826). See also Duncan and Winter, Nature 322:738-40 (1988); US 5,648,260; US 5,624,821; and WO 1994 / 029351, which relates to other instances of Fc region variants.

[0280] To increase the serum half-life of an antibody, a salvage receptor-binding epitope can be incorporated into the antibody (especially an antibody fragment), for example, as described in U.S. Patent 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.

[0281] 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 present invention, although only one or two such compositions (e.g., immunoconjugates) are mentioned herein as non-limiting examples. All such applications are covered by the present invention.

[0282] Chelating agents

[0283] As described herein, in some embodiments, the chelating agent (i.e., R) 1 The chelating factor is coupled to a tumor-targeting moiety (e.g., a polypeptide comprising the antigen-binding region and the immunoglobulin heavy chain constant region as described herein). The chelating moiety allows the tumor-targeting moiety to be loaded with an appropriate radioisotope, such as a beta-emitter or alpha-emitter. The chelating factor can be coupled to the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof. Such coupling can suitably be achieved by covalently attaching one or more amino acids to the immunoconjugate, the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof.

[0284] In one embodiment, the chelating agent of the immunoconjugate is covalently linked to the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof. In one embodiment, the chelating agent is directly covalently linked to the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof (e.g., without using a spacer, stretcher, or linker). In one embodiment, the chelating agent is covalently linked to the antigen-binding arm via a linker that is covalently linked to both the chelating agent and 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 olefin chain). Chelating factors can be linked or coupled to the immunoconjugate, as described in Sadiki, A. et al., “Site-specific conjugation of native antibody.” Antibody Therapeutics 2020, 3, 271-284.

[0285] In some embodiments, the immunoconjugate is formed by attaching a chelating factor-linker in a site-specific manner (directed to a specific amino acid or glycan residue). In some embodiments, site-specific conjugation involves the directed functionalization of a specific lysine residue in a framework region having a chelating factor-linker. In other embodiments, this residue may be functionalized with different reactive functional groups and then reacted with the chelating factor-linker in a second step to obtain the immunoconjugate. In some embodiments, this reactive functional group is a thiopropionate group.

[0286] In some embodiments, non-natural cysteine ​​residues are engineered into the antibody framework as sites for thiol-directed conjugation to obtain immunoconjugates. In some embodiments, other non-natural amino acids or amino acid sequences are engineered into the framework to serve as attachment sites for chelating factor-linkers or second reactive groups, which conjugate to the attachment sites to obtain immunoconjugates.

[0287] In some embodiments, non-natural amino acids containing cross-linking groups are engineered into the framework for attachment of chelating factors-connectors. In some embodiments, such non-natural amino acids contain azides.

[0288] In some embodiments, the chelating factor-linker is attached to glutamine residues via the action of transglutaminase. In other embodiments, a second reactive group is attached via transglutaminase, adding the chelating factor-linker to the transglutaminase to obtain an immunoconjugate.

[0289] In some embodiments, one or more N-glycans are modified with reactive functional groups by the action of glycosidases, and then chelating factor-connectors are conjugated to the sites to attach chelating factor-connectors. In some embodiments, the glycans are modified by the action of β-galactosidases. In some embodiments, the glycans are modified with glycosides containing azides for attaching appropriately functionalized chelating factor-connectors.

[0290] In one embodiment, the immunoconjugate contains more than one chelating agent, which may be the same or different.

[0291] In one embodiment, an immunoconjugate having more than one chelating agent has more than one chelating agent attached to the same antigen-binding arm.

[0292] In one embodiment, the immunoconjugate having more than one chelating agent and less than eleven chelating agents has more than two chelating agents, more than three chelating agents, more than four chelating agents, more than five chelating agents, more than six chelating agents, more than seven chelating agents, more than eight chelating agents, or more than nine chelating agents. In one embodiment, the chelating agents are identical. In one embodiment, each antigen-binding arm is directly or indirectly linked to more than one chelating agent.

[0293] In one embodiment, the chelating agent comprises a radioisotope chelating component and a functional group that allows covalent bonding with an antigen-binding arm. In one embodiment, the functional group is directly attached to the radioisotope chelating component. In one embodiment, the chelating agent further comprises a connector between the functional group and the radioisotope chelating component.

[0294] In one embodiment, the radioisotope chelating component comprises DOTA or a DOTA derivative. In one embodiment, the radioisotope chelating component comprises DOTAGA. In one embodiment, the radioisotope chelating component comprises macropa or a macropa derivative. In one embodiment, the radioisotope chelating component comprises Py4Pa or a Py4Pa derivative.

[0295] In a preferred embodiment, the chelating agent of the immunoconjugate does not attach to the antigen-binding region in the antigen-binding arm of the immunoconjugate.

[0296] In one embodiment, the chelating agent of the immunoconjugate is non-covalently associated with the antigen-binding arm. In a preferred embodiment, the chelating factor does not associate with the antigen-binding region in the antigen-binding arm of the immunoconjugate.

[0297] 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.

[0298] In some embodiments, the chelating agent is a radioisotope chelating agent. In some embodiments, the radioisotope chelating agent is selected from the list of the following: tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), α-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTAGA), or (Py4Pa). In some embodiments, the radioisotope chelating agent is DOTA. In some embodiments, the radioisotope chelating agent is DOTAGA. In some embodiments, the radioisotope chelating agent is Py4Pa. In some embodiments, the radioisotope, wherein the radioisotope chelating agent is directly coupled to the antigen-binding region and / or the immunoglobulin heavy chain constant region. In some embodiments, the radioisotope chelating agent is coupled to the antigen-binding region or the immunoglobulin heavy chain constant region via a linker. In some embodiments, the linker is selected from: 6-maleimide hexanoyl (MC), maleimide propionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and those obtained by conjugation with the following linker reagents: 4-(2-pyridylthio)valerate N-succinimide ester (SPP), 4-(N-maleimide methyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), 4-(2-pyridyldithio)butyrate N-succinimide ester (SPDB), (4-iodo-acetyl)aminobenzoic acid N-succinimide ester (SIAB), polyethylene glycol (PEG), polyethylene glycol polymer (PEGn), and S-2-(4-isothiocyanobenzyl) (SCN). In some embodiments, the linker is selected from polyethylene glycol (PEG), polyethylene glycol polymer (PEG), and S-2-(4-isothiocyanobenzyl) (SCN). In some embodiments, the linker is PEG5. In some embodiments, the linker is SCN. In some embodiments, the radioisotope chelating agent is a linker-chelating factor selected from the list of the following compositions: TFP-Ad-PEG5-DOTAGA, p-SCN-Bn-DOTA, p-SCN-Ph-Et-Py4Pa, and TFP-Ad-PEG5-Ac-Py4Pa.

[0299] In some embodiments, the chelating factor is conjugated to the chelating factor at a predetermined ratio of peptide (i.e., antigen-binding region and / or immunoglobulin heavy chain constant region). In some embodiments, a radioisotope chelating agent is conjugated to the antigen-binding region and / or immunoglobulin heavy chain constant region at a ratio of 1:1 to 8:1. In some embodiments, a radioisotope chelating agent is conjugated to the antigen-binding region and / or immunoglobulin heavy chain constant region at a ratio of 1:1 to 6:1. In some embodiments, a radioisotope chelating agent is conjugated to the antigen-binding region and / or immunoglobulin heavy chain constant region at a ratio of 2:1 to 6:1.

[0300] For example, using bifunctional chelating factors to conjugate radioisotopes to the radioisotope delivery platform of the present invention produces the immunoconjugates of the present invention. (See, for example, Scheinberg D, McDevitt M, Curr Radiopharm 4:306-20 (2011)). Examples of bifunctional chelating factors 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.

[0301] Examples of bifunctional chelating factors are 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), and related analogues. These chelating factors are suitable for coordinating metal ions, such as α- and β-emitting radionuclides.

[0302] In some embodiments, the chelating agent of the immunoconjugate or radioimmunoconjugate of the present invention is selected from the group comprising: bifunctional chelating factors, 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 (ThieleNA 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 (Wickstrom). K et al., Int. J. Rad. Onc. Biol. Phys. 105:410 (2019) (For reviews of these and other bifunctional chelating factors, see, for example, Price EW and Orvig C Chem. Soc. Rev., 2014, 43:260 (2014) and Brechbiel MW QJ Nucl. Med. Mol. Imaging 52:166 (2008)).

[0303] In some embodiments, the chelating agent of the immunoconjugates or radioimmunoconjugates of the present invention is selected from: bifunctional chelating factors, 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 (Wickstrom). K et al., Int. J. Rad. Onc. Biol. Phys. 105:410 (2019) (For reviews of these and other bifunctional chelating factors, see, for example, Price EW and Orvig C Chem. Soc. Rev., 2014, 43:260 (2014) and Brechbiel MWQ. J. Nucl. Med. Mol. Imaging 52:166 (2008)).

[0304] for 225 Ac immunoconjugates have a variety of acyclic and cyclic ligands known in the art as suitable chelating factors (see, for example, 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)).

[0305] In some implementations, the chelating factor is a chelating factor suitable for chelating α-emitters. Some chelating factors 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.

[0306] In some embodiments, the chelating factors suitable for alpha emitter chelation are selected from a list of the following compositions: 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-tetraazacyclodecadecane Dialkyl-1,4,7-triyl)triacetic acid; Py4Pa, 6,6',6”,6”'-(((pyridine-2,6-diylbis(methylene))bis(azanetriyl))tetra(methylene))tetrapicolinic acid; Py4Pa-NCS, 6,6'-((((4-isothiocyanopyridine-2,6-diyl)bis(methylene))bis((carboxymethyl)azanetriyl))bis(methylene))dipicolinic acid; Crown, 2,2',2”,2”'-(1,10-dioxa-4,7,13,16-tetraazacyclo-decane Octadecan-4,7,13,16-tetrayl)tetraacetic acid; Macropa, 6,6'-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))dipicolinic acid; Macropa-NCS, 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)-4-isothiocyanopicolinic 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-pyridyl)methyl]-9-hydroxy-3-methyl-2,4-di-2-pyridyl-1,5-dimethyl ester; Nonunpa, 6,6'-(((oxybis(ethane-2,1-diyl))bis((carboxymethyl)azanyl))bis(methylene))dipicolinic acid; and combinations thereof.

[0307] In some embodiments, the chelating factor is a chelating factor suitable for chelating β or γ emitters. In some embodiments, the chelating factor suitable for chelating β or γ emitters is selected from a list of the following compositions: 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-tetra(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane); DOTPA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid; DO3AM-acetic acid, (2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4 ,7,10-Tetraazacyclododecane-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-tetra(acetamidomethylenephosphonic acid); CB-TE2A, 1,4,8,11-Tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid; NOA, 1,4,7-triazacyclononane-1,4,7-triacetic acid; N OTP, 1,4,7-triazacyclononane-1,4,7-tris(methylenephosphonic acid); TETPA, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid; TETA, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid; PEPA, 1,4,7,10,13-pentaazacyclopentadecane-N,N',N”,N”',N””-pentaacetic acid; H4Octapa, N,N'-bis(6-carboxy-2-pyridinylmethyl)-ethylenediamine-N,N'-diacetic acid; H2Dedpa, 1,2-[[6-(carboxy)-pyridin-2-yl] [-methylamino]ethane; H6phospa, N,N'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane; TTHA, triethylenetetramine-N,N,N',N”,N”',N”'-hexaacetic acid; DO2P, tetraazacyclododecanedimethylphosphonic acid; HP-DO3A, hydroxypropyltetraazacyclododecanetriacetic acid; EDTA, ethylenediaminetetraacetic acid; DTPA, diethylenetriaminepentaacetic acid; DTPA-BMA, diethylenetriaminepentaacetic acid-bismethylamide; HOPO, octadentate hydroxypyridinones;3,2,3-LI(HOPO), N,N'-(butane-1,4-diyl)bis(1-hydroxy-N-(3-(1-hydroxy-6-oxo-1,6-dihydropyridine-2-carboxamido)propyl)-6-oxo-1,6-dihydropyridine-2-carboxamide); 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) Neunpa, 6,6'-(((N-alkyldiylbis(ethane-2,1-diyl))bis((carboxymethyl)azonyl))bis(methylene))dipicolinic acid; Neunpa-NCS, 6,6'-((((4-isothiocyanophenylethyl)azonyl)bis(ethane-2,1-diyl))bis((carboxymethyl)azonyl))bis(methylene))dipicolinic acid; Octa Pa, 6,6'-((ethane-1,2-diylbis((carboxymethyl)azanidinediyl))bis(methylene))dipicolinic acid; Octox, 2,2'-(ethane-1,2-diylbis(((8-hydroxyquinoline-2-yl)methyl)azanidinediyl))diacetic acid; PyPa, 6,6'-(((pyridin-2,6-diylbis(methylene))bis((carboxymethyl)azanidinediyl))bis(methylene))dipicolinic acid; Porphyrin, 21,22,23,24-tetraazapentacyclo[16.2.1.13,6.18,11.113,16] Twenty-four-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-pentazatetrasans-2,10,13,21,24-pentanone; 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.

[0308] In some implementation schemes, R 1 Contains a chelate moiety selected from the list of the following: DOTA, DO3A, DO3Apic, DOTAGA, DOTAGA anhydride, Py4Pa, Py4Pa-NCS, Crown, Macropa, Macropa-NCS, HEHA, CHXoctapa, Bispa, and Noneunpa; or a radionuclide complex thereof.

[0309] In some implementation schemes, R 1 Contains chelate moieties selected from the list of the following: DOTMA, DOTPA, DO3Apic, DO3AM-acetic acid, DOTP, DOTMP, DOTA-4AMP, CB-TE2A, NOA, 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 radionuclide complexes thereof.

[0310] In some implementation schemes, R 1 It is the chelate portion selected from the list consisting of the following:

[0311] 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA);

[0312] 1,4,7,10-Tetraazacyclododecane-1,4,7-triacetic acid (DO3A);

[0313] 1,4,7,10-Tetraazacyclododecane-1,7-diacetic acid (DO2A);

[0314] α,α',α”,α”'-Tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTMA);

[0315] 1,4,7,10-Tetra(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM);

[0316] 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrapropionic acid (DOTPA);

[0317] 2,2',2"-(10-(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid;

[0318] 10-((6-carboxypyridin-2-yl)methyl)-1,4,7,10-tetra-azacyclododecane-1,4,7-triacetic acid (DO3Apic);

[0319] 6,6'-(((pyridin-2,6-diylbis(methylene))bis((carboxymethyl)azanidinediyl))-bis(methylene))dipicolinic acid (H4pypa);

[0320] 6,6',6",6"'-(((pyridine-2,6-diylbis(methylene))bis(azanetriyl))tetra(methylene))-tetrapicolinic acid (H4py4pa); and

[0321] 3,6,9,12-Tetra(carboxymethyl)-3,6,9,12-tetraazatetradecanoic acid (TTHA);

[0322] Or its radionuclide complex.

[0323] In some implementation schemes, R 1 It contains a chelating portion having one of the following structures:

[0324]

[0325]

[0326]

[0327]

[0328]

[0329] In some implementation schemes, R 1 Includes DOTA and is attached to the tumor-targeting portion at the indicated location:

[0330]

[0331] In some implementation schemes, R 1 Includes DOTA and is attached to the tumor-targeting portion at the indicated location:

[0332]

[0333] In some implementation schemes, R 1 Includes DOTA and is attached to the tumor-targeting portion at the indicated location:

[0334]

[0335] It should be understood that the immunoconjugates described herein contain one or more R 1 Partial. In some embodiments, the immunoconjugates described herein comprise one or more R... 1 Part, where each R 1 Some parts are the same.

[0336] Radionuclides

[0337] In some embodiments, the radionuclide in the immunoconjugates described herein is an Auger electron-emitting radionuclide. In some embodiments, the radionuclide is an alpha-emitting radionuclide. In some embodiments, the radionuclide is a beta-emitting radionuclide. In some embodiments, the radionuclide is a gamma-emitting radionuclide. In some embodiments, the type of radionuclide used in non-peptide-targeted therapeutic compounds can be customized according to the specific type of cancer, the type of targeting portion (e.g., non-peptide ligands), etc. A radionuclide undergoing alpha decay emits an alpha particle (a helium ion with a +2 charge) from its nucleus. Due to alpha decay, the daughter radionuclide has 2 fewer protons and 2 fewer neutrons than the parent radionuclide. This means that in alpha decay, the number of protons decreases by 2, while the number of nucleons decreases by 4. A radionuclide undergoing beta decay emits a beta particle (an electron) from its nucleus. During beta decay, one of the neutrons becomes a proton and an electron. The proton remains in the nucleus, while the electron is emitted as a beta particle. This means that in beta decay, the nucleus loses a neutron but gains a proton. In gamma decay, atomic nuclei in an excited state (higher energy state) emit gamma-ray photons to transition to a lower energy state. The number of protons and nucleons remains unchanged during gamma decay. Gamma-ray emission is often accompanied by the emission of alpha and beta particles.

[0338] Auger electrons (AEs) are very low-energy electrons that decay from radioactive nuclides (e.g., electron capture electrons) through electron capture (EC). 111 In(Indium-1ll) 67 Ga(gallium-67), 99m Tc (Technetium-99m), 195m Pt (Platinum-195m) 125 I (Iodine-125) and 123 I(I-123) emission. This energy is deposited over a nanometer to micrometer distance, resulting in highly linear energy transfer, which can cause lethal damage to cancer cells. Therefore, AE-emission radiotherapy agents hold great potential for cancer treatment.

[0339] Beta particles are electrons emitted from the atomic nucleus. They typically have a relatively long range in tissues (approximately 1-5 mm) and are the most frequently used.

[0340] Alpha particles are helium nuclei (two protons and two neutrons) emitted from the nucleus of radioactive atoms. Depending on their emission energy, they can propagate 50-100 μm through tissues. They are positively charged and several orders of magnitude larger than electrons. The amount of energy deposited by an alpha particle per path length (terminated 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 electrons. Alpha particle trajectories lead to numerous complex and largely irreparable DNA double-strand breaks. The absorbed dose required to achieve cytotoxicity is related to the number of alpha particles that penetrate the cell nucleus. By this measure, cytotoxicity can be achieved with 1 to 20 alpha particles penetrating the cell nucleus. The resulting high efficiency, combined with the short range of alpha particles (which reduces toxicity to normal organs), has attracted considerable attention for the development of alpha particle emitters. Commonly 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 Th (Thorium-227).

[0341] In some implementations, the radionuclide is a diagnostic or therapeutic radionuclide.

[0342] Representative radionuclides

[0343] isotope <![CDATA[t 1 / 2 (h)]]> decay mode <![CDATA[ 66 Here]]> 9.5 β+ (56%), EC (44%) <![CDATA[ 67 Here]]> 78.2 EC (100%) <![CDATA[ 68 Here]]> 1.1 β+ (90%), EC (10%) <![CDATA[ 111 In]]> 67.2 EC (100%) <![CDATA[ lll InIn]]> 495d EC(100:o) <![CDATA[ 114 In(child body)]]> 73s β-(100％) <![CDATA[ 177 Monday]]> 159.4 β-(100％) <![CDATA[ 89 Zr]]> 78.5 β+ (23%), EC (77%) <![CDATA[ 112 With]]> 1.1 α(36％),β-(64％) <![CDATA[ 213 With]]> 0.76 α(2.2％),β-(97.8％) <![CDATA[ 212 Pb (daughter body is) 212 Bi)]]> 10.6 β-(100％) <![CDATA[ 225 And]]> 240 α(100％)

[0344] In some embodiments, the radionuclide is an Auger electron-emitting radionuclide. In some embodiments, the radionuclide is an Auger electron-emitting radionuclide, which is... 111 In、 67 Ga、 68 Ga、 99m Tc or 195m Pt. In some implementations, the radionuclide is an Auger electron-emitting radionuclide, which is 111 In、 67 Ga、 68 Ga or 99m Tc.

[0345] In some embodiments, the radionuclide is an alpha-emitting radionuclide. In some embodiments, the radionuclide is an alpha-emitting radionuclide, which is... 225 Ac、 213 Bi、 223 Ra or 212 Pb. In some embodiments, the radionuclide is an alpha-emitting radionuclide, which is 225Ac. In some embodiments, the radionuclide is a beta-emitting radionuclide. In some embodiments, the radionuclide is a beta-emitting radionuclide, which 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(Gold-198) 169 Er (Erbium-169) 165 Dy (Dysprosium-165), 99m Tc, 89 Zr or 52 Mn (Manganese-52). In some embodiments, the radionuclide is a beta-emitting radionuclide, which is... 90 Y、 177 Lu、 99m Tc or 89 Zr.

[0346] In some embodiments, the radionuclide is a gamma-emitting radionuclide. In some embodiments, the radionuclide is a gamma-emitting radionuclide, which is... 60 Co (Cobalt-60), 103 Pd(Palladium-103) 137 Cs(Cesium-137), ( 169 Yb)(Ytterbium-169), 192 Ir (Iridium-192) 212 Bi、 213 Bi or 226Ra.

[0347] connector

[0348] In some implementations, the tumor-targeting portion and the radionuclide chelating factor portion R are used. 1 The connector. In some embodiments, L is a hydrophobic connector. In some other embodiments, L is a hydrophilic connector. In some embodiments, the connector is flexible. In some embodiments, the connector is rigid. In some embodiments, the connector is linear. In other embodiments, the connector is branched. In some embodiments, the branched connector allows for the joining of at most one R. 1In some embodiments, the linker allows conjugation to cell-penetrating peptides (CPPs) to enhance cell penetration. 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 portions, one or more rigid portions, or a combination thereof.

[0349] The connector length can be determined based on the radionuclide chelating factor R. 1 The linear number of atoms between the linker and the tumor-targeting portion is considered, where the cyclic portion is calculated by using the shortest path around the ring. In some embodiments, the linear stretch of the linker is between 1-100 atoms, 1-50 atoms, 1-40 atoms, and 1-30 atoms; in other embodiments, it is between 1-20 atoms; in still other embodiments, it is between 1-15 atoms; in still other embodiments, it is between 1-10 atoms; and in still other embodiments, it is between 1-5 atoms. In some embodiments, the length of the linker is a range, with the lower limit selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and the upper limit selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, and 30. In some implementations, the length of the connector is a range, with the lower limit selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and the upper limit selected from 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0350] Factors to consider for the connector include its impact on the physical or pharmacokinetic properties of the resulting radioimmunoconjugate, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (to a greater or lesser degree of stability and planned degradation), rigidity, flexibility, immunogenicity, and regulation of antibody binding.

[0351] The optimal linker should connect the carrier mAb to the radioactive chelating factor without impairing the function of either, providing stable bonding during cycling and degradation under specific conditions. Cyclic stability has proven to be a particularly important factor for the success of this strategy.

[0352] In malignant tumors, the accessibility of tumor cells enables targeting by specific antibodies. Antigens expressed on tumor cells but not on normal healthy cells allow for selective targeting of tumor cells without harming non-tumor cells. Short-lived radiolabeled mAbs that emit short-range, high-linear-energy transfer particles represent an attractive approach to treating tumors. These short-range particles may be able to kill single cells without harming bystander cells. High-energy alpha emitters used in radioimmunotherapy include... 211 At(astatine-211)、 212 Bi、 213 Bihe 225 Ac. The latter isotope is an isotope generator in vivo because it has a long (10-day) half-life and decays via alpha emission through three short-lived atoms, each of which produces an alpha particle.

[0353] In some cases, tumor targeting 225 The limitation of the widespread use of Ac radioimmunotherapy is the excessive radiation exposure of normal tissues to the radioimmunoconjugates. Apart from antibodies targeting leukemia, the majority (>99%) of the injected antibody dose remains in circulation in the bloodstream or is unrelated to the target tumor. The prolonged circulation time of non-targeted antibodies (typically days to weeks) results in radiation exposure to normal organs (primarily the liver and kidneys) that break down or retain proteins and peptides.

[0354] Another major potential obstacle to the safe use of alpha emitters in vivo is the sequential release of three unchelated alpha emitter daughter atoms following the initial decay of the initial radionuclide. Ingestion organs (such as the kidneys or liver) can cause toxicity. An attractive approach to overcoming the long circulation time of untargeted radioimmunoconjugates is to develop connectors that allow conditional release of the chelated radioactive payload. Introducing cleavage sites at the bond between the radioconjugate and the mAb aims to allow the release of low-molecular-weight radioconjugates from their carrier mAb, and subsequently, rapid clearance once the radioconjugate has accumulated in metabolic organs. The chelated radiometal is then rapidly excreted through the kidneys, thereby reducing toxicity. This strategy has been attempted using disulfide, ester, tartramide, 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, Vriesendor pHM. "Effects of linker chemistry on the pharmacokinetics of radioimmunoconjugates." QJ Nucl Med. 1998; 42:250–261). The optimal linker should connect the carrier mAb to the radiochelating factor without impairing the function of either, providing stable binding in circulation and degrading under specific conditions. Stability in circulation has proven to be a particularly important factor for the success of this strategy.

[0355] Peptide linkers are generally more stable than esters and disulfides in serum and have been the most successful in the design of radioimmunoconjugates and antibody-drug conjugates (ADCs). The most extensively studied peptide linkers are sensitive to cathepsins. More precisely, ADCs comprising the dipeptide valine-citrulline have been shown to enter target cells and migrate to lysosomes, where they specifically release their drugs under the action of cysteine ​​proteases. The most advanced strategy for releasing radionuclides from radioimmunoconjugates is based on cathepsin-sensitive peptide linkers (DeNardo GL et al., “Preclinicale 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 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 radioimmunotherapy(RIT) in prostate cancer patients: comparison of radiation dosimetry for 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid(DOTA)-peptide versus 2IT-DOTA monoclonal antibody linkage for RIT." Clin Cancer Res. 2003; 9:3938S–3944S).In some cases, the use of cathepsin B unstable radioconjugates (e.g., glycylglycylglycyl-L-p-isothiocyanate phenylalanine-) has demonstrated reduced liver dose and observed a slight increase in tumor dose (DeNardo GL et al., Clin Cancer Res. 2003; 9:3865S–3872S; DeNardo GL et al., Clin Cancer Res. 1998; 4:2483–2490).

[0356] 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 in the linker are non-natural amino acids.

[0357] In some embodiments, the linker may 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 α-helix forming sequences, proline-rich sequences, and regions rich in double and / or triple bonds.

[0358] In some embodiments, the linker comprises a peptide linker. In some embodiments, the length of the peptide linker can be between 3 and 20 amino acids, such as a repeat of a single amino acid residue (e.g., polyglycine) or a combination of amino acid residues, to obtain a peptide linker that confers favorable pharmacokinetics.

[0359] In some embodiments, the linker comprises a peptide linker. The peptide linker contains L-amino acids and / or D-amino acids. In some embodiments, D-amino acids are preferred to minimize immunogenicity and non-specific cleavage by background peptidases or proteases. Cellular uptake of oligo-D-arginine sequences is known to be as good as or better than that of oligo-L-arginine.

[0360] In some implementations, the linker is cleavable. In some implementations, the linker is designed to cleave under specific conditions or environments near such target cells, tissues, or regions. Cleavable linkers rely on the inherent properties of the cell's cytoplasmic compartments to selectively release cytotoxic drugs. Such linkers primarily include chemically cleavable linkers that respond to low pH values ​​(acid-labile linkers) or reducing environments (disulfide linkers), and enzyme-cleavable linkers (peptide linkers or β-glucuronide linkers) that are susceptible to certain lysosomal enzymes.

[0361] 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 enzymatically cleavable (e.g., protease-sensitive, peptidase-sensitive) linker. In some embodiments, the linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. For example, pH-sensitive linkers can be hydrolyzed under acidic conditions. For example, the linker can be an acid-labile linker that is hydrolyzable in lysosomes (e.g., hydrazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.). Such linkers are relatively stable under neutral pH conditions (such as those in blood) but unstable below pH 7.0 (such as pH 6.5 to 4.5, approximate pH values ​​for lysosomes and / or endosomes).

[0362] In some embodiments, the linker is cleaved by intracellular peptidases or proteases, including but not limited to lysosomal or endosomal proteases. In some embodiments, the linker is cleaved by glycosidases (e.g., glucuronidase). β-glucuronide linkers can be readily 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.

[0363] In some embodiments, the linker component comprises an amino acid unit. In one such embodiment, the amino acid unit allows the linker to be cleaved by a protease, 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, upon internalization. Exemplary tripeptides include: glycine-valine-citrulline (Gly-val-cit) and glycine-glycine (Gly-gly-gly).

[0364] Amino acid units can contain naturally occurring amino acid residues, as well as minor amino acids and non-natural amino acid analogs, such as citrulline. Amino acid units can be engineered and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasminogen lysins.

[0365] In some embodiments, the linker is cleaved by a protease, matrix metalloproteinase, 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.

[0366] In some embodiments, the linker is cleaved by MMPs. The hydrolytic activity of matrix metalloproteinases (MMPs) has been 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 metalloproteinases MMP-2, MMP-9, or MMP-7 (MMPs associated with cancer and inflammation).

[0367] In some implementations, the linker is cleaved by proteolytic enzymes or a reducing environment, such as that found near cancer cells. This environment or such enzymes are typically not found near normal cells.

[0368] In some embodiments, the linker is cleaved by serine proteases (including, but not limited to, thrombin and cathepsins). 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.

[0369] In some embodiments, the linker is cleaved in a necrotic environment. Necrosis often results in the release of enzymes or other intracellular contents that can be used to initiate linker cleavage. In some embodiments, linker cleavage occurs via necrotic enzymes (e.g., calpains).

[0370] In some implementations, the connector contains one or more disulfide bonds.

[0371] Alternatively, the linker can be a non-peptide linker. Typical examples of these types of linkers can be those based on straight-chain or branched hydrocarbons or polyethylene glycol of varying lengths. These can introduce other groups to affect solubility, rigidity, isoelectric point, such as aromatic or non-aromatic rings, halogens, ketones, aldehydes, esters, sulfonyl groups, phosphate groups, etc.

[0372] In some embodiments, the linker component is either “self-immolative” or “non-self-immolative.” A “non-self-immolative” spacer sequence unit is a spacer sequence unit in which some or all of the spacer sequence units remain bound to the conjugate moiety upon enzymatic (e.g., proteolytic) cleavage. Examples of non-self-immolative spacer sequence units include, but are not limited to, glycine spacer sequence units and glycine-glycine spacer sequence units. Other combinations of peptide spacer sequences susceptible to sequence-specific enzymatic cleavage are also considered. For example, enzymatic cleavage of a glycine-glycine spacer sequence unit by a tumor cell-associated protease would result 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 then subjected to a separate hydrolysis step in tumor cells, thus cleaving the glycine-glycine spacer sequence unit from the chelating factor moiety.

[0373] The “self-destructive” spacer sequence unit allows for the release of the drug moiety without a separate hydrolysis step. In some embodiments, the spacer sequence 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, methyl carbamate, or carbonate is formed between the benzyl alcohol and the cytotoxic agent (see, for example, Hamann et al., (2005) Expert Opin. Ther. Patents (2005) 15:1087-103). In one embodiment, the spacer sequence unit is a p-aminobenzyloxycarbonyl (PAB). In some embodiments, the phenylene moiety of the p-aminobenzyl unit is substituted with Qm, where Q is a halogen, nitro, cyano, -OH, or -OR. 5 -CO2H, -NHR 5 -C(=O)NHR 5 -NHC(=O)R 5 and substituted C1-C6 alkyl groups, wherein the substituted C1-C6 alkyl groups are substituted with -OH, -CO2H, or -NHR. 5 -C(=O)NHR 5 and -NHC(=O)R 5Substitution; and m is an integer ranging from 0 to 4 (e.g., see Yam B. Poudel et al., ACS Medicinal Chemistry Letters 2020 11(11), 2190-2194). Examples of self-destructive spacer sequence units also include, but are not limited to, aromatic compounds that are electronically similar to p-aminobenzyl alcohol (see, for example, US2005 / 0256030 A1), such as 2-aminoimidazolium-5-methanol derivatives (Hay et al., (1999) Bioorg. Med. Chem. Lett. 9:2237) and o- or p-aminobenzyl acetal. Spacer sequences that undergo cyclization upon amide bond hydrolysis can be used, such as substituted and unsubstituted 4-aminobutyramides (Rodrigues et al., Chemistry Biology, 1995, 2, 223); appropriately substituted bicyclic [2.2.1] and bicyclic [2.2.2] cyclic systems (Storm et al., J. Amer. Chem. Soc., 1972, 94: 5815); and 2-aminophenylpropionamide (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-destructive spacer sequence that can be used in ADCs.

[0374] In some embodiments, the immunoconjugate includes a linker, such as, for example, a dendritic type linker for covalently attaching more than one drug moiety to an antibody via a branched, multifunctional linker portion (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 molar ratio of drug to antibody, i.e., the loading, which is related to the potency of the ADC. Therefore, in cases where the cysteine-engineered antibody carries only one reactive cysteine ​​thiol group, multiple drug moieties can be attached via dendritic linkers.

[0375] In some implementations, the tumor-targeting portion is connected to the chelating portion (R). 1 The linker of its radionuclide complex or the radionuclide complex contains -CH2CH2C(=O)X 1 -LL c -, where L is -L 1 -L 2 -L 3 -L 4 -L 5 -;

[0376] X 1 For -NR a -、-NR a S(=O)2-、-NR a S(=O)2NR a - or one or more independently selected natural or non-natural amino acids, wherein any free amine of the amino acid is optionally independently controlled by R 5 or -C(=O)R 5 Substitution, and optionally, any free carboxylic acid of any amino acid is replaced by -C(=O)NH-R. 5 replace;

[0377] Each R a Independently selected from hydrogen and C1-C4 alkyl groups;

[0378] L 1 C1-C, whether unsubstituted or substituted 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, unsubstituted or substituted C2-C 20 alkenyl, unsubstituted or substituted C2-C 20 Ethyne group, C4-C 20 Polyethylene glycol, -(X 3 CH2CH2) t - Unsubstituted or substituted cycloalkylene, unsubstituted or substituted heteroalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene;

[0379] Each X 3 Independently selected from O and NR 4 ;

[0380] Each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0381] L 2 C1-C that is absent, unsubstituted, or substituted 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, -C(=O)- (unsubstituted or substituted C1-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)NR4 -(CH2CH2X 3 ) m -(CH2) p -、-NR 4 C(=O)-(CH2CH2X 3 ) m -(CH2) p -or-(CH2CH2X) 3 ) m -(CH2) p -;

[0382] Each R 4 Independently selected from hydrogen and C1-C6 alkyl groups;

[0383] Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0384] Each p is independently 0, 1, or 2;

[0385] L 3 The group is absent, or one or more independently selected from the group consisting of: natural or non-natural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates; wherein any free amine (-NH2) of the amino acid is optionally independently controlled by R. 5 or -C(=O)R 5 Substitution, wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 Substitution, wherein the substituted benzyl group is selected from halogen, -OH-, -OR 5 -CO2H, -NHR 5 -C(=O)NHR 5 -NHC(=O)R 5 The substituted C1-C6 alkyl groups are replaced by -OH, -CO2H, or -NHR groups. 5 -C(=O)NHR 5 and -NHC(=O)R 5 replace;

[0386] L 4 C1-C that is absent, unsubstituted, or substituted 10 Alkylene, -C(=O)- (unsubstituted or substituted C1-C) 10 alkylene)-,-C(=O)NR 4 -(unsubstituted or substituted C1-C) 10 alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C1-C) 10alkylene)-, -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 -;

[0387] Each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0388] Each q is independently 0, 1, or 2;

[0389] L 5 It does not exist; -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 -;

[0390] Each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0391] Each q is independently 0, 1, or 2;

[0392] Each R a Independently selected from hydrogen and C1-C4 alkyl groups;

[0393] Each R b Independently selected from hydrogen and C1-C4 alkyl groups;

[0394] Wherein, the heteroalkylene group is an alkylene group in which one carbon atom is replaced by one of the following groups: -O-, -S-, -S(=O)-, -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(=NR 5 )NR a -or-NR a C(=O)O-; where -L 1 -、-L 2 -、-L 3 -、-L 4 -and-L 5 When any one of - is replaced, then -L 1 -、-L 2 -、-L 3 -、-L 4 -and-L 5 - Selected by 1, 2, 3 or 4 from halogen, -OH, -OR 5 -CO2H, -NHR 5 -C(=O)NHR 5 -NHC(=O)R 5 The substituted C1-C6 alkyl groups are replaced by -OH, -CO2H, or -NHR groups. 5 -C(=O)NHR 5 and -NHC(=O)R 5 replace;

[0395] Each R 5 Independently selected from C1-C 10 Alkyl, C4-C30 Polyethylene glycol, unsubstituted or substituted arylene groups, and unsubstituted or substituted heteroarylene groups;

[0396] L c For -C(=O)-, -NHC(=S)-, -X-phenyl-NHC(=S)-, -S-, -C(=O)CH2-,

[0397] X represents non-existent, -O-, -S-, -S(=O)-, -S(=O)2-, -NRa-, -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-;Each R a It is independently selected from hydrogen and C1-C4 alkyl groups.

[0398] In some implementations, L c For -C(=O)-. In some implementations, L c For -NHC(=S)-. In some implementations, L c It is -X-phenyl-NHC(=S)-. In some embodiments, X is absent or -NHC(=S)NH-. In some embodiments, L c For -NHC(=S)NH-. In some implementations, L c -S-, -C(=O)CH2-, In some implementations, L c For -C(=O)CH2-. In some implementations, L c for

[0399] In some implementations, L 1 C1-C, whether unsubstituted or substituted 20 Alkylene, unsubstituted or substituted C1-C 20 Heteroalkylene, C4-C 20 Polyethylene glycol, unsubstituted or substituted C3-C8 cycloalkylene, unsubstituted or substituted monocyclic C3-C8 heteroalkylene, unsubstituted or substituted phenylene, unsubstituted or substituted monocyclic heteroaryl.

[0400] In some implementations, L1 Unsubstituted or substituted C1-C6 alkylene groups, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 Polyethylene glycol, unsubstituted or substituted cyclohexene, or unsubstituted or substituted phenylene.

[0401] In some implementation schemes, X 1 For one or more independently selected natural or non-natural amino acids, wherein any free amine of the amino acid is optionally independently controlled by R. 5 or -C(=O)R 5 Substitution, and optionally, any free carboxylic acid of any amino acid is replaced by -C(=O)NH-R. 5 Replace; and L 1 For -NR 4 CH2CH2-(OCH2CH2) t -; t is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0402] In some implementations, L 2 For non-existent, -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.

[0403] In some implementations, L 3 The group is absent, or one or more independently selected from the group consisting of: natural or non-natural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates; wherein any free amine (-NH2) of the amino acid is optionally independently controlled by R. 5 or -C(=O)R 5 Substitution, and optionally, any free carboxylic acid of any amino acid is replaced by -C(=O)NH-R. 5 replace.

[0404] In some implementations, L 3 The group is absent, or one or more independently selected from the following groups: 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; wherein any free amine of the amino acid is optionally R 5 or -C(=O)(R) 5 ) is replaced, and any free carboxylic acid of the amino acid is optionally replaced by -C(=O)NH(R) 5 )replace.

[0405] In some implementations, L 3 The amino acid 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-(p-aminobenzylcarbamate) or combinations thereof; wherein any free amine (-NH2) of the amino acid is optionally surrounded by -C (=O) (unsubstituted or substituted C1-C). 20 Alkylene or -C(=O)-C4-C 20 Polyethylene glycol substitution; wherein any free carboxylic acid (-CO2H) of any amino acid is optionally replaced by -C(=O)NH-(2,4,6-trimethyl-3-bromophenyl), -C(=O)NH-(unsubstituted or substituted C1-C 20 alkylene) or -C(=O)NH-(C4-C 20 (Polyethylene glycol) replacement.

[0406] In some implementations, L 3 for:

[0407]

[0408]

[0409] In some implementations, L 4 The following are not present: -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)NR4 -(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; each q is independently 1 or 2.

[0410] In some implementations, L 5 It 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 -; Each p is independently 0, 1, or 2.

[0411] In some implementations, L 5 For -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 -; Each q is independently 1 or 2.

[0412] In some implementations, L 1Unsubstituted or substituted C1-C6 alkylene groups, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 Polyethylene glycol, unsubstituted or substituted cyclohexene, or unsubstituted or substituted phenylene; L 2 For non-existent, -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; L 4 The following are not present: -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 -;L 5 For -NR 4 C(=O)-(CH2) n -、-C(=O)-(CH2CH2O) n -(CH2) q -、-C(=O)NR 4 -(CH2CH2O) n -(CH2) q -or-NR4 C(=O)-(CH2CH2O) n -(CH2) q -; each n is independently 1, 2, 3, 4, 5 or 6; each q is independently 1 or 2.

[0413] In some implementations, L 1 Unsubstituted or substituted C1-C6 alkylene groups, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 Polyethylene glycol, unsubstituted or substituted cyclohexene, or unsubstituted or substituted phenylene.

[0414] L 2 For non-existent, -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; L 4 It does not exist; L 5 For -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 -; each n is independently 1, 2, 3, 4, 5 or 6; each q is independently 1 or 2.

[0415] In some implementation schemes, X 1The amino acids are alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, leucine, lysine, phenylalanine, serine, tyrosine, valine, or combinations thereof; wherein any free amine of the amino acid is optionally R 5 or -C(=O)(R) 5 ) is replaced, and any free carboxylic acid of the amino acid is optionally replaced by -C(=O)NH(R) 5 Replace; for each R 5 Independently, unsubstituted or substituted C1-C 10 Alkylene, C4-C 20 Polyethylene glycol or unsubstituted or substituted phenyl groups, wherein the substituted phenyl groups are substituted by 1, 2, 3, 4 or 5 groups independently selected from F, Cl, Br, I, -CH3 and CF3; L 1 For -NR 4 CH2CH2-(OCH2CH2) t -; t is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; L 2 For non-existent, -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 -

[0416] Each m is independently 1, 2, 3, 4, 5, or 6; each p is independently 1 or 2; L 3 It does not exist; L 4 It does not exist; L 5 For -NR 4 C(=O)-(CH2) n -、-C(=O)-(CH2CH2O) n -(CH2) q -、-C(=O)NR 4 -(CH2CH2O) n -(CH2) q -or-NR4 C(=O)-(CH2CH2O) n -(CH2) q -; each n is independently 1, 2, 3, 4, 5 or 6; each q is independently 1 or 2.

[0417] In some implementations, L 1 Unsubstituted or substituted C1-C6 alkylene groups, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 Polyethylene glycol, unsubstituted or substituted cyclohexene, or unsubstituted or substituted phenylene; L 2 For non-existent, -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; L 3 For one or more independently selected groups chosen from the following: 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; wherein any free amine of the amino acid is optionally R 5 or -C(=O)(R) 5 ) is replaced, and any free carboxylic acid of the amino acid is optionally replaced by -C(=O)NH(R) 5 Replace; for each R 5 Independently, unsubstituted or substituted C1-C 10 Alkylene, C4-C 20 Polyethylene glycol or unsubstituted or substituted phenyl groups, wherein the substituted phenyl groups are substituted by 1, 2, 3, 4 or 5 groups independently selected from F, Cl, Br, I, -CH3 and CF3; L 4 It does not exist; L 5 For -NR4 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 -; each n is independently 1, 2, 3, 4, 5 or 6; each q is independently 1 or 2.

[0418] In some embodiments, the heteroalkylene group is one of the carbon atoms surrounded by -S(=O)(=NH)- or -S(=O)(=NR). 5 -, -P(=O)OH-, -NHC(=N-CN)NH- or -NHC(=NR)NH- 5 )NH-substituted alkylene groups.

[0419] In some implementations, -CH2CH2C(=O)X 1 -L- means:

[0420]

[0421]

[0422]

[0423]

[0424]

[0425] Joined part (R) 2 )

[0426] In addition, the lacing part (R) 2 This is used to facilitate attachment to the tumor-targeting region. In some implementations, R... 2 In order to be able to target the tumor-specific R 3 The reaction part of the amine (-NH2) or thiol (-SH). In some embodiments, R 2 In order to be able to target the tumor-specific R 3 The amine (-NH2) reaction part of the side chain of the lysine residue.

[0427] In some implementation schemes, R 2 In order to be able to target the tumor-specific R 3The amine (-NH2) reaction part, and includes tetrafluorophenyl ester, pentafluorophenyl ester, dinitrophenyl ester, succinimide ester, sulfosuccinimide ester or isothiocyanate.

[0428] In some implementation schemes, R 2 In order to be able to target the tumor-specific R 3 The portion of the amine (-NH2) reaction, and includes:

[0429]

[0430] X represents non-existent, -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-;Each R a It is independently selected from hydrogen and C1-C4 alkyl groups.

[0431] In some implementation schemes, R 2 In order to be able to target the tumor-specific R 3 The portion of the amine (-NH2) reaction, and includes:

[0432] X is non-existent or -NR a C(=S)NR a -; Each R a X is independently selected from hydrogen and C1-C4 alkyl groups. In some embodiments, X is absent or is -NHC(=S)NH-.

[0433] In some implementation schemes, R 2 In order to be able to target the tumor-specific R 3 The portion of the amine (-NH2) reaction, and includes: X is either non-existent or -NHC(=S)NH-.

[0434] In some implementation schemes, R 2 In order to be able to target the tumor-specific R 3 The portion of the amine (-NH2) reaction, and includes:

[0435] In some implementation schemes, R 2In order to be able to target the tumor-specific R 3 The portion of the amine (-NH2) reaction, and includes: X is either non-existent or -NHC(=S)NH-.

[0436] Alternatively or additionally, isothiocyanate linkers, such as p-SCN-Bn-DOTA, can be used, which relate to lysine residues within the immunoconjugates of the present invention.

[0437] In some implementation schemes, R 2 In order to be able to target the tumor-specific R 3 The thiol (-SH) reaction part of the side chain of the cysteine ​​residue, and comprising: maleimide group, haloacetamide group, haloacetyl group, haloacetate group, pyridylthio group, vinyl carbonyl group, aziridine propane group, disulfide group, acetylene group, hydroxysuccinimide group or thiol group.

[0438] In some implementation schemes, R 2 In order to be able to target the tumor-specific R 3 The portion of the thiol (-SH) reaction, and contains m can be 0, 1, 2, 3, 4, or 5.

[0439] In some implementation schemes, R 2 In order to be able to target the tumor-specific R 3 The portion of the thiol (-SH) reaction, and contains

[0440] In some implementations, the connector can be bridged by cysteine-based functional groups such as... Alternatively, DBM (dibromomaleimide) can be conjugated to the antibody. These linkers can re-stabilize intrachain disulfide bonds after reduction and conjugation (Bird M et al., Antibody-Drug Conjugates, pp. 113-129 (2019) and Behrens CR et al., Mol. Pharmaceutics 12:3986 (2015)). Exemplary rebridging extended sequence elements are shown below (where wavy lines indicate sites covalently attached to the immunoconjugate):

[0441]

[0442] For further details regarding the connectors 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. April 2016; 17(4): 561; each of which is incorporated herein by reference into the disclosure of such connectors.

[0443] Exemplary immune conjugates

[0444] In some implementations, the immunoconjugate is a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0445]

[0446] in:

[0447] R 1 It is a chelated portion or a radionuclide complex thereof;

[0448] R 2 In order to be able to target the tumor-specific R 3 The portion of the reaction between amines (-NH2) or thiols (-SH);

[0449] X 1 For -NR a -、-NR a S(=O)2-、-NR a S(=O)2NR a - or one or more independently selected natural or non-natural amino acids, wherein any free amine of the amino acid is optionally independently controlled by R 5 or -C(=O)R 5 Substitution, and optionally, any free carboxylic acid of any amino acid is replaced by -C(=O)NH-R. 5 replace;

[0450] Each R a Independently selected from hydrogen and C1-C4 alkyl groups;

[0451] L is the connector, which is -L. 1 -L 2 -L3 -L 4 -L 5 -;

[0452] L 1 C1-C, whether unsubstituted or substituted 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, unsubstituted or substituted C2-C 20 alkenyl, unsubstituted or substituted C2-C 20 Ethyne group, C4-C 20 Polyethylene glycol, -(X 3 CH2CH2) t - Unsubstituted or substituted cycloalkylene, unsubstituted or substituted heteroalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene;

[0453] Each X 3 Independently selected from O and NR 4 ;

[0454] Each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0455] L 2 C1-C that is absent, unsubstituted, or substituted 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, -C(=O)- (unsubstituted or substituted C1-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 -;

[0456] Each R 4Independently selected from hydrogen and C1-C6 alkyl groups;

[0457] Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0458] Each p is independently 0, 1, or 2;

[0459] L 3 The group is absent, or one or more independently selected from the group consisting of: natural or non-natural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates; wherein any free amine (-NH2) of the amino acid is optionally independently controlled by R. 5 or -C(=O)R 5 Substitution, wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 Substitution, wherein the substituted benzyl group is selected from halogen, -OH, -OR. 5 -CO2H, -NHR 5 -C(=O)NHR 5 -NHC(=O)R 5 The substituted C1-C6 alkyl groups are replaced by -OH, -CO2H, or -NHR groups. 5 -C(=O)NHR 5 and -NHC(=O)R 5 replace;

[0460] L 4 C1-C that is absent, unsubstituted, or substituted 10 Alkylene, -C(=O)- (unsubstituted or substituted C1-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 -;

[0461] Each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0462] Each q is independently 0, 1, or 2;

[0463] L 5 This does not exist; -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 -;

[0464] Each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0465] Each q is independently 0, 1, or 2;

[0466] Each R a Independently selected from hydrogen and C1-C4 alkyl groups;

[0467] Each R b Independently selected from hydrogen and C1-C4 alkyl groups;

[0468] Wherein, the heteroalkylene group is an alkylene group in which one carbon atom is replaced by one of the following groups: -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NH)-, -S(=O)(=NR5 )-、-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 C(=O)O-;

[0469] Where -L 1 -、-L 2 -、-L 3 -、-L 4 -and-L 5 When any one of - is replaced, then -L 1 -、-L 2 -、-L 3 -、-L 4 -and-L 5 - Selected by 1, 2, 3 or 4 from halogen, -OH, -OR 5 -CO2H, -NHR 5 -C(=O)NHR 5 -NHC(=O)R 5 The substituted C1-C6 alkyl groups are replaced by -OH, -CO2H, or -NHR groups. 5 -C(=O)NHR 5 and -NHC(=O)R 5 replace;

[0470] Each R 5 Independently selected from C1-C 10 Alkyl, C4-C 30 Polyethylene glycol, and unsubstituted or substituted arylene groups, unsubstituted or substituted heteroarylene groups;

[0471] The premise is that when L 3 If it does not exist, then there exists at least one R. 5 ;or

[0472] The premise is X 1 -N (C1-C4 alkyl), -NR a S(=O)2-、-NR a S(=O)2NR a- or one or more independently selected natural or non-natural amino acids.

[0473] In some embodiments, the immunoconjugate is a compound of formula (II), formula (III) or formula (IV) or a pharmaceutically acceptable salt thereof:

[0474]

[0475] in:

[0476] R 1 It is a chelated portion or a radionuclide complex thereof;

[0477] -NH-R 3 For tumor targeting;

[0478] X represents non-existent, -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-;

[0479] Each R a Independently selected from hydrogen and C1-C4 alkyl groups;

[0480] X 1 For -NR a -、-NR a S(=O)2-、-NR a S(=O)2NR a - or one or more independently selected natural or non-natural amino acids, wherein any free amine of the amino acid is optionally independently controlled by R 5 or -C(=O)R 5 Substitution, and optionally, any free carboxylic acid of any amino acid is replaced by -C(=O)NH-R. 5 replace;

[0481] Each R a Independently selected from hydrogen and C1-C4 alkyl groups;

[0482] L is the connector, which is -L. 1 -L 2 -L 3 -L 4 -L5 -;

[0483] L 1 C1-C, whether unsubstituted or substituted 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, unsubstituted or substituted C2-C 20 alkenyl, unsubstituted or substituted C2-C 20 Ethyne group, C4-C 20 Polyethylene glycol, -(X 3 CH2CH2) t - Unsubstituted or substituted cycloalkylene, unsubstituted or substituted heteroalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene;

[0484] Each X 3 Independently selected from O and NR 4 ;

[0485] Each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0486] L 2 C1-C that is absent, unsubstituted, or substituted 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, -C(=O)- (unsubstituted or substituted C1-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 -;

[0487] Each R 4 Independently selected from hydrogen and C1-C6 alkyl groups;

[0488] Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0489] Each p is independently 0, 1, or 2;

[0490] L 3 The group is absent, or one or more independently selected from the group consisting of: natural or non-natural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates; wherein any free amine (-NH2) of the amino acid is optionally independently controlled by R. 5 or -C(=O)R 5 Substitution, wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 Substitution, wherein the substituted benzyl group is selected from halogen, -OH, -OR. 5 -CO2H, -NHR 5 -C(=O)NHR 5 -NHC(=O)R 5 The substituted C1-C6 alkyl groups are replaced by -OH, -CO2H, or -NHR groups. 5 -C(=O)NHR 5 and -NHC(=O)R 5 replace;

[0491] L 4 C1-C that is absent, unsubstituted, or substituted 10 Alkylene, -C(=O)- (unsubstituted or substituted C1-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 -;

[0492] Each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0493] Each q is independently 0, 1, or 2;

[0494] L 5 This does not exist; -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 -;

[0495] Each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0496] Each q is independently 0, 1, or 2;

[0497] Each R a Independently selected from hydrogen and C1-C4 alkyl groups;

[0498] Each R b Independently selected from hydrogen and C1-C4 alkyl groups;

[0499] Wherein, the heteroalkylene group is an alkylene group in which one carbon atom is replaced by one of the following groups: -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NH)-, -S(=O)(=NR 5 )-、-NR 5 -、-P(=O)ORb -、-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 C(=O)O-;

[0500] Where -L 1 -、-L 2 -、-L 3 -、-L 4 -and-L 5 When any one of - is replaced, then -L 1 -、-L 2 -、-L 3 -、-L 4 -and-L 5 - Selected by 1, 2, 3 or 4 from halogen, -OH, -OR 5 -CO2H, -NHR 5 -C(=O)NHR 5 -NHC(=O)R 5 The substituted C1-C6 alkyl groups are replaced by -OH, -CO2H, or -NHR groups. 5 -C(=O)NHR 5 and -NHC(=O)R 5 replace;

[0501] Each R 5 Independently selected from C1-C 10 Alkyl, C4-C 30 Polyethylene glycol and unsubstituted or substituted arylene groups, unsubstituted or substituted heteroarylene groups;

[0502] The premise is that when L 3 If it does not exist, then there exists at least one R. 5 ;or

[0503] The premise is X 1 -N (C1-C4 alkyl), -NR a S(=O)2-、-NR a S(=O)2NR a -Or one or more independently selected natural or non-natural amino acids.

[0504] In some embodiments, the immunoconjugate is a compound of formula (II) or a pharmaceutically acceptable salt thereof:

[0505]

[0506] In some embodiments, the immunoconjugate is a compound of formula (III) or a pharmaceutically acceptable salt thereof:

[0507]

[0508] In some implementations, the immunoconjugate is a compound of formula (IV) or a pharmaceutically acceptable salt thereof:

[0509]

[0510] In some implementation schemes, R 2 In order to be able to target the tumor-specific R 3 The part of the amine (-NH2) reaction of lysine.

[0511] In some embodiments, the compound of formula (II) has the structure of formula (IIa) or a pharmaceutically acceptable salt thereof:

[0512]

[0513] Wherein: -NHCH2CH2CH2CH2- is the tumor-targeting fraction R 3 The side chain of lysine residues.

[0514] In some embodiments, the compound of formula (III) has the structure of formula (IIIa) or a pharmaceutically acceptable salt thereof:

[0515]

[0516] Wherein: -NHCH2CH2CH2CH2- is the tumor-targeting fraction R 3 The side chain of lysine residues.

[0517] In some implementation schemes, R 2 In order to be able to target the tumor-specific R 3 The part of the thiol (-SH) reaction of cysteine.

[0518] In some embodiments, the compound of formula (IV) has the structure of formula (IVa) or a pharmaceutically acceptable salt thereof:

[0519]

[0520] Wherein: -NHCH2CH2CH2CH2- is the tumor-targeting fraction R3 The side chain of lysine residues.

[0521] In some embodiments, the immunoconjugate is a compound of formula (V), (VI), (VII), or (VIII) or a pharmaceutically acceptable salt thereof:

[0522]

[0523] in:

[0524] R 1 It is a chelated portion or a radionuclide complex thereof;

[0525] -SR 3 For tumor targeting;

[0526] X 1 For -NR a -、-NR a S(=O)2-、-NR a S(=O)2NR a - or one or more independently selected natural or non-natural amino acids, wherein any free amine of the amino acid is optionally independently controlled by R 5 or -C(=O)R 5 Substitution, and optionally, any free carboxylic acid of any amino acid is replaced by -C(=O)NH-R. 5 replace;

[0527] Each R a Independently selected from hydrogen and C1-C4 alkyl groups;

[0528] L is the connector, which is -L. l -L 2 -L 3 -L 4 -L 5 -;

[0529] L 1 C1-C, whether unsubstituted or substituted 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, unsubstituted or substituted C2-C 20 alkenyl, unsubstituted or substituted C2-C 20 Ethyne group, C4-C 20 Polyethylene glycol, -(X 3 CH2CH2) t - Unsubstituted or substituted cycloalkylene, unsubstituted or substituted heteroalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene;

[0530] Each X 3Independently selected from O and NR 4 ;

[0531] Each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0532] L 2 C1-C that is absent, unsubstituted, or substituted 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, -C(=O)- (unsubstituted or substituted C1-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 -;

[0533] Each R 4 Independently selected from hydrogen and C1-C6 alkyl groups;

[0534] Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0535] Each p is independently 0, 1, or 2;

[0536] L 3 The group is absent, or one or more independently selected from the group consisting of: natural or non-natural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates; wherein any free amine (-NH2) of the amino acid is optionally independently controlled by R. 5 or -C(=O)R 5 Substitution, wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 Substitution, wherein the substituted benzyl group is selected from halogen, -OH, -OR.5 -CO2H, -NHR 5 -C(=O)NHR 5 -NHC(=O)R 5 The substituted C1-C6 alkyl groups are replaced by -OH, -CO2H, or -NHR groups. 5 -C(=O)NHR 5 and -NHC(=O)R 5 replace;

[0537] L 4 C1-C that is absent, unsubstituted, or substituted 10 Alkylene, -C(=O)- (unsubstituted or substituted C1-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 -;

[0538] Each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0539] Each q is independently 0, 1, or 2;

[0540] L 5 This does not exist; -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 -;

[0541] Each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0542] Each q is independently 0, 1, or 2;

[0543] Each R a Independently selected from hydrogen and C1-C4 alkyl groups;

[0544] Each R b Independently selected from hydrogen and C1-C4 alkyl groups;

[0545] Wherein, the heteroalkylene group is an alkylene group in which one carbon atom is replaced by one of the following groups: -O-, -S-, -S(=O)-, -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(=NR 5 )NR a -or-NR a C(=O)O-;

[0546] Where -L 1 -、-L 2 -、-L3 -、-L 4 -and-L 5 When any one of - is replaced, then -L 1 -、-L 2 -、-L 3 -、-L 4 -and-L 5 - Selected by 1, 2, 3 or 4 from halogen, -OH, -OR 5 -CO2H, -NHR 5 -C(=O)NHR 5 -NHC(=O)R 5 The substituted C1-C6 alkyl groups are replaced by -OH, -CO2H, or -NHR groups. 5 -C(=O)NHR 5 and -NHC(=O)R 5 replace;

[0547] Each R 5 Independently selected from C1-C 10 Alkyl, C4-C 30 Polyethylene glycol and unsubstituted or substituted arylene groups, unsubstituted or substituted heteroarylene groups;

[0548] The premise is that when L 3 If it does not exist, then there exists at least one R. 5 ;or

[0549] The premise is X 1 -N (C1-C4 alkyl), -NR a S(=O)2-、-NR a S(=O)2NR a -Or one or more independently selected natural or non-natural amino acids.

[0550] In some implementations, the immunoconjugate is a compound of formula (V) or a pharmaceutically acceptable salt thereof:

[0551]

[0552] In some implementations, the immunoconjugate is a compound of formula (VI) or a pharmaceutically acceptable salt thereof:

[0553]

[0554] In some implementations, the immunoconjugate is a compound of formula (VII) or a pharmaceutically acceptable salt thereof:

[0555]

[0556] In some implementations, the immunoconjugate is a compound of formula (VIII) or a pharmaceutically acceptable salt thereof:

[0557]

[0558] In some embodiments, the compound of formula (V) has the structure of formula (Va) or a pharmaceutically acceptable salt thereof:

[0559]

[0560] Where: -SCH2- is the tumor-targeting fraction R 3 Thiols (-SH) on the side chains of cysteine ​​residues.

[0561] In some embodiments, the compound of formula (VI) has the structure of formula (VIa) or a pharmaceutically acceptable salt thereof:

[0562]

[0563] Where: -SCH2- is the tumor-targeting fraction R 3 Thiols (-SH) on the side chains of cysteine ​​residues.

[0564] In some embodiments, the compound of formula (VI) has the structure of formula (VIa) or a pharmaceutically acceptable salt thereof:

[0565]

[0566] Where: -SCH2- is the tumor-targeting fraction R 3 Thiols (-SH) on the side chains of cysteine ​​residues.

[0567] In some embodiments, the compound of formula (VII) has the structure of formula (VIIa) or a pharmaceutically acceptable salt thereof:

[0568]

[0569] Where: -SCH2- is the tumor-targeting fraction R 3 Thiols (-SH) on the side chains of cysteine ​​residues.

[0570] In some embodiments, the compound of formula (VIII) has the structure of formula (VIIIa) or a pharmaceutically acceptable salt thereof:

[0571]

[0572] Where: -SCH2- is the tumor-targeting fraction R 3 Thiols (-SH) on the side chains of cysteine ​​residues.

[0573] In some implementation schemes, R 1 The chelate is selected from the list of the following: DOTA, DO3A, DOTAGA, DOTAGA anhydride, Py4Pa, Py4Pa-NCS, Crown, Macropa, Macropa-NCS, HEHA, CHXoctapa, Bispa, and Noneunpa; or radionuclides thereof.

[0574] In some implementation schemes, R 1 The chelate moieties selected from the list of the following components are: DOTMA, DOTPA, DO3AM-acetic acid, DOTP, DOTMP, DOTA-4AMP, CB-TE2A, NOA, 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 their radionuclide complexes.

[0575] In some implementation schemes, R 1 The chelate is selected from the following:

[0576] 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA);

[0577] 1,4,7,10-Tetraazacyclododecane-1,4,7-triacetic acid (DO3A);

[0578] 1,4,7,10-Tetraazacyclododecane-1,7-diacetic acid (DO2A);

[0579] α,α',α”,α”'-Tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTMA);

[0580] 1,4,7,10-Tetra(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM);

[0581] 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrapropionic acid (DOTPA);

[0582] 2,2',2"-(10-(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid;

[0583] 6,6'-(((pyridin-2,6-diylbis(methylene))bis((carboxymethyl)azanidinediyl))-bis(methylene))dipicolinic acid (H4pypa);

[0584] 6,6',6",6"'-(((pyridine-2,6-diylbis(methylene))bis(azanetriyl))tetra(methylene))-tetrapicolinic acid (H4py4pa);

[0585] 3,6,9,12-Tetra(carboxymethyl)-3,6,9,12-tetraazatetradecanoic acid (TTHA);

[0586] Or its radionuclide complex.

[0587] In some implementation schemes, R 1 The chelate is selected from the following: Or its radionuclide complex.

[0588] In some implementation schemes, R 1 The chelate is selected from the following: Or its radionuclide complex.

[0589] In some implementation schemes, R 1 The chelate is selected from the following: Or its radionuclide complex.

[0590] In some implementation schemes, R 2 In order to be able to target the tumor-specific R 3 The amine (-NH2) reaction part, and includes tetrafluorophenyl ester, pentafluorophenyl ester, dinitrophenyl ester, succinimide ester, sulfosuccinimide ester or isothiocyanate.

[0591] In some implementation schemes, R 2 In order to be able to target the tumor-specific R 3 The portion of the amine (-NH2) reaction, and includes:

[0592]

[0593] X represents non-existent, -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)NRa -or-NR a C(=O)O-; and each R a It is independently selected from hydrogen and C1-C4 alkyl groups.

[0594] In some implementations, X is non-existent, -O-, -S-, -S(=O)-, -S(=O)2-, -NR a -、-C(=O)-、-NR a C(=O)-or-C(=O)NR a In some implementations, X is -O-, -S-, -S(=O)-, -S(=O)2-, or -NR. a - In some implementations, X is -O-. In some implementations, X is -S-. In some implementations, X is -NR. a C(=O)NR a -、-NR a C(=S)NR a -or-NR a C(=O)O-. In some implementations, X is -OC(=O)NR. a -or-NR a C(=O)O-. In some implementations, X is -NR. a C(=O)NR a - In some implementations, X is -NR a C(=S)NR a - In some implementations, X is non-existent.

[0595] In some implementation schemes, R 2 In order to be able to target the tumor-specific R 3 The portion of the thiol (-SH) reaction, and containing maleimide group, haloacetamide group, haloacetyl group, haloacetate group, pyridylthio group, vinyl carbonyl group, aziridine propane group, disulfide group, acetylene group, hydroxysuccinimide group or thiol group.

[0596] In some implementations, the tumor-targeting component R 3 It is a polypeptide containing an antigen-binding region and an immunoglobulin heavy chain constant region, wherein the molecular weight of the polypeptide is between 60 kDa and 110 kDa.

[0597] In some implementation schemes, R 2 In order to be able to target the tumor-specific R 3 The portion of the thiol (-SH) reaction, and contains m can be 0, 1, 2, 3, 4, or 5.

[0598] In some implementation schemes, X 1 For -NR a -、-NR a S(=O)2- or one or more independently selected natural or non-natural amino acids, wherein any free amine of the amino acid is optionally independently converted by R 5 or -C(=O)R 5 Substitution, and optionally, any free carboxylic acid of any amino acid is replaced by -C(=O)NH-R. 5 Replacement. In some implementations, X 1 It can be -NH-, -N(CH3)-, -N(CH2CH3)-, -NHS(=O)2-, -N(CH3)S(=O)2-, or -N(CH2CH3)S(=O)2-. In some embodiments, X 1 It can be -NH-, -N(CH3)-, or -N(CH2CH3)-. In some embodiments, X 1 It is -NHS(=O)2-, -N(CH3)S(=O)2-, or -N(CH2CH3)S(=O)2-. In some embodiments, X 1 For -NH-. In some implementations, X 1 It is -N(CH3)-. In some implementations, X 1 It is -N(CH2CH3)-. In some implementations, X 1 For -NHS(=O)2-. In some implementations, X 1 It is -N(CH3)S(=O)2-. In some implementations, X 1 It is -N(CH2CH3)S(=O)2-.

[0599] In some implementation schemes, X 1 For one or more independently selected natural or non-natural amino acids, wherein any free amine of the amino acid is optionally independently controlled by R. 5 or -C(=O)R 5 Substitution, and optionally, any free carboxylic acid of any amino acid is replaced by -C(=O)NH-R. 5 Replace; and L 1 For -NR 4 CH2CH2-(OCH2CH2) t -; where t is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0600] In some implementation schemes, X 1The amino acids are lysine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, serine, tyrosine, valine, citrulline, methionine-valine-lysine, glycine-phenylalanine-glycine, tyrosine-arginine-valine, arginine-valine, or combinations thereof; wherein any free amine (-NH2) of the amino acid is optionally and independently converted by R. 5 or -C(=O)R 5 Substitution, and optionally, any free carboxylic acid of any amino acid is replaced by -C(=O)NH-R. 5 replace.

[0601] In some implementations, L 1 Unsubstituted or substituted C1-C6 alkylene groups, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 Polyethylene glycol, unsubstituted or substituted cyclohexene, or unsubstituted or substituted phenylene; L 2 For non-existent, -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 -; where each m is independently 1, 2, 3, 4, 5, or 6; and each p is independently 1 or 2; L 4 The following are not present: -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 4C(=O)-(CH2CH2O) n -(CH2) q -or-(CH2CH2O) n -(CH2) q -;and L 5 For -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 -; where each n is independently 1, 2, 3, 4, 5 or 6; and each q is independently 1 or 2.

[0602] In some implementations, L 1 Unsubstituted or substituted C1-C6 alkylene groups, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 Polyethylene glycol, unsubstituted or substituted cyclohexene, or unsubstituted or substituted phenylene; L 2 For non-existent, -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 -; where each m is independently 1, 2, 3, 4, 5, or 6; and each p is independently 1 or 2; L 4 It does not exist; and L 5 For -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 -; where each n is independently 1, 2, 3, 4, 5 or 6; and each q is independently 1 or 2.

[0603] In some implementation schemes, X 1 The amino acids are alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, leucine, lysine, phenylalanine, serine, tyrosine, valine, or combinations thereof; wherein any free amine of the amino acid is optionally R 5 or -C(=O)(R) 5 ) is replaced, and any free carboxylic acid of the amino acid is optionally replaced by -C(=O)NH(R) 5 Replace; where each R 5 Independently, unsubstituted or substituted C1-C 10 Alkylene, C4-C 20 Polyethylene glycol or unsubstituted or substituted phenyl groups, wherein the substituted phenyl groups are substituted by 1, 2, 3, 4 or 5 groups independently selected from F, Cl, Br, I, -CH3 and CF3; L 1 For -NR 4 CH2CH2-(OCH2CH2) t -; t is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; L 2 For non-existent, -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 -; where each m is independently 1, 2, 3, 4, 5, or 6; and each p is independently 1 or 2; L3 It does not exist; L 4 It does not exist; and L 5 For -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 -; where each n is independently 1, 2, 3, 4, 5 or 6; and each q is independently 1 or 2.

[0604] In some implementations, L 1 Unsubstituted or substituted C1-C6 alkylene groups, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 Polyethylene glycol, unsubstituted or substituted cyclohexene, or unsubstituted or substituted phenylene; L 2 For non-existent, -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 -; where each m is independently 1, 2, 3, 4, 5, or 6; and each p is independently 1 or 2; L 3 The amino acids are alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, tyrosine, valine, or combinations thereof; wherein any free amine of the amino acid is optionally R 5 or -C(=O)(R) 5 ) is replaced, and any free carboxylic acid of the amino acid is optionally replaced by -C(=O)NH(R)5 Replace; where each R 5 Independently, unsubstituted or substituted C1-C 10 Alkylene, C4-C 20 Polyethylene glycol or unsubstituted or substituted phenyl groups, wherein the substituted phenyl groups are substituted by 1, 2, 3, 4 or 5 groups independently selected from F, Cl, Br, I, -CH3 and CF3; L 4 It does not exist; L 5 For -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 -; where each n is independently 1, 2, 3, 4, 5 or 6; and each q is independently 1 or 2.

[0605] In some implementations, L 1 C1-C, whether unsubstituted or substituted 20 Alkylene, unsubstituted or substituted C1-C 20 Heteroalkylene, C4-C 20 Polyethylene glycol, unsubstituted or substituted C3-C8 cycloalkylene groups, unsubstituted or substituted monocyclic C3-C8 heterocyclic alkylene groups, unsubstituted or substituted phenylene groups, and unsubstituted or substituted monocyclic heteroaryl groups. In some embodiments, L 1 Unsubstituted or substituted C1-C6 alkylene groups, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 Polyethylene glycol, unsubstituted or substituted cyclohexene, or unsubstituted or substituted phenylene. In some embodiments, L 1 It does not exist.

[0606] In some implementations, L 2 For non-existent, -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 -; where each m is independently 1, 2, 3, 4, 5, or 6; and each p is independently 1 or 2. In some implementations, L 2 It does not exist.

[0607] In some implementations, L 3 The amino acids are: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, tyrosine, valine, or combinations thereof; any free amine of the amino acid may optionally be R. 5 or -C(=O)(R) 5 ) is replaced, and any free carboxylic acid of the amino acid is optionally replaced by -C(=O)NH(R) 5 )replace.

[0608] In some implementations, L 3 The amino acid 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-(p-aminobenzylcarbamate) or combinations thereof; wherein any free amine (-NH2) of the amino acid is optionally surrounded by -C (=O) (unsubstituted or substituted C1-C). 20 Alkylene or -C(=O)-C4-C 20 The polyethylene glycol is substituted; and any free carboxylic acid (-CO2H) of any amino acid therein is optionally replaced by -C(=O)NH-(2,4,6-trimethyl-3-bromophenyl), -C(=O)NH-(unsubstituted or substituted C1-C 20 alkylene) or -C(=O)NH-(C4-C 20 (Polyethylene glycol) replacement. In some implementations, L 3 It does not exist.

[0609] In some implementations, L 4 C1-C that is absent, unsubstituted, or substituted 10 Alkylene, -C(=O)- (unsubstituted or substituted C1-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 -; where 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 implementations, L 4 The following are not present: -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 implementations, L 4 It does not exist.

[0610] In some implementations, L 5 It does not exist; -C(=O)-(CH2) n -、-C(=O)NR 4 -(CH2) n -、-NR4 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 -; where 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 implementations, L 5 It 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 implementations, L 5 For -NR 4 C(=O)-(CH2) n -、-C(=O)-(CH2CH2O) n -(CH2) q -、-C(=O)NR4 -(CH2CH2O) n -(CH2) q -or-NR 4 C(=O)-(CH2CH2O) n -(CH2) q -; Each q is independently 1 or 2. In some implementations, L 5 It does not exist.

[0611] In some embodiments, the heteroalkylene group is one of the carbon atoms surrounded by -S(=O)(=NH)- or -S(=O)(=NR). 5 -, -P(=O)OH-, -NHC(=N-CN)NH- or -NHC(=NR)NH- 5 )NH-substituted alkylene groups. In some embodiments, the heteroalkylene group is one in which one carbon atom is replaced by -S(=O)(=NH)- or -S(=O)(=NR)-. 5 The heteroalkylene group is a alkylene group in which one carbon atom is replaced by -P(=O)OH-. In some embodiments, the heteroalkylene group is a alkylene group in which one carbon atom is replaced by -NHC(=N-CN)NH- or -NHC(=NR)NH-. 5 )NH-substituted alkylene groups.

[0612] In some implementations, each X 3 Independently represented by O. In some implementations, each X... 3 Independent for NR 4 .

[0613] In some implementations, each R 4 It is hydrogen. In some implementations, each R... 4 It is a C1-C6 alkyl group.

[0614] In some implementations, each R 5 Independently selected from C1-C 10 Alkyl, C4-C 30 Polyethylene glycol and unsubstituted or substituted arylene groups, unsubstituted or substituted heteroarylene groups. In some embodiments, each R 5 Independently selected from C1-C 10 Alkyl or C4-C 30 Polyethylene glycol. In some implementations, each R 5 It is independently selected from unsubstituted or substituted aryl groups or unsubstituted or substituted heteroaryl groups.

[0615] In some implementations, each R a Independently selected from hydrogen and C1-C4 alkyl groups. In some embodiments, each Ra Independently hydrogen. In some implementations, each R a It is independently a C1-C4 alkyl group.

[0616] In some implementations, each R b Independently selected from hydrogen and C1-C4 alkyl groups. In some embodiments, each R b Independently hydrogen. In some implementations, each R b It is independently a C1-C4 alkyl group.

[0617] Exemplary compounds used to prepare the immunoconjugates described herein include those described in Table A.

[0618] Table A.

[0619]

[0620]

[0621]

[0622]

[0623]

[0624]

[0625]

[0626] Radioimmunoconjugates

[0627] In one embodiment, the present invention provides an immunoconjugate. In one embodiment, when so labeled, connected, or loaded with an α-emitter, the immunoconjugate is capable of delivering the α-emitter in vivo. In one embodiment, when so labeled, connected, or loaded, the immunoconjugate is also capable of delivering other radioisotopes (β-emitters and / or γ-emitters) and / or other atoms in vivo. In one embodiment, when so labeled, connected, or loaded, the immunoconjugate is capable of delivering imaging metals (e.g., 111 In、 89 Zr、 64 Cu、 68 Ga or 134 Ce).

[0628] The immunoconjugates disclosed herein can be loaded with radioisotopes to achieve therapeutic or diagnostic effects. In some embodiments, the chelating factor may also contain a radioisotope. In some embodiments, the radioisotope is an alpha emitter. In some embodiments, the radioisotope is an alpha emitter selected from the list of the following: 225 Ac、 223 Ra、 224 Ra、 227 Th、 212 Pb, 212 Bihe 213 Bi. In some implementations, the radioactive isotope is 225 Ac. In some embodiments, the radioactive isotope is a beta emitter. In some embodiments, the radioactive isotope is a beta emitter selected from: 177 Lu、 90 Y、 67 Cu and 153 Sm.

[0629] This document also describes a method for preparing a radioimmunoconjugate, the method comprising loading or conjugating the immunoconjugate of this disclosure to a radioisotope. In some embodiments, the radioisotope is an alpha emitter. In some embodiments, the radioisotope is an alpha emitter selected from the list of the following: 225 Ac、 223 Ra、 224 Ra、 227 Th、 212 Pb, 212 Bihe 213 Bi. In some embodiments, the radioactive isotope is 225-Ac. In some embodiments, the radioactive isotope is a beta emitter. In some embodiments, the radioactive isotope is a beta emitter selected from: 177 Lu、 90 Y、 67 Cu and 153 Sm.

[0630] In one aspect, the present invention provides a radioimmunoconjugate comprising the immunoconjugate of the present invention and an alpha-emitting radioisotope. In one embodiment, the alpha-emitting radioisotope of the radioimmunoconjugate is selected from the group consisting of: 225 Ac、 223 Ra、 224 Ra、 227 Th、 212 Pb, 212 Bihe 213 Bi. In one embodiment, the alpha-emitting radioisotope of the radioimmunoconjugate is selected from: 225Ac、 223 Ra、 224 Ra、 227 Th、 212 Pb, 212 Bihe 213 Bi. In one embodiment, the alpha-emitted radioisotope of the radioimmunoconjugate is 225 Ac. In one embodiment, the alpha-emitted radioisotope of the radioimmunoconjugate is 223 Ra. In one embodiment, the alpha-emitted radioisotope of the radioimmunoconjugate is... 224 Ra. In one embodiment, the alpha-emitted radioisotope of the radioimmunoconjugate is... 227 Th. In one embodiment, the alpha-emitted radioisotope of the radioimmunoconjugate is 212 Pb. In one embodiment, the alpha-emitted radioisotope of the radioimmunoconjugate is... 212 Bi. In one embodiment, the alpha-emitted radioisotope of the radioimmunoconjugate is 213 Bi.

[0631] In some embodiments, the immunoconjugate of the present invention is combined with a radioisotope to provide the radioimmunoconjugate of the present invention. In some embodiments, the radioisotope is... 225 Ac、 86 Y、 90 Y、 177 Lu、 186 Re、 188 Re、 89 Sr、 153 Sm、 213 Bi、 213 -Po、 212 Bi、 223 Ra、 224 Ra、 227 Th、 149 Tb, 68 Ga、 64 Cu、 67 Cu、 89 Zr、 137 Cs、 212 Pb or 103 Pd. In some embodiments, the radioactive isotope is an alpha emitter, such as, for example... 225 Ac、 223 Ra、 224 Ra、 227 Th、 212 Pb, 212 Bihe 213Bi. In some embodiments, the radioactive isotope is a beta particle emitter, such as, for example 177 Lu、 90 Y、 67 Cu、 153 Sm. In some embodiments, the radioactive isotope is both an alpha particle emitter and a beta and / or gamma particle emitter. In some embodiments, the radioactive isotope 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 both alpha and beta emitters, and thus contains them. In some embodiments, the radioactive isotope is obtained from, for example, 68 Ga、 64 Cu、 89 Zr、 111 In、 134 Ce is selected for use in radiographic imaging.

[0632] The immunoconjugates and radioimmunoconjugates of the present invention may contain other cargo or payloads besides radioisotopes, including various cytotoxic agents, such as, for example, small molecule chemotherapeutic agents, cytotoxic antibiotics, alkylating agents, antimetabolites, topoisomerase inhibitors, and / or tubulin inhibitors. For example, the immunoconjugates of the present invention can be used to deliver non-radioisotope cytotoxic agents to target cells. Non-limiting examples of cytotoxic agents include aziridines, cisplatins, tetrazines, procarbazine, hexamethylmelamine, and vinca alkaloids. Alkaloids, taxanes, camptothecins, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, aclarubicin, anthracyclines, actinomycin, bleomycin, plicamycin, mitomycin, daunorubicin, epirubicin, idarubicin Dolastatins, maytansines, docetaxel, adriamycin, calicheamicin, auristatin, pyrrolobenzodiazepine, carboplatin, 5-fluorouracil (5-FU), capecitabine, mitomycin C, paclitaxel, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), rifampicin, cisplatin, methotrexate, and gemcitabine.

[0633] In some embodiments, the radioimmunoconjugates of the present invention comprise a radioisotope selected from the group consisting of: 225 Ac、 86 Y、 90 Y、 177 Lu、 186 Re、 188 Re、89 Sr、 153 Sm、 213 Bi、 213 Po、 211 At、 212 Bi、 223 Ra、 224 Ra、 227 Th、 149 Tb, 68 Ga、 64 Cu、 67 Cu、 89 Zr、 137 Cs、 212 Pb and 103 Pd.

[0634] In some embodiments, the radioimmunoconjugates of the present invention comprise a radioisotope selected from the following: 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.

[0635] In some embodiments, the radioactive isotope is a radioactive isotope that emits alpha particles, and it contains... 225 Ac、 223 Ra、 224 Ra、 227 Th、 212 Pb, 212 Bi or +Bi.

[0636] In some implementations, the radioactive isotope is selected from the following radioactive isotopes that emit alpha particles: 225 Ac、 223 Ra、 224 Ra、 227 Th、 212 Pb,212 Bihe 213 Bi.

[0637] Further embodiments of the immune conjugate, antigen-binding region, and heavy chain variable region are described below:

[0638] In some embodiments, the immunoconjugate comprises a dimerizing domain or motif. In other embodiments, the dimerizing domain or motif is located in a variant-constant region, a linker, or a hinge region.

[0639] Those skilled in the art can engineer the multimeric immune conjugates of the present invention using methods and approaches known in the art. For example, engineered cysteine ​​residues can form covalent bonds, thereby stabilizing the spontaneously assembled multimeric structure (see, for example, Glockshuber R et al., Biochemistry 29:1362-7 (1990)). For example, disulfide-bonded stable structures can be generated by introducing cysteine ​​residues at specific positions, such as Cys-biantibodies, scFv' polymers, VHH polymers, VNAR polymers, and IgNAR polymers, for example 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).

[0640] Alternatively, two or more polypeptide chains can be linked together using polypeptide domains that are self-associated or polymerized (see, for example, US 6,329,507). For example, multivalent proteins containing immunoglobulin domains have been constructed using the addition of a carboxyl-terminal polymerizing domain, such as, for example, scFv, autonomous V. H Structural domain, V H H, V NARAnd IgNAR. Examples of self-associating domains known to those skilled in the art include: immunoglobulin constant domains (such as knots-into-holes; electrostatic switching and IgG / IgA chain exchange); immunoglobulin Fab chains (e.g., (Fab-scFv)2 and (Fab'scFv)2); immunoglobulin Fc domains (e.g., (sc biantibody-Fc)2, (scFv-Fc)2, and scFv-Fc-scFv); immunoglobulin CHX domains; immunoglobulin CH1-3 regions; immunoglobulin CH3 domains (e.g., (sc biantibody-CH3)2, LD microantibodies, and Flex-microantibodies); immunoglobulin CH4 domains; CHC L-domain; amphiphilic helical bundles (e.g., scFv-HLX); helical-turn-helical domains (e.g., scFv-dHlx); helical coils including leucine zippers and chondrocyte oligomeric matrix proteins (e.g., scZIP); combinations of cAMP-dependent protein kinase (PKA) dimerization and docking domains (DDD) with A kinase anchor protein (AKAP) anchoring domains (AD) (also known as "dock-and-lock" or "DNL"); streptavidin; shigatoxin B multimerization domains; tetramerization regions derived from p53; and barnase-barstar interaction domains (Pack). P, Plückthun 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); Müller 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 MolCellBiol 5:959-70 (2004); Zhang J et al., J MolBiol 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) ;

[0641] Those skilled in the art can engineer the multimeric immune conjugates of the present invention using various scFv-based peptide interactions known in the art, such as, for example, scFv-based dimers, trimers, tetramers, etc. For example, the length of the linker in the scFv can affect spontaneous assembly based on non-covalent, multimeric, multivalent structures. Generally, linkers of twelve amino acids or less, including the absence of any linkers, promote the polymerization of scFv-containing peptides or proteins into higher molecular weight substances via intermolecular domain exchange, which is more favorable than intra-chain domain pairing (see, for example, Dolezal O et al., Protein Eng 16:47-56 (2003)). However, scFvs that do not have a linker at all or have a linker of an exemplary length of 15 amino acid residues can be polymerized (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 polymeric structures produced and / or purified using techniques known in the art and / or described herein.

[0642] In some embodiments, amino acid sequence variants of the immunoconjugates described herein are considered. For example, it may be necessary to improve the binding affinity, stability, and / or other biological properties of the immunoconjugates of the present invention (e.g., altering the half-life or therapeutic window, reducing immunogenicity, or increasing ease of manufacture). Amino acid sequence variants of the immunoconjugates can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the immunoconjugate, or by synthesizing the desired immunoconjugate or polypeptide. Such modifications include, for example, fusion of immunoglobulin domains or polypeptide sequences; substitution of hinge, linker, and / or chelating factor components; and substitution of radioactive isotopes. 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 performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as a certain binding affinity level for antigen binding, a certain K+ level, etc. D Level and / or a certain Koff level.

[0643] This document provides antigen-binding antibody fragments and CDR sets. For example, such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, when compared to full-length natural antibodies (e.g., full-length camel VHH IgG2 or IgG3). Some fragments may lack amino acid residues or domains that are not essential for the desired biological activity of the antibody or for reducing the overall size of the immunoconjugates of this invention.

[0644] In some embodiments, variants of the immunoconjugates of the present invention are made larger by incorporating additional structures. In some embodiments, the immunoconjugate is linked to a heterologous or easily detectable portion. In some other embodiments, the linking comprises a protein fusion. In some other embodiments, the heterologous portion is a cytotoxic agent. In some embodiments, a carboxyl-terminal lysine residue is added to provide a site-specific attachment site. Amino acid sequence insertions include N-terminal and / or carboxyl-terminal fusions of polypeptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include immunoconjugates having an N-terminal methionine residue. Other insertion variants of the immunoconjugate include the fusion of the N-terminus or C-terminus of the immunoconjugate with an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the immunoconjugate.

[0645] The nucleic acids encoding the immunoconjugates of the present invention can be modified to produce chimeric or fusion immunoconjugate peptides, for example, by substituting the constant domains of the human heavy and light chains (replacing the homologous murine sequences with CH and CO (US Patent No. 4,816,567; and Morrison et al., Proc Natl Acad Sci USA 81:6851 (1984))) or by fusing all or part of the coding sequence of an immunoglobulin peptide with that of a non-immunoglobulin peptide (heterologous peptide). The non-immunoglobulin peptide sequences can substitute the constant domains of the immunoconjugates, or they can substitute the variable domain of an antigen-binding site of the immunoconjugate to produce chimeric bivalent immunoconjugates comprising an antigen-binding site specific to one antigen and another antigen-binding site specific to a different antigen.

[0646] Variations of antibody constructs used as antigen-binding domains in the invention described herein can be generated, for example, using any techniques and guidance on conserved and non-conserved mutations as set forth in, for example, U.S. Patent No. 5,364,934. Variations can be substitutions, deletions, or insertions of one or more codons encoding an immunoconjugate or polypeptide, resulting in an amino acid sequence change compared to the native sequence antibody or polypeptide. Optionally, the variation is achieved by substituting at least one amino acid in one or more domains of the immunoconjugate for any other amino acid. By comparing the sequence of the immunoconjugate with the sequence of a homologous known protein molecule and minimizing the number of amino acid sequence changes performed in regions of high homology, guidance can be found to determine which amino acid residues can be inserted, substituted, or deleted without adversely affecting the desired activity. Amino acid substitution can result in replacing one amino acid with another amino acid having similar structure and / or chemical properties, such as replacing leucine with serine, i.e., a conserved amino acid substitution. Insertions or deletions can optionally range from about 1 to 5 amino acids. Permissible variations can be determined by systematically inserting, deleting, or substituting amino acids in the sequence and testing the activity exhibited by the full-length or mature native sequence of the resulting variant.

[0647] In specific implementations, the conserved 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, more substantial changes are introduced (named exemplary substitutions in Table C, or further described below with reference to amino acid categories), and products are screened.

[0648] Table C.

[0649] Original residues Exemplary replacement Preferred replacement Ala(A) val; leu; ile val Arg(R) lys; gln; asn lys Asn(N) gin; his; lys; arg gln Asp(D) glu glu

[0650] Original residues Exemplary replacement Preferred replacement Cys(C) ser ser Gin(Q) asn asn Glu(E) asp asp <![CDATA[Gl y (G)]]> pro,ala ala His(H) asn; gln; lys; arg arg Ile(I) leu;val;met;ala;phe;orleucine leu Leu(L) Leucine; ile; val; met; ala; phe ile Lys(K) arg; gln; asn arg <![CDATA[M e of@)]]> leu,phe,ile leu Phe(F) leu,val,ile,ala,tyr leu Pro(P) ala ala Ser(S) thr thr Thr(T) ser ser Trp(W) tyr;phe tyr <![CDATA[T y r(Y)]]> trp; phe; thr; ser phe Val(V) ile; leu; met; phe; ala; leucine leu

[0651] The substantial modification of the functional or immunological marker of the immunoconjugates of the present invention is accomplished through selective substitution, which differs significantly in maintaining: (a) the structure of the polypeptide backbone in the substituted region, for example, a sheet-like or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the packing volume of the side chains. Based on common side chain properties, naturally occurring residues are divided into the following groups:

[0652] (1) Hydrophobic: ortholeucine, met, ala, val, leu, ile;

[0653] (2) Neutral hydrophilicity: cys, ser, thr;

[0654] (3) Acidic: asp, glu;

[0655] (4) Alkaline: asn, gln, his, lys, arg;

[0656] (5) Residues that affect chain orientation: gly, pro; and

[0657] (6) Aromatics: trp, tyr, phe.

[0658] Non-conservative substitutions would require exchanging members of one of these categories for another. Such substituted residues can also be introduced into conserved substitution sites, or more preferably, the remaining (non-conservative) sites.

[0659] Mutation can be performed 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)), box mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction-selective mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniques can be used to produce DNA molecules encoding the immunoconjugate variants of the present invention.

[0660] In some embodiments, immunoconjugate variants with one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include the immunoglobulin variable domains HVR and FR, as well as regions within the immunoglobulin constant domains. Amino acid substitutions can be introduced into the immunoconjugate of interest, and desired activities of the product can be screened, such as enhanced / retained antigen binding, reduced / retained immunogenicity, enhanced / retained antibody-dependent cytotoxicity (ADCC), enhanced / retained complement-dependent cytotoxicity (CDC), enhanced / retained target inhibition, and / or enhanced / retained antibody-dependent cell-mediated phagocytosis (ADCP). Similarly, amino acid substitutions can be introduced into the immunoconjugate of interest, and the activity of the product can be reduced or eliminated, such as ADCC, CDC, target inhibition, and / or ADCP.

[0661] One type of substitution variant involves replacing one or more hypervariable residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting variant selected for further research will have modifications (e.g., enhancements) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and / or will substantially retain certain biological properties of the parent antibody. Illustrative substitution variants are affinity-matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. In short, one or more HVR residues are mutated, and the variant antibody is displayed on a phage and screened for its specific biological activity (e.g., binding affinity).

[0662] Alterations (e.g., substitutions) can be made in the HVR, for example, to improve the affinity of the immunoconjugate. Such alterations can be made in HVR “hotspots” (i.e., residues encoded by codons that undergo mutations at a high frequency during somatic maturation) (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or SDR (a-CDR), testing the binding affinity of the resulting variant VH or VL. For example, Hoogenboom et al. described affinity maturation by constructing a secondary library and reselecting from it in Methods in Molecular Biology 178:1-37 (edited by O'Brien et al., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand truncation, or oligonucleotide directed mutagenesis). The secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves HVR-targeting methods, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. Alanine scan mutagenesis or modeling can be used, for example, to specifically identify HVR residues involved in antigen binding. CDR-H3 and CDR-L3 are particularly frequently targeted.

[0663] In some embodiments, substitution, insertion, or deletion can occur within one or more HVRs, as long as such changes do not significantly reduce the ability of the immunoconjugate to bind to the antigen. For example, conserved changes that do not significantly reduce binding affinity (e.g., conserved substitutions as described herein) can be made in the HVRs. Such changes can occur outside of HVR “hotspots” or SDRs. In some embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0664] A method used to identify antibody residues or regions that can be targeted by mutagenesis is called "alanine scan mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Additional substitutions can be introduced at the amino acid position to demonstrate functional sensitivity to the initial substitution.

[0665] Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact sites between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine if they contain the desired properties.

[0666] In some embodiments, the immunoconjugates of the present invention comprise an antibody construct (used herein as an antigen-binding region) comprising a humanized immunoglobulin domain.

[0667] Humanized forms of non-human (e.g., camel, mouse, or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of the antibody) containing minimal sequences derived from non-human immunoglobulins. Humanized antibodies comprise human immunoglobulins (receptor antibodies) in which residues from the receptor complementarity-determining region (CDR) are replaced by residues from a non-human species (e.g., camel, mouse, rat, or rabbit) (donor antibody) having the desired specificity, affinity, and capability. In some cases, Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibodies may also contain residues not found in the receptor antibody or in the introduced CDR or framework sequence. Generally, humanized antibodies will contain at least one, and typically substantially all, of two variable domains, wherein all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are those of human immunoglobulin common sequences. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region of human immunoglobulins (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)).

[0668] Methods for humanizing nonhuman antibodies are well known in the art. Generally, humanized antibodies have one or more amino acid residues introduced from a nonhuman source. These nonhuman amino acid residues are often referred to as “input” residues, which are typically derived from the “input” variable domain. Humanization can be largely performed by following the approach of Winter and colleagues (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 replacing the corresponding sequence of the human antibody with a rodent CDR or CDR sequence. Thus, such “humanized” antibodies are chimeric antibodies (US Patent No. 4,816,567), in which substantially less than the complete human variable domain is replaced by a corresponding sequence from a nonhuman species. In the process, humanized antibodies are usually human antibodies, in which some of the CDR residues and possibly some FR residues are replaced by residues from similar sites in rodent antibodies.

[0669] According to another approach, antigen binding can be restored during antibody humanization by selectively repairing hypervariable regions (see, for example, U.S. Application Serial No. 11 / 061,841, filed February 18, 2005). This approach involves incorporating a nonhuman hypervariable region into the receptor framework and further introducing one or more amino acid substitutions within one or more hypervariable regions without modifying the receptor framework sequence. Alternatively, the introduction of one or more amino acid substitutions may be accompanied by modifications to the receptor framework sequence.

[0670] Generally, serine can be used to replace any cysteine ​​residues that are not involved in maintaining the correct conformation of the immunoconjugates of the present invention to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds can be added to the immunoconjugates of the present invention to improve their stability (especially when the antibody is an antibody fragment, such as an Fv fragment or a VHH fragment).

[0671] In some embodiments, it may be necessary 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 are located at accessible sites of the immunoconjugate. By substituting those residues with cysteine, a reactive thiol group is thereby positioned at an accessible site of the immunoconjugate and can be used to conjugate the immunoconjugate to other parts, such as a pharmaceutical part or a linker-pharmaceutical part. In some embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat number) of the light chain; Al18 (EU number) of the heavy chain; and S400 (EU number) of the Fc region of the heavy chain. Cysteine-engineered antibodies can be generated as described, for example, in US 7,521,541.

[0672] Technicians will understand that amino acid changes can alter the post-translational processes of immune conjugates, such as changing the number or location of glycosylation sites or altering membrane anchoring characteristics.

[0673] In some embodiments, the immunoconjugates provided herein are modified to increase or decrease the degree of glycosylation and / or alter the glycosylation pattern. "Altering the native glycosylation pattern" is intended, for the purposes of this document, to mean the deletion of one or more carbohydrate moieties found in the parental immunoconjugates of the present invention (by removing potential glycosylation sites or by chemical and / or enzymatic deletion), and / or the addition of one or more glycosylation sites not present in the native sequence immunoconjugates of the present invention. Furthermore, the phrase includes qualitative changes in the glycosylation of native proteins, relating to changes in the characteristics and proportions of the various carbohydrate moieties present.

[0674] Glycosylation of antibodies and other peptides is typically N-linked or O-linked. N-linking refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in the peptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyl amino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine can also be used.

[0675] Adding or deleting glycosylation sites to immunoconjugates can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites. Adding glycosylation sites to the immunoconjugates of the present invention can be conveniently achieved by altering the amino acid sequence to contain one or more of the aforementioned tripeptide sequences (for N-linked glycosylation sites). Alterations can also be made by adding or substituting one or more serine or threonine residues into or into the sequence of the original immunoconjugate of the present invention (for O-linked glycosylation sites). The amino acid sequence of the immunoconjugates of the present invention can optionally be altered by changes at the DNA level, particularly by mutating the DNA encoding the immunoconjugates of the present invention at preselected bases to generate codons that will be translated into the desired amino acids.

[0676] In the case of immunoconjugates containing an Fc region, the carbohydrates attached thereto can be modified. Naturally occurring antibodies produced by mammalian cells typically contain branched bitennary oligosaccharides of Asn297 attached to the CH2 domain of the Fc region via N-linkage (see, for example, Wright et al., TIBTECH 15:26-32 (1997)). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc attached to the “backbone” of the bitennary oligosaccharide structure. In some embodiments, the oligosaccharides in the immunoconjugates of the present invention can be modified to produce immunoconjugate variants with improved properties.

[0677] Another means of increasing the amount of carbohydrate moiety on the immunoconjugates of the present invention is through chemical or enzymatic coupling of glycosides with polypeptides. Such methods are described in the art, for example in WO87 / 05330, published on September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0678] The removal of the carbohydrate moiety present on the immunoconjugates of the present invention can be accomplished by chemical or enzymatic means or by mutational substitution of codons encoding amino acid residues that act as glycosylation targets. Chemical deglycosylation techniques are known in the art and have been described, for example, by Hakimuddin et al., Arch. Biochem. Biophys., 259:52 (1987) and Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of the carbohydrate moiety on the polypeptide can be achieved by using various endonucleases and exonucleases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).

[0679] In some embodiments, immunoconjugate variants are provided that have a carbohydrate structure lacking (directly or indirectly) fucose attached 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 at Asn297 within the glycan chain relative to the sum of all glycan structures attached to Asn297 (e.g., complex, mixed, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 (Eu number of the Fc region residue) in the Fc region; however, due to the small sequence variation in the antibody, Asn297 may 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 enhanced ADCC function (see, for example, US 2003 / 0157108; US 2004 / 0093621). Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003 / 0157108; WO2000 / 61739; WO 2001 / 29246; US 2003 / 01 15614; US 2002 / 0164328; US 2004 / 0093621; US2004 / 0132140; US 2004 / 0110704; US 2004 / 01 10282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 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 defucosylation antibodies include Lecl3CHO cells with protein fucosylation defects (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US2003 / 0157108; WO 2004 / 056312, Adams et al., especially in Example 11) and knockout cell lines, such as α-1,6-fucosylation gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); WO2003 / 085107).

[0680] Immunoconjugate variants also have bisected oligosaccharides, for example, in which the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such immunoconjugate variants may have reduced fucosylation and / or enhanced ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878, US 6,602,684, and US 2005 / 0123546. Immunoconjugate variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such immunoconjugate variants may have enhanced CDC function. Such antibody variants are described, for example, in WO 1997 / 030087, WO 1998 / 058964, and WO 1999 / 022764.

[0681] Immunoconjugates, derivatives and other modifications

[0682] 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 the target amino acid residues of the immunoconjugates of the present invention with an organic derivatizing agent capable of reacting with selected side chains or N-terminal or C-terminal residues of the immunoconjugates. Derivatization using bifunctional agents can be used, for example, to crosslink 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 crosslinking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane; glutaraldehyde; N-hydroxysuccinimide esters, such as esters with 4-azidosalicylic acid; homodifunctional imides, including disuccinimide esters, such as 3,3'-dithiobis(succinimide propionate); bifunctional maleimides, such as bis-N-maleimide-1,8-octane; and agents such as methyl-3-[(p-azidophenyl)dithio]propionimide ester.

[0683] Other modifications include: deaminoation of glutamine acyl and asparagine acyl residues to the corresponding glutamine acyl and asparagine acyl residues, respectively; hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of serine or threonine residues; methylation of the α-amino groups of the lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)); acetylation of N-terminal amines; and amidation of any C-terminal carboxyl group.

[0684] In some embodiments, the immunoconjugates provided herein can be further modified to contain additional non-protein moieties known and readily available in the art. Suitable derivatization moieties for 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), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the immunoconjugate may vary, and if more than one polymer is attached, they may be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the immunoconjugate to be enhanced, and whether the immunoconjugate derivative will be used in therapy under defined conditions.

[0685] The PEG-derived immunoconjugates of the present invention may contain a linker comprising one or more -CH2CH2O- and can be used to modify the biodistribution and pharmacokinetics of the immunoconjugates. PEG may be prepared in polymeric or discrete oligomeric forms. The bifunctionalized forms of these polymers may link the immunoconjugates to chelating agents and / or provide additional size and / or solubility to the overall molecule. In some embodiments, the PEG-derived immunoconjugates exhibit reduced immunogenicity compared to their underived parent molecules.

[0686] Method for generating the immunoconjugate of the present invention

[0687] This invention provides a composition comprising one or more of the immunoconjugates as described in any of the above embodiments or herein. In another aspect, this invention provides an isolated nucleic acid encoding a radioisotope delivery platform as described herein. This invention also provides nucleic acids encoding protein components of the immunoconjugates of this invention, expression vectors comprising the aforementioned nucleic acids, and host cells comprising the aforementioned expression vectors.

[0688] In another aspect, the present invention provides a host cell comprising nucleic acids and / or vectors as provided herein. In some embodiments, the host cell of the present invention is isolated or purified. In some embodiments, the host cell of the present invention is in a 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 cell is a eukaryotic cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is *Escherichia coli*.

[0689] The following describes illustrative techniques for producing the immunoconjugates and radioimmunoconjugates of the present invention for use according to the methods of the present invention. In some embodiments, the present invention provides a method for preparing the immunoconjugates of the present invention, the method comprising culturing host cells as provided herein under conditions suitable for an expression vector encoding a radioisotope delivery platform and recovering or purifying the radioisotope delivery platform. In some embodiments, the method further comprises radiolabeling the radioisotope delivery platform with a suitable isotope (such as, for example, an alpha or beta particle emitter).

[0690] Antigen-binding domains, immune conjugates, and the generation and identification of nucleic acids

[0691] Antigen-binding domains that can be used as antigen-binding regions in this paper can be identified in antibodies, which are monoclonal and / or polyclonal antibodies. The DNA encoding the monoclonal antibody can be easily isolated and sequenced using standard procedures. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells (such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody proteins) to obtain the synthesis of monoclonal antibodies in recombinant host cells (see, for example, Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Plückthun, Immunol Revs. 130:151-188 (1992)).

[0692] In some embodiments, the antigen-binding domains or fragments thereof of the immunoconjugates of the present invention are isolated by screening phage libraries containing phages displaying various antibody variable region fragments (Fv, scFv, or VHH) fused to a phage coat protein. Such phage libraries are screened for binding to a desired target antigen or epitope. Clones expressing an Fv fragment, scFv, or VHH capable of binding the desired antigen are adsorbed onto 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 antigen adsorption / elution cycles.

[0693] In some implementations, antibodies or antibody fragments thereof are isolated from antibody phage libraries generated using techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J Mol Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the generation of high-affinity (nM range) human antibodies via chain truncation (Marks et al., Bio / Technology, 10:779-783 (1992)), as well as combined infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nuc Acids Res. 21:2265-2266 (1993)). Variable domains can be functionally displayed on phages as single-chain Fv (scFv) fragments (in which VH and VL are covalently linked by a short flexible peptide) or as Fab fragments (in which they are each fused to a constant domain and interact non-covalently), as described by Winter et al., Ann. Rev. Immunol., 12:433-455 (1994).

[0694] The VH and VL gene libraries can be cloned individually by polymerase chain reaction (PCR) and randomly recombined in a phage library, and their antigen-binding clones can then be searched as described by Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Initial libraries for screening can be constructed from non-immunogenic sources to provide antibodies with high affinity for the antigens (see, for example, Griffiths et al., EMBO J, 12:725-734 (1993)). Another example is the synthetic construction of initial libraries by cloning an unrearranged V gene fragment from stem cells and encoding a highly variable CDR3 region using PCR primers containing random sequences, followed by in vitro rearrangement, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

[0695] Library screening can be performed using a variety of techniques known in the art. For example, target antigens can be used to coat the wells of an adsorption plate, expressed on host cells attached to the adsorption plate, used for cell sorting, conjugated with biotin to capture with streptavidin-coated beads, or used in any other panning method for displaying libraries. The selection of antibodies with slow dissociation kinetics (and strong binding affinity) can be facilitated by using prolonged washing and monovalent phage display (as described in Bass et al., Proteins, 8:309-314 (1990) and WO 1992 / 09690) and low antigen coating density (as described in Marks et al., Biotechnol., 10:779-783 (1992)).

[0696] 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 genes of interest or proteins encoded by them. cDNA or genomic libraries can be screened with selected probes 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 method for isolating genes encoding the immunoconjugates of this invention is to use PCR methods (Sambrook et al. (as described above); Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)).

[0697] The DNA encoding the immunoconjugates of the present invention can be obtained from a cDNA library prepared from tissues believed to contain the mRNA of the immunoconjugates of the present invention and express it at a detectable level. Therefore, the human immunoconjugate DNA of the present invention can be readily obtained from cDNA libraries prepared from human tissues. The genes encoding the immunoconjugates of the present invention can also be obtained from genomic libraries or through known synthetic procedures (e.g., automated nucleic acid synthesis). For some embodiments, the desired polynucleotide sequence encoding the antibody can be isolated from antibody-producing cells such as hybridoma cells and sequenced.

[0698] Sequences identified in such library screening methods can be compared and aligned with other known sequences residing in public databases such as GenBank or other private sequence databases. Sequence identity (at the amino acid or nucleotide level) within a defined region of a molecule or across its full length can be determined using methods known in the art and as described herein. Any of the antibody CDRs or heavy chain variable fragments of the present invention can be obtained by designing an antigen screening procedure suitable for selecting phage clones of interest, and then constructing an antibody clone using the variable domain and / or CDR sequence from the phage clone of interest and a suitable constant region (Fc) sequence as described in Kabat et al., 1991 (as above).

[0699] Generation of immune conjugates; host cells and expression vectors of the present invention

[0700] The following description primarily relates to the production of antibody constructs of the present invention by culturing cells transformed or transfected with a vector containing nucleic acids encoding the immunoconjugates of the present invention. Of course, alternative methods well known in the art are contemplated for preparing the antibody constructs of the present invention. For example, suitable amino acid sequences or portions thereof can be produced by direct peptide synthesis using solid-phase techniques (e.g., Stewar et al., Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, CA (1969); Merrifield, J, Am. Chem. Soc., 85:2149-54 (1963)). In vitro protein synthesis can be performed manually or automatically. For example, automated synthesis can be performed using an Applied Biosystems peptide synthesizer (Foster City, CA) following the manufacturer's instructions. The various portions of the immunoconjugates of the present invention can be chemically synthesized individually and combined using chemical or enzymatic methods to produce the desired immunoconjugates of the present invention.

[0701] Antibody constructs can be produced using recombinant methods and compositions, for example, as described in US 4,816,567. In one embodiment, an isolated nucleic acid encoding the antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising antibody VH and / or VL (e.g., antibody light chain and / or heavy chain). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acids is provided. In some embodiments, the host cell comprises (e.g., transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising antibody VH. In some other embodiments, the host cell comprises: (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising antibody VL and an amino acid sequence comprising antibody VH, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising antibody VL and a second vector comprising a nucleic acid encoding an amino acid sequence comprising antibody VH. In one embodiment, the host cell is a eukaryotic cell, for example, Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., YO, NSO, Sp20 cells). In one embodiment, a method for preparing the immunoconjugate of the present invention is provided, wherein the method includes culturing a host cell containing a nucleic acid encoding an antibody as provided above under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0702] For the recombinant generation of the immunoconjugates of the present invention, nucleic acids encoding antibody constructs, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures, e.g., by using oligonucleotide probes capable of specifically binding to genes encoding antibody heavy and / or light chains. Nucleic acid molecules encoding the amino acid sequences (including sequence variants) of the immunoconjugates of the present invention can be prepared by a variety of methods known to those skilled in the art. These methods include, but are not limited to, preparation by isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of variant or non-variant forms of the antibody construct prepared earlier.

[0703] Manipulating host cells for the production of immune conjugates

[0704] Host cells are transfected or transformed with the expression or cloning vectors described herein to achieve the production of the conjugates of the present invention, and cultured in a suitably modified conventional nutrient medium to induce promoters, select transformants, or amplify genes encoding desired sequences. Culture conditions, such as medium, temperature, pH, etc., can be selected by a technician without excessive experimentation. Generally, the principles, procedures, and practical techniques for maximizing the production capacity of cell cultures can be found in *Mammalian Cell Biotechnology: A Practical Approach*, edited by M. Butler (IRL Press, 1991) and Sambrook et al. (as above).

[0705] 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 generated in bacteria, especially when glycosylation and Fc effector function are not required. For information on antibody fragment and peptide expression 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 (edited by BKCLo, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in *Escherichia coli*. Following expression, the immunoconjugate can be separated from the bacterial cell paste in soluble fractions and can be further purified.

[0706] In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable cloning or expression hosts for vectors encoding immune conjugates, including fungal and yeast strains whose glycosylation pathways have been “humanized”, resulting in the production of antibodies with partial or complete human glycosylation patterns (see, for example, Gerngross, Nat. Biotech. 22:1409-1414 (2004); Li et al., Nat. Biotech. 24:210-215 (2006)).

[0707] Host cells suitable for the expression of glycosylated immunoconjugates are also derived from multicellular organisms (e.g., invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that are suitable for use in combination with insect cells, particularly for transfection of fall armyworm (Spodoptera frugiperda) cells. Plant cell cultures have also been used as hosts (see, for example, US 5,959,177; US 6,040,498; US 6,420,548; US 7,125,978; and US 6,417,429).

[0708] Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are: monkey kidney CV 1 line transformed from SV40 (COS-7); human embryonic kidney line, 293 or 293 cells, as described, for example, in Graham et al., J Gen Viral. 36:59 (1977); juvenile hamster kidney cells (BHK); mouse Sertoli cells, TM4 cells, as described, for example, in Mather, Biol. Reprod. 23:243-251 (1980); monkey kidney cells (CV 1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MOCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep02); mouse mammary tumors (MMT). 060562); TRI cells, such as those described, for example, in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFK CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77:4216 (1980)); and myeloma cell lines such as YO, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for the production of immune conjugates, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0709] Methods for eukaryotic cell transfection and prokaryotic cell transformation are known to those skilled in the art, referring to the introduction of DNA into the host in an extrachromosomal form or via chromosomal integrities, making the DNA replicable, such as CaCl2, CaPO4, liposome-mediated, polyethylene glycol / DMSO, and electroporation. Depending on the host cells used, standard techniques suitable for those cells are employed for transformation. Calcium treatment with calcium chloride or electroporation, as described by Sambrook et al. (as above), is commonly used for prokaryotes. *Agrobacterium tumefaciens* infection has been used to transform 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, calcium phosphate precipitation, as described by Graham and van der Eb, *Virology*, 52:456-457 (1978), can be used. General aspects of transfection into mammalian cell-host systems have been described in U.S. Patent No. 4,399,216. Transformation into yeast is typically performed according to the methods described in 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 can also be used, such as by nuclear microinjection, electroporation, fusion of bacterial protoplasts with intact cells, or polycations such as polyglobulin and polyornithine. 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).

[0710] prokaryotic host cells

[0711] Suitable prokaryotes include, but are not limited to, archaebacteria and eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, including Escherichia coli. Various strains of Escherichia coli 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, such as Escherichia (e.g., Escherichia coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescans), and Shigella, as well as Bacillus (e.g., B. subtilis and B. licheniformis) (e.g., DD published on April 12, 1989). The examples disclosed in 266,710 include *Bacillus licheniformis* 41P, *Pseudomonas* (such as *P. aeruginosa*), *Rhizobia*, *Vitreoscilla*, *Paracoccus*, and *Streptomyces*. These examples are illustrative and not limiting. *Escherichia coli* strain W3110 is a favorable host or parental host because it is a common host strain for the fermentation of recombinant DNA products. Preferably, the host cells secrete a minimal amount 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 number 27,325) can be modified to achieve gene mutations encoding endogenous proteins in the host. Examples of such hosts include: Escherichia coli W3110 strain 1A2 with the complete genotype tonA; Escherichia coli W3110 strain 9E4 with the complete genotype tonA ptr3; Escherichia coli W3110 strain 27C7 (ATCC 55,244) with the complete genotype tonA ptr3 phoA E15(argF-lac)169degP ompT kanr; and Escherichia coli W3110 strain 37D6 with the complete genotype tonA ptr3 phoAE15(argF-lac)169degP ompT rbs7. ilvG kanr; Escherichia coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin-resistant degP deletion mutation; Escherichia coli W3110 strain 33D3 with the genotype W3110ΔfhuA(ΔtonA)ptr3 lac Iq lacL8ΔompTΔ(nmpc-fepE)degP41 kanR (US Patent No. 5,639,635); and Escherichia coli strains with the mutant periplasmic protease disclosed in US Patent No. 4,946,783, issued August 7, 1990. Other strains and their derivatives, such as Escherichia coli 294 (ATCC 31,446), Escherichia coli B, Escherichia coli λ1776 (ATCC 31,537), and Escherichia coli RV308 (ATCC 31,608), are also suitable. These examples are illustrative and not limiting. Methods for constructing derivatives of any of the bacteria with defined genotypes described above are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). The reproducibility of the replicon in bacterial cells is typically considered when selecting suitable bacteria. For example, *Escherichia coli*, *Serratia*, or *Salmonella* species may be suitable as hosts when well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to provide the replicon. Typically, the host cell should secrete a minimal amount of proteolytic enzymes, and additional protease inhibitors may be desirablely incorporated into the cell culture. Alternatively, in vitro cloning methods, such as PCR or other nucleic acid polymerase reactions, are suitable.

[0712] Full-length antibodies, antibody fragments, and antibody fusion proteins can be produced in bacteria, especially when glycosylation and Fc effector function are not required. Full-length antibodies have a longer half-life in circulation. Production in *E. coli* is faster and more cost-effective. For expression of antibody fragments and peptides in bacteria, see, for example, US5,648,237, US5,789,199, and US5,840,523, which describe translation initiation regions (TIRs) and signal sequences for optimizing expression and secretion. After expression, the immunoconjugate can be separated from the *E. coli* cell paste in soluble fractions and purified according to isotype, for example, by protein A or G columns. Final purification can be performed similarly to the process used to purify antibodies expressed, for example, in CHO cells.

[0713] eukaryotic host cells

[0714] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for vectors encoding the immune conjugates of this 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); hosts of *Kluyveromyces* (US Patent No. 4,943,529; Fleer et al., Bio / Technology, 9:968-75 (1991)), such as *Kluyveromyces lactis* (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 (1983)), *Kluyveromyces fragilis* (ATCC 12,424), and *Kluyveromyces bulgaricus* (ATCC 12,424). 16,045), Kluyveromyces wickeramii (ATCC 24,178), Kluyveromyces waltii (ATCC 56,500), Kluyveromyces drosophilarum (ATCC 36,906; Van den Berg et al., Bio / Technology, 8:135 (1990)), Kluyveromyces thermomyotrophicus (Kluyveromyces thermolones) and Kluyveromyces marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol).,28:265-278(1988)); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., ProcNatl Acad Sci USA 76:5259-5263(1979)); Schwanniomyces, such as Schwanniomyces occidentalis (EP 394,538 published on October 31, 1990); and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium (WO published on January 10, 1991). 91 / 00357) and Aspergillus hosts, such as Aspergillus nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289 (1983); Tilburn et al., Gene, 26:205-221 (1983); Yelton et al., Proc Natl Acad Sci USA 81:1470-1474 (1984)) and Aspergillus niger (Kelly and Hynes, EMBO J., 4:475-479 (1985)). The term "methylotropic yeast" applies here and includes, but is 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 as examples of this type of yeast can be found in C. Anthony, *The Biochemistry of Methylotrophs*, 269 (1982).

[0715] Suitable host cells for expressing 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; and plant cells, such as cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco. Numerous baculovirus strains and variants, as well as corresponding licensed insect host cells from hosts such as fall armyworm (Spodoptera frugiperda) (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm). A variety of viral strains for transfection are publicly available, such as the L-1 variant of the alfalfa silver-striped armyworm (Autographa californica) NPV and the Bm-5 strain of the silkworm NPV, and such viruses can be used as viruses according to the present invention, particularly for transfection of fall armyworm cells.

[0716] However, the greatest interest lies in vertebrate cells, and the proliferation of vertebrate cells in cultures (tissue cultures) has become a routine procedure. Examples of useful mammalian host cell lines include: monkey kidney CV1 line transformed from SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (subcloned for use in suspension culture as 293 or 293 cells, Graham et al., J. Gen Virol. 36:59 (1977)); juvenile 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 setroli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); and human cervical cancer cells (HELA, ATCC CCL 1651). 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCCCCL51); TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatocellular carcinoma lineage (Hep G2).

[0717] The host cells are transformed with the expression or cloning vectors described above to achieve the production of the conjugates of the present invention, and cultured in a conventional nutrient medium with appropriate modifications in order to induce promoters, select transformants, or amplify genes encoding the desired sequences.

[0718] Selection and use of reproducible media

[0719] To reconstitute the radioisotope delivery platform of this invention, the nucleic acid encoding it (e.g., cDNA or genomic DNA) is isolated and inserted into a reproducible vector for further cloning (DNA amplification) or for expression. DNA encoding immunoconjugates can be readily isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding antibody heavy and light chains). Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, suitable host cells are of prokaryotic or eukaryotic (typically mammalian) origin.

[0720] Vectors can be in the form of, for example, plasmids, granules, viral particles, or bacteriophages. Appropriate nucleic acid sequences can be inserted into vectors using various procedures. Generally, DNA is inserted into appropriate restriction endonuclease sites using techniques known in the art. Vector components typically include, but are not limited to, one or more of a signal sequence, origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. The construction of suitable vectors containing one or more of these components employs standard ligation techniques known to those skilled in the art.

[0721] The immunoconjugates of the present invention can be generated not only through direct recombination but also as fusion peptides with heterologous polypeptides, which can be signal sequences or other polypeptides having specific cleavage sites at the N-terminus of mature proteins or polypeptides. Generally, the signal sequence can be a component of a vector, or it can be a portion of the DNA encoding the immunoconjugates of the present invention inserted into the vector. The signal sequence can be a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, lpp, or a heat-stable enterotoxin II leader sequence. For yeast secretion, the signal sequence can be, for example, a yeast invertase leader sequence, an α-factor leader sequence (including α-factor leader sequences of *Saccharomyces* and *Kluyveromyces*, the latter described in U.S. Patent No. 5,010,182), or an acid phosphatase leader sequence, a *Candida albicans* glucosylamylase leader sequence (EP 362,179, published April 4, 1990), or the signal described in WO 90 / 13646, published November 15, 1990. In mammalian cell expression, mammalian signal sequences can be used to guide protein secretion, such as signal sequences of secreted peptides from the same or related species and viral secretion leader sequences.

[0722] Culture host cells to generate a radioisotope delivery platform

[0723] The host cells used to generate the immune conjugates of the present invention can be cultured in a variety of culture media and under various culture conditions.

[0724] prokaryotic host cell culture

[0725] Prokaryotic cells used to produce the polypeptides of the present invention are grown in a culture medium known in the art and suitable for culturing selected host cells. Examples of suitable culture media include Luria broth (LB) with necessary nutritional supplements. In some embodiments, the culture medium also contains a selector chosen 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 culture medium for the growth of cells expressing an ampicillin resistance gene.

[0726] In addition to sources of carbon, nitrogen, and inorganic phosphates, any necessary supplements may be included at appropriate concentrations, introduced alone or in mixture with another supplement or culture medium (such as a complex nitrogen source). Optionally, the culture medium may contain one or more reducing agents selected from the following: glutathione, cysteine, cystamine, thioglycolate, dithioerythritol, and dithiothreitol.

[0727] The prokaryotic host cells are cultured at a suitable temperature. For example, for the growth of *Escherichia coli*, the preferred temperature range is from about 20°C to about 39°C, more preferably from about 25°C to about 37°C, and even more preferably from about 30°C. The pH of the culture medium can be any pH ranging from about 5 to about 9, depending primarily on the host organism. For *Escherichia coli*, the pH is preferably from about 6.8 to about 7.4, more preferably from about 7.0.

[0728] If an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for promoter activation. In one aspect of the invention, the PhoA promoter is used to control the transcription of the polypeptide. Therefore, transformed host cells are cultured in a phosphate-restricted medium for induction. In some embodiments, the phosphate-restricted medium is CRAP medium (see, for example, Simmons et al., J. Immunol. Methods (2002), 263:133-47). Depending on the vector construct used, a variety of other inducers, such as those known in the art, may be used.

[0729] In one embodiment, the expressed peptides of the present invention are secreted into the periplasm of host cells and recovered from the periplasm. Protein recovery typically involves disrupting the microorganisms, usually 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 proteins can be further purified, for example, by affinity resin chromatography. Alternatively, the proteins can be transferred to a culture medium and separated therein. Cells can be removed from the culture, and the culture supernatant can be filtered and concentrated for further purification of the resulting proteins. The expressed peptides can be further separated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blotting.

[0730] In one aspect of the invention, immunoconjugates are produced in large quantities via fermentation. Various large-scale fed-batch fermentation programs can be used for the production of recombinant proteins. Large-scale fermentations have a capacity of at least 1000 liters, preferably from about 1,000 to 100,000 liters. These fermenters use agitator impellers to distribute oxygen and nutrients, particularly glucose (a preferred carbon / energy source). Small-scale fermentation typically refers to fermentation in fermenters with a volume capacity not exceeding about 100 liters, and ranging from about 1 liter to about 100 liters.

[0731] In fermentation processes, induction of protein expression is typically initiated after cells have grown to the desired density (e.g., an OD550 of approximately 180-220) under suitable conditions (at which stage the cells are in an early stationary phase). Depending on the vector construct used, a variety of inducers can be used, such as those known in the art and described above. A relatively short period of cell growth can be allowed before induction. Cells are typically induced for approximately 12-50 hours, but longer or shorter induction times can be used.

[0732] To improve the yield and quality of the peptides of this invention, various fermentation conditions can be modified. For example, to improve the correct assembly and folding of the secreted immunoconjugated peptides, host prokaryotic cells can be co-transformed using vectors that overexpress chaperone proteins (such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and / or DsbG) or FkpA (a peptidyl prolyl cis-trans isomerase with chaperone protein activity). Chaperone proteins have been shown to facilitate the correct folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al., (1999) J Bio Chem 274:19601-5; US Patent No. 6,083,715; US Patent 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.

[0733] To minimize the proteolysis of expressed heterologous proteins (especially those sensitive to proteolysis), certain host strains lacking proteolytic enzymes can be used in this invention. For example, host cell strains can be modified to induce mutations in one or more 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 described, for example, in Joly et al., (1998), ibid.; U.S. Patent No. 5,264,365; U.S. Patent No. 5,508,192; Hara et al., *Microbial Drug Resistance*, 2:63-72 (1996).

[0734] In one embodiment, an Escherichia coli strain lacking proteolytic enzymes and transformed with a plasmid overexpressing one or more chaperone proteins is used as the host cell in the expression system of the present invention.

[0735] eukaryotic host cell culture

[0736] Commercially available culture media, such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Durbecco's Modified Eagle Medium (DMEM, Sigma), are suitable for culturing host cells. Additionally, any of the culture media 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; WO 90 / 03430; WO87 / 00195; or U.S. Patent No. 30,985 can be used as the culture medium for host cells. Any of these culture media can be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), and antibiotics (such as GENTAMYCIN). TM The formula may include drugs, trace elements (defined as inorganic compounds typically present in final concentrations in the micromolar range), and glucose or an equivalent energy source. It may also include any other necessary supplements in appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those previously used with the host cells selected for expression and are obvious to those skilled in the art.

[0737] Purification of the immunoglobulin-derived structures of the present invention

[0738] The immunoconjugates of the present invention can be recovered from culture medium or from host cell lysates. If membrane-bound, they can be detached from the membrane using a suitable detergent solution (e.g., Triton-X 100) or by enzymatic lysis. Cells used to express the immunoconjugates of the present invention can be disrupted by various physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysis agents.

[0739] Purification of the immunoconjugates of the present invention from recombinant cellular proteins or peptides may be necessary. The following procedures are examples of suitable purification procedures: fractionation on an ion-exchange column; ethanol precipitation; reversed-phase HPLC; chromatography on silica or cation-exchange resins such as DEAE; chromatographic focusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A agarose gel column to remove contaminants such as IgG; and metal chelating column to bind the immunoconjugates of the present invention in epitope-labeled form. Various protein purification methods can be employed, and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The chosen purification steps will depend, for example, on the nature of the production method used and the specific immunoconjugate of the present invention produced.

[0740] When using recombinant technologies, immunoconjugates can be generated intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the immunoconjugate is generated intracellularly, particulate debris, host cell fragments, or lysed fragments are removed as a first step, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-7 (1992) describe a procedure for isolating antibodies secreted into the periplasmic space of *E. coli*. Briefly, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and benzoyl sulfonyl fluoride (PMSF) for approximately 30 min. Cell debris can be removed by centrifugation. When the immunoconjugate is secreted into the culture medium, the supernatant from such expression systems is typically concentrated first using a commercially available protein concentrator filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors (such as PMSF) can be included in any of the preceding steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0741] Cell-prepared immunoconjugate compositions can be purified using, for example, hydroxyapatite 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 type 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. 5:1567-1575 (1986)). The most common matrix for affinity ligand attachment is agarose, but other matrices can also be used. Mechanically stable matrices (such as controlled-pore glass or poly(divinyl)styrene) allow for faster flow rates and shorter processing times compared to agarose. When the immunoconjugate contains a CH3 domain, Bakerbond ABX TM Resin (JTBaker, Phillipsburg, NJ) can be used for purification. Other protein purification techniques can also be used depending on the immunoconjugate to be recovered, such as ion exchange column fractionation, ethanol precipitation, reversed-phase HPLC, silica chromatography, and heparin SEPHAROSE. TM Chromatography, anion or cation exchange resin (such as polyaspartic acid column) chromatography, chromatographic focusing, SDS-PAGE and ammonium sulfate precipitation.

[0742] Following any initial purification step, a low-pH hydrophobic interaction chromatography method can be used to elute the mixture containing the immunoconjugates of interest and contaminants with an elution buffer at a pH of about 2.5–4.5 and typically at a low salt concentration (e.g., about 0–0.25 M salt).

[0743] Immunoconjugates (including antibody-drug conjugates (ADCs))

[0744] In another aspect of the invention, the immunoconjugate of the invention according to any of the above embodiments or described herein is conjugated to a heterologous portion or agent, such as, for example, those described below and including any other exogenous material as described herein.

[0745] In one embodiment, the present invention provides an immunoconjugate comprising an antibody construct of the present invention conjugated with one or more therapeutic agents or radioisotopes.

[0746] In some embodiments, the immunoconjugate comprises an antibody construct, as described herein, conjugated with a radioactive atom to form a radioconjugate. As described herein, a variety of radioisotopes can be used to produce the radioconjugates of this invention.

[0747] Immunoconjugates or antibody constructs can be prepared using a variety of bifunctional protein conjugates, such as N-succinimide-3-(2-pyridyldithio)propionate (SPDP), succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC), iminothiocyclopentane (IT), bifunctional derivatives of imide esters (such as hexamethylenediimide H), active esters (such as disuccinimide octanoate), aldehydes (such as glutaraldehyde), diazid compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), diazide derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and difluorinated compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies (see, for example, WO 1994 / 11026). The linker can be a “cleavable linker” that facilitates the release of cytotoxic drugs into the cell. For example, acid-instable linkers, protease-sensitive linkers, photostable 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).

[0748] The immunoconjugates or ADCs described herein are explicitly considered, but not limited to, such conjugates prepared with crosslinking agents, including but not limited to BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfon-EMCS, sulfon-GMBS, sulfon-KMUS, sulfon-MBS, sulfon-SIAB, sulfon-SMCC, and sulfon-SMPB, as well as SVSB (succinimide-(4-vinyl sulfone)benzoate), which are commercially available (e.g., available from Pierce Biotechnology, Inc., Rockford, IL., US).

[0749] As will be recognized by those skilled in the art, some of the methods described above can also be used to prepare radioimmunoconjugates and targeted imaging complexes (however, the text only refers to immunoconjugates or antibody constructs), and such preparation methods are also covered within the scope of this invention.

[0750] Immunoconjugation using chelating factors and / or linkers [07...

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: in: R 1 It is a chelated portion or a radionuclide complex thereof; R 2 In order to be able to target the tumor-specific R 3 The amine (-NH2) reaction part, and includes tetrafluorophenyl ester, pentafluorophenyl ester, dinitrophenyl ester, succinimide ester, sulfosuccinimide ester or isothiocyanate; X 1 is -NR a -, -NR a S(=O)2-, -NR a S(=O)2NR a -; L is the connector, which is -L. 1 -L 2 -L 3 -L 4 -L 5 -; L 1 C1-C, whether unsubstituted or substituted 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, unsubstituted or substituted C2-C 20 alkenyl, unsubstituted or substituted C2-C 20 Ethyne group, C4-C 20 Polyethylene glycol, -(X 3 CH2CH2) t - Unsubstituted or substituted cycloalkylene, unsubstituted or substituted heteroalkylene, unsubstituted or substituted arylene, or unsubstituted or substituted heteroarylene; Each X 3 Independently selected from O and NR 4 ; Each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; L 2 C1-C is non-existent, unsubstituted, or substituted. 10 Alkylene, unsubstituted or substituted C1-C 10 Heteroalkylene, -C(=O)- (unsubstituted or substituted C1-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 -; Each R 4 Independently selected from hydrogen and C1-C6 alkyl groups; 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 The group is absent, or one or more independently selected from the group consisting of: natural or non-natural amino acids, and optionally amino (unsubstituted or substituted benzyl) carbamates; wherein any free amine (-NH2) of the amino acid is optionally independently controlled by R. 5 or -C(=O)R 5 Substitution, wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 Substitution, wherein the substituted benzyl group is selected from halogen, -OH, -OR. 5 -CO2H, -NHR 5 -C(=O)NHR 5 -NHC(=O)R 5 The substituted C1-C6 alkyl groups are substituted with -OH, -CO2H, or -NHR groups. 5 -C(=O)NHR 5 or -NHC(=O)R 5 replace; L 4 C1-C is non-existent, unsubstituted, or substituted. 10 Alkylene, -C(=O)- (unsubstituted or substituted C1-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; Each q is independently 0, 1, or 2; L 5 This does not exist; -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; Each q is independently 0, 1, or 2; Each R a Independently selected from hydrogen and C1-C4 alkyl groups; Each R b Independently selected from hydrogen and C1-C4 alkyl groups; The heteroalkylene group is an alkylene group in which one carbon atom is replaced by one of the following groups: -O-, -S-, -S(=O)-, -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(=NR 5 )NR a -or- NR a C(=O)O-; Where -L 1 -、-L 2 -、-L 3 -、-L 4 -and-L 5 When any one of - is replaced, then -L 1 -、-L 2 -、-L 3 -、-L 4 -and-L 5 - Selected by 1, 2, 3 or 4 from halogen, -OH, -OR 5 -CO2H, -NHR 5 -C(=O)NHR 5 -NHC(=O)R 5 The substituted C1-C6 alkyl groups are substituted with -OH, -CO2H, or -NHR groups. 5 -C(=O)NHR 5 or -NHC(=O)R 5 replace; Each R 5 Independently selected from C1-C 10 Alkyl, C4-C 30 Polyethylene glycol, and unsubstituted or substituted arylene groups, and unsubstituted or substituted heteroarylene groups; The premise is that when L 3 If it does not exist, then there exists at least one R. 5 ;or The premise is X 1 -N (C1-C4 alkyl), -NR a S(=O)2-、-NR a S(=O)2NR a - Or one or more independently selected natural or non-natural amino acids.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein: R 2 In order to be able to target the tumor-targeting portion R 3 The part of the amine (-NH2) reaction, and contains or X represents non-existent, -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-; and Each R a It is independently selected from hydrogen and C1-C4 alkyl groups.

3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (II) has the structure of formula (IIa) or a pharmaceutically acceptable salt thereof: Formula (IIa) in: -NHCH2CH2CH2CH2- is the tumor-targeting portion R. 3 The side chain of lysine residues.

4. The compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof. in: R 1 The chelate moieties 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 their radionuclide complexes; or R 1 The chelate moieties selected from the list of those consisting of: DOTMA, DOTPA, DO3AM-acetic acid, DO3Apic, DOTP, DOTMP, DOTA-4AMP, CB-TE2A, NOA, 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 their radionuclide complexes; or R 1 The chelate is selected from the following: 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-Tetra(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'-(((pyridin-2,6-diylbis(methylene))bis((carboxymethyl)azanidinediyl))-bis(methylene))dipicolinic acid (H4pypa); 6,6',6",6"'-(((pyridine-2,6-diylbis(methylene))bis(azanetriyl))tetra(methylene))-tetrapicolinic acid (H4py4pa); 10-((6-carboxypyridin-2-yl)methyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3Apic); and 3,6,9,12-Tetra(carboxymethyl)-3,6,9,12-tetraazatetradecanoic acid (TTHA); or radionuclide complexes thereof.

5. The compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof, wherein: R 1 The chelate is selected from the following: Or its radionuclide complex.

6. The compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, wherein: X 1 For -NR a -、-NR a S(=O)2- or one or more independently selected natural or non-natural amino acids, wherein any free amine of the amino acid is optionally independently converted by R 5 or -C(=O)R 5 Substitution, and optionally, any free carboxylic acid of any amino acid is replaced by -C(=O)NH-R. 5 replace.

7. The compound of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein: X 1 It can be -NH-, -N(CH3)-, -N(CH2CH3)-, -NHS(=O)2-, -N(CH3)S(=O)2- or -N(CH2CH3)S(=O)2-.

8. The compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, wherein: L 1 Unsubstituted or substituted C1-C6 alkylene groups, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 Polyethylene glycol, unsubstituted or substituted cyclohexene, or unsubstituted or substituted phenylene.

9. The compound of any one of claims 1-8 or a pharmaceutically acceptable salt thereof, wherein: L 2 For non-existent, -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.

10. The compound of any one of claims 1-9, or a pharmaceutically acceptable salt thereof, wherein: L 3 The group is absent, or one or more independently selected from the following groups: 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; wherein any free amine of the amino acid is optionally R 5 or -C(=O)(R) 5 ) is replaced, and any free carboxylic acid of the amino acid is optionally replaced by -C(=O)NH(R) 5 )replace.

11. The compound of any one of claims 1-9, or a pharmaceutically acceptable salt thereof, wherein: L 3 for or 12. The compound of any one of claims 1-11, or a pharmaceutically acceptable salt thereof, wherein: L 4 The following are not present: -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; Each q is independently either 1 or 2.

13. The compound of any one of claims 1-12, or a pharmaceutically acceptable salt thereof, wherein: L 5 It 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 -; Each p is independently 0, 1, or 2.

14. The compound of any one of claims 1-12, or a pharmaceutically acceptable salt thereof, wherein: L 5 For -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 -; where each q is independently 1 or 2.

15. The compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, wherein: L 1 Unsubstituted or substituted C1-C6 alkylene groups, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 Polyethylene glycol, unsubstituted or substituted cyclohexene, or unsubstituted or substituted phenylene; L 2 For non-existent, -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; L 4 The following are not present: -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 -; L 5 For -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 -; Each n is independently 1, 2, 3, 4, 5, or 6; Each q is independently either 1 or 2.

16. The compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, wherein: L 1 Unsubstituted or substituted C1-C6 alkylene groups, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 Polyethylene glycol, unsubstituted or substituted cyclohexene, or unsubstituted or substituted phenylene; L 2 For non-existent, -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; L 4 It does not exist; L 5 For -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 -; Each n is independently 1, 2, 3, 4, 5, or 6; Each q is independently either 1 or 2.

17. The compound of any one of claims 1-7, or a pharmaceutically acceptable salt thereof, wherein: L 1 For -NR 4 CH2CH2-(OCH2CH2) t -; t is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; L 2 For non-existent, -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) m -(CH2) P -、-NR 4 C(=O)-(CH2CH2O) m -(CH2) P -or-(CH2CH2O) m -(CH2) P -; Each m is independently 1, 2, 3, 4, 5, or 6; Each p is independently 1 or 2; L 3 It does not exist; L 4 It does not exist; L 5 For -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 -; Each n is independently 1, 2, 3, 4, 5, or 6; Each q is independently either 1 or 2.

18. The compound of any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, wherein: R 1 The chelate is selected from the following: Or its radionuclide complex; X 1 It can be -NH-, -N(CH3)- or -N(CH2CH3)-; R 2 In order to be able to target the tumor-targeting portion R 3 The amine (-NH2) reaction of lysine, and includes L 1 Unsubstituted or substituted C1-C6 alkylene groups, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 Polyethylene glycol, unsubstituted or substituted cyclohexene, or unsubstituted or substituted phenylene.

19. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein: L 2 It does not exist; L 3 It does not exist; and L 4 It does not exist.

20. The compound of claim 11 or a pharmaceutically acceptable salt thereof, wherein: L 5 For -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 -; Each n is independently 1, 2, 3, 4, 5, or 6; Each q is independently either 1 or 2.

21. The compound of claim 18 or a pharmaceutically acceptable salt thereof, wherein: R 2 In order to be able to target the tumor-targeting portion R 3 The amine (-NH2) reaction of lysine, and includes L 1 Unsubstituted or substituted C1-C6 alkylene groups, unsubstituted or substituted C1-C 10 Heteroalkylene, C4-C 20 Polyethylene glycol, unsubstituted or substituted cyclohexene, or unsubstituted or substituted phenylene; L 2 It does not exist; L 3 It does not exist; L 4 It does not exist; and L 5 For -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 -; Each n is independently 1, 2, 3, 4, 5, or 6; Each q is independently either 1 or 2.

22. The compound of any one of claims 1-21 or a pharmaceutically acceptable salt thereof, wherein: Each R 4 It is a C1-C6 alkyl group.

23. The compound of any one of claims 1-22 or a pharmaceutically acceptable salt thereof, wherein: X 1 It is -N(CH2CH3)-.

24. The compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, wherein -CH2CH2C(=O)X 1 -L- is or 25. The compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, wherein... yes Or its radionuclide complex.

26. The compound of any one of claims 1-25 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.

27. The compound of any one of claims 1-26, or a pharmaceutically acceptable salt thereof, wherein: The radionuclide is an Auger electron emission radionuclide, specifically indium-111 (…). 111 In), Gallium-67 ( 67 Ga), gallium-68( 68 Ga), Technetium-99m( 99m Tc) or Platinum-195m 195m Pt); The radionuclide is an alpha-emitting radionuclide, specifically actinium-225 (α-emission radionuclide). 225 Ac), Bismuth-213 ( 213 Bi), radium ( 223 Ra) or lead-212 ( 212 Pb); The radionuclide is a beta-emitting radionuclide, specifically yttrium-90 (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 Mn); or The radionuclide is a gamma-emitting radionuclide, specifically cobalt-60 (cobalt-60). 60 Co), Palladium-103 103 Pd), Cesium-137 137 Cs), Ytterbium-169 169 Yb), Iridium-192 192 Ir) or radium-226 226 Ra).

28. The compound of any one of claims 1-27 or a pharmaceutically acceptable salt thereof, wherein: The radionuclide is actinium-225 ( 225 Ac).

29. A pharmaceutical composition comprising a compound as described in any one of claims 1-28 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

30. The pharmaceutical composition of claim 29, wherein the pharmaceutical composition is formulated for administration to mammals via intravenous administration.

31. A method of treating a tumor in a mammal, comprising administering to the mammal a compound as described in any one of claims 1 to 28, thereby treating the tumor.

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