VHH antibody conjugates

Abstract

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 and 110 kDa. The immunoconjugates can be used to deliver alpha and beta emitters to treat tumors or cancers.

Description

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Application No. 63 / 373,183, filed on August 22, 2022, which is hereby incorporated by reference in its entirety. Background Art

[0003] The exquisite specificity of antibodies (such as IgG) for their antigens makes antibodies a prime targeting platform for therapeutic agents; however, the typical serum half-life of IgG is at least three weeks, and the sensitivity of IgG to radioisotopes (including alpha-emitting isotopes such as actinium-225 ( 225 Ac) and beta-emitting isotopes such as lutetium-177 ( 177 Lu) and Yttrium-90( 90 Y)) delivery is disadvantageous, especially due to prolonged exposure and chronic off-target toxicity. The emergence of engineered smaller antibody forms (e.g., monomeric scFv, heavy chain antibodies only or single domain antibody fragments) provides a sharp specificity (Bates A, Power, C, Antibodies (Basel) 8:28 (2019)) to full-size (full-size) antibodies (e.g., IgG (about 150kDa)) with a smaller form (e.g., 15 to 30kDa) and a much shorter serum half-life (e.g., 30 minutes to 2 hours). Unfortunately, due to poor retention and tumor uptake, these short half-lives do not allow enough time for effective target binding, and in addition, the plasma clearance of these small antibody forms by the renal system may lead to isotope accumulation in renal tissue and problematic off-target toxicity.

[0004] 225Ac is one of the most cytotoxic radioisotopes among α-emitting radioisotopes, and a single decay event can effectively destroy cancer cells by causing double-strand DNA breaks and subsequent cell death. The potency of α-emitting radioisotopes makes them attractive as cell-killing agents, capable of overcoming acquired resistance observed in response to other therapies. Unfortunately, however, due to decay events at different locations in the body, there remain many challenges regarding whole-body administration and achieving the desired dosimetry in target versus non-target tissues. A key to the application of α-emitting radionuclides as targeted therapeutic agents is the ability to modulate the distribution of daughter nuclides in the body in order to limit toxicity. This in turn is related to the production time of the parent nuclide, the time of administration of the therapeutic agent, the decay pathways and half-lives of the daughter nuclides, the circulation time, and the biodistribution and pharmacokinetics of the delivery vehicle. Unfortunately, the emission of α-particles typically also generates recoil energy that is large enough to decouple the daughter nuclide from the chelator, potentially separating the daughter nuclide from its targeting vehicle and leading to subsequent redistribution of the "free" daughter nuclide, which may induce multiple toxicities. See, for example, Robertson A et al., Curr Radiopharm 11:156 (2018). Thus, the renal toxicity caused by 225 Ac recoil daughter nuclides (such as 213-Bi) has so far emerged as 225 a major limiting factor in the therapeutic use of Ac (see, for example, Jaggi J et al., Cancer Res. 65:4888 (2005)).

[0005] Another confounding issue regarding the use of antibodies and antibody fragments with α-emitting radioisotopes in therapy is that interfering with radioactive decay may particularly damage the antibody component and targeting sequence, even prior to therapy. Radiolysis of the antibody fragment may occur prior to the administration of the α-emitter-labeled antibody fragment to a patient, thereby reducing the amount targeted (see, e.g., Larsen R, Bruland O, J Labelled Cmpd Radiopharm. 36:1009-18 (1995)), and at the higher specific activities required for therapeutic administration, immunoreactivity may rapidly decline with radiochemical quality. Salako et al., J Nucl Med. 39(4):667-670 (1998). For example, at doses of 1,000 gray (Gy) or higher, the high ionization density released by the α-emitter impairs the immunoreactivity of the isotope-labeled Fab fragment via radiolysis. Similarly, significant radiolysis of α-emitting isotope-labeled antibodies has been observed at doses exceeding 1,200 Gy (Zalutsky M et al., J Nucl Med. 42(10):1508-15 (2001)). Thus, the identification of a suitable targeting delivery vehicle for α-emitting radioisotopes is not straightforward.

[0006] In addition, there are additional problems with targeted radiosonde delivery platforms (including α-emitting and β-emitting radioisotopes), and the design of such platforms requires the simultaneous optimization of, for example, immunogenicity, specificity, tissue penetration, stability, ease of manufacture, and an acceptable therapeutic window. Summary of the Invention

[0007] The present invention relates to immunoconjugates or radioimmunoconjugates, compositions comprising them, and methods of using such immunoconjugates and compositions. The immunoconjugates and compositions of the present invention have numerous uses, e.g., for delivering a radioisotope to kill target cells (e.g., cancer cells expressing a target antigen to which the radioimmunoconjugate binds); for detecting and characterizing malignant cells (e.g., target antigen expression) in an object; and for diagnosing and treating various diseases and conditions, such as, for example, cancer, tumors, and other growth abnormalities involving antigen-expressing cells.

[0008] Through the selection and specific combination of the components of the specific delivery platform, the present invention solves many challenges inherent in the targeted delivery of α-particle emitters in vivo. The radioactive isotope delivery platform for emitting α-particles of the present invention provides a shorter half-life than conventional IgG, but a longer half-life than smaller monomeric antibody fragments. Such a half-life allows for a reduction in toxicity due to α-emitters while keeping the antibody fragment in the body for a long enough time to exert therapeutic activity. For example, the radioactive isotope delivery platform for emitting α-particles of the present disclosure exhibits enhanced tumor targeting and reduced accumulation in radiosensitive tissues such as bone marrow and kidney. Additionally, surprisingly, the radioactive isotope delivery platform for emitting α-particles of the present invention exhibits excellent tumor binding and labeling properties for tumors with different antigen densities, which may be a limitation for some uses of some immunoconjugates.

[0009] In one aspect, the present invention describes an immunoconjugate comprising: a) an antigen-binding region; b) an immunoglobulin heavy chain constant region; and c) a chelator; wherein the molecular weight of the immunoconjugate is between 60 and 110 kDa. In certain embodiments, the antigen-binding region comprises an scFv polypeptide or a VHH polypeptide. In certain embodiments, the antigen-binding region comprises an scFv polypeptide. In certain embodiments, the antigen-binding region comprises a VHH polypeptide. In certain embodiments, the antigen-binding region is humanized.

[0010] In some embodiments, the present invention describes a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0011]

[0012] Wherein:

[0013] R 1 is a chelating moiety or its radionuclide complex;

[0014] X 1 is -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-, -(unsubstituted or substituted C 1 -C6 -(alkylene)-X 2 -、-(unsubstituted or substituted C 2 -C 10 -(alkenylene)-X 2 -、-(unsubstituted or substituted C 2 -C 10 -(alkynylene)-X 2 - or -(C 4 -C 20 -(polyethylene glycol)-X 2 -;

[0015] X 2 is absent, -C(=O)-, -NR a C(=O)-, -C(=O)NR a - or -C(=O)X 4 -;

[0016] Each R a is independently selected from hydrogen and C 1 -C 4 alkyl;

[0017] X 4 is -NR a -, -NR a S(=O) 2 -, -NR a S(=O) 2 NR a - or one or more independently selected natural or unnatural amino acids, wherein any free amine of the amino acid is optionally and independently substituted by R 5 or -C(=O)R 5 and wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 ;

[0018] Each R 5 is independently selected from C 1 -C 10 alkyl, C 4 -C 30 polyethylene glycol and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene;

[0019] L is an optional linker;

[0020] R 2 is a moiety capable of reacting with the amine (-NH 3 ) or thiol (-SH) of the tumor targeting moiety R 2 ); and

[0021] v is 1, 2, 3 or 4.

[0022] In some embodiments, v is 1.

[0023] In some embodiments, R 2 is a moiety capable of reacting with the amine (-NH 3 ) of the tumor targeting moiety R 2 and includes tetrafluorophenyl ester, pentafluorophenyl ester, dinitrophenyl ester, succinimidyl ester, sulfosuccinimidyl ester, or isothiocyanate.

[0024] In some embodiments, R 2 is a moiety capable of reacting with the amine (-NH 3 ) of the tumor targeting moiety R 2 and includes:

[0025]

[0026] X is absent, -O-, -S-, -S(=O)-, -S(=O) 2- , -NR a -, -C(=O)-, -NR a C(=O)-, -C(=O)NR a -, -C(=O)O-, -OC(=O)-, -OC(=O)NR a -, -NR a C(=O)NR a -, -NR a C(=S)NR a -, -NR a C(=O)O-; and

[0027] each R a is independently selected from hydrogen and C 1 -C 4 alkyl.

[0028] In some embodiments, immunoconjugates are described herein having the structure of a compound of formula (II), formula (III), or formula (IV) or a pharmaceutically acceptable salt thereof:

[0029]

[0030] Wherein:

[0031] R 1 is a chelating moiety or a radionuclide complex thereof;

[0032] X 1 is -O-, -S-, -S(=O)-, -S(=O) 2- , -NR a -, -C(=O)-, -NRa 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-, -(unsubstituted or substituted C 1 -C 6 -alkylene)-X 2 -, -(unsubstituted or substituted C 2 -C 10 -alkenylene)-X 2 -, -(unsubstituted or substituted C 2 -C 10 -alkynylene)-X 2 - or -(C 4 -C 20 -polyethylene glycol)-X 2 -;

[0033] X 2 is absent, -C(=O)-, -NR a C(=O)-, -C(=O)NR a - or -C(=O)X 4 -;

[0034] Each R a is independently selected from hydrogen and C 1 -C 4 -alkyl;

[0035] X 4 is -NR a -, -NR a S(=O) 2 -, -NR a S(=O) 2 NR a - or one or more independently selected natural or non-natural amino acids, wherein any free amine of the amino acid is optionally independently substituted by R 5 or -C(=O)R 5 and wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 ;

[0036] Each R 5 is independently selected from C 1 -C 10 -alkyl, C 4 -C 30Polyethylene glycol and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene;

[0037] L is an optional linker;

[0038] -NH-R 3 is a tumor targeting moiety; and

[0039] v is 1, 2, 3 or 4.

[0040] In some embodiments, R 2 is a moiety capable of reacting with the thiol (-SH) of the tumor targeting moiety R 3 and includes a maleimide group, a haloacetamide group, a haloacetyl group, a haloacetic acid group, a pyridylthio group, a vinylcarbonyl group, an aziridinyl group, a disulfide group, an acetylene group, a hydroxysuccinimide group or a thiol group.

[0041] In some embodiments, R 2 is a moiety capable of reacting with the thiol (-SH) of the tumor targeting moiety R 3 and includes m is 0, 1, 2, 3, 4 or 5.

[0042] In some embodiments, immunoconjugates are described herein having the structure of a compound of formula (V), formula (VI), formula (VII) or formula (VIII) or a pharmaceutically acceptable salt thereof:

[0043]

[0044] Wherein:

[0045] R 1 is a chelating moiety or a radionuclide complex thereof;

[0046] X 1 is -O-, -S-, -S(=O)-, -S(=O) 2- , -NR a -, -C(=O)-, -NR a C(=O)-, -

[0047] C(=O)NR a -, -C(=O)O-, -OC(=O)-, -OC(=O)NR a -, -NR a C(=O)NR a -,

[0048] -NR a C(=S)NRa -, -NR a C(=O)O-, -(unsubstituted or substituted C 1 -C 6 -alkylene)-X 2 -, -(unsubstituted or substituted C 2 -C 10 -alkenylene)-X 2 -, -(unsubstituted or substituted C 2 -C 10 -alkynylene)-X 2 - or -(C 4 -C 20 -polyethylene glycol)-X 2 -;

[0049] X 2 is absent, -C(=O)-, -NR a C(=O)-, -C(=O)NR a - or -C(=O)X 4 -;

[0050] Each R a is independently selected from hydrogen and C 1 -C 4 -alkyl;

[0051] X 4 is -NR a -, -NR a S(=O) 2 -, -NR a S(=O) 2 NR a - or one or more independently selected natural or unnatural amino acids, wherein any free amine of the amino acid is optionally and independently substituted by R 5 or -C(=O)R 5 and wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 -;

[0052] Each R 5 is independently selected from C 1 -C 10 -alkyl, C 4 -C 30 -polyethylene glycol and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene;

[0053] L is an optional linker;

[0054] -S-R 3 is a tumor targeting moiety; and

[0055] v is 1, 2, 3 or 4.

[0056] In some embodiments, R 2 is a moiety capable of reacting with the amine (-NH 3 ) of the lysine of the tumor targeting moiety R 2 .

[0057] In some embodiments, the immunoconjugate of formula (II) has the structure of formula (IIa) or a pharmaceutically acceptable salt thereof:

[0058]

[0059] wherein: -NHCH 2 CH 2 CH 2 CH 2 - is the side chain of the lysine residue of the tumor targeting moiety R 3 .

[0060] In some embodiments, the immunoconjugate of formula (III) has the structure of formula (IIIa) or a pharmaceutically acceptable salt thereof:

[0061]

[0062] wherein: -NHCH 2 CH 2 CH 2 CH 2 - is the side chain of the lysine residue of the tumor targeting moiety R 3 .

[0063] In some embodiments, the immunoconjugate of formula (IV) has the structure of formula (IVa) or a pharmaceutically acceptable salt thereof:

[0064]

[0065] wherein: -NHCH 2 CH 2 CH 2 CH 2 - is the side chain of the lysine residue of the tumor targeting moiety R 3 .

[0066] In some embodiments, R 2 is a moiety capable of reacting with the thiol (-SH) of the cysteine of the tumor targeting moiety R 3 .

[0067] In some embodiments, the immunoconjugate of formula (V) has the structure of formula (Va) or a pharmaceutically acceptable salt thereof:

[0068]

[0069] Wherein: -SCH 2 - is the thiol (-SH) of the side chain of the cysteine residue of the tumor targeting moiety R 3 of.

[0070] In some embodiments, the immunoconjugate of formula (VI) has the structure of formula (VIa) or a pharmaceutically acceptable salt thereof:

[0071]

[0072] Wherein: -SCH 2 - is the thiol (-SH) of the side chain of the cysteine residue of the tumor targeting moiety R 3 of.

[0073] In some embodiments, the immunoconjugate of formula (VII) has the structure of formula (VIIa) or a pharmaceutically acceptable salt thereof:

[0074]

[0075] Wherein: -SCH 2 - is the thiol (-SH) of the side chain of the cysteine residue of the tumor targeting moiety R 3 of.

[0076] In some embodiments, the immunoconjugate of formula (VIII) has the structure of formula (VIIIa) or a pharmaceutically acceptable salt thereof:

[0077]

[0078] Wherein: -SCH 2 - is the thiol (-SH) of the side chain of the cysteine residue of the tumor targeting moiety R 3 of.

[0079] In some embodiments, the tumor targeting moiety R 3 is a polypeptide comprising an antigen binding region and an immunoglobulin heavy chain constant region, wherein the molecular weight of the polypeptide is between 60 and 110 kDa.

[0080] In some embodiments, the antigen-binding region comprises a scFv polypeptide or a VHH polypeptide. In some embodiments, the immunoglobulin heavy chain constant region comprises the CH2 domain of an immunoglobulin, the CH3 domain of an immunoglobulin, or the 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, 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 an alteration of one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region or alters the binding of the immunoconjugate to the neonatal Fc receptor (FcRn); or the immunoglobulin heavy chain constant region comprises an alteration of one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region and alters the binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In some embodiments, the immunoglobulin heavy chain constant region comprises an alteration of one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region; or the immunoglobulin heavy chain constant region comprises an alteration of one or more amino acid residues that alters the binding of the immunoconjugate to the neonatal Fc receptor (FcRn); or both. In some embodiments, the alteration of 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-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), or a combination thereof.

[0081] In certain embodiments, the antigen-binding region specifically binds to HER2 or DLL3. In certain embodiments, the antigen-binding region specifically binds to HER2. In certain embodiments, the antigen-binding region of the immunoconjugate comprises: a) 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 certain embodiments, the antigen-binding region of the immunoconjugate comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO:20 and binds to HER2. In certain embodiments, the antigen-binding region specifically binds to DLL3. In certain embodiments, the antigen-binding region of the immunoconjugate comprises: a) 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 to DLL3. In certain embodiments, the antigen-binding region of the immunoconjugate comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO:30 and binds to DLL3. In certain embodiments, the immunoglobulin heavy chain constant region comprises the CH2 domain of the immunoglobulin, the CH3 domain of the immunoglobulin or the CH2 and CH3 domains of the immunoglobulin. In certain embodiments, the immunoglobulin heavy chain constant region comprises the CH2 and CH3 domains of the immunoglobulin. In certain embodiments, the immunoglobulin heavy chain constant region is a human immunoglobulin heavy chain constant region. In certain embodiments, the immunoglobulin heavy chain constant region is of the IgA, IgG1, IgG2, IgG3 or IgG4 isotype. In certain embodiments, the immunoglobulin heavy chain constant region is of the IgG1 isotype. In certain embodiments, the immunoglobulin heavy chain constant region is of the IgG4 isotype. In certain embodiments, the immunoglobulin heavy chain constant region comprises an alteration of one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region or alters the binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises an alteration of one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region and alters the binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In certain 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 certain embodiments, the immunoglobulin heavy chain constant region comprises alterations to one or more amino acid residues that alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In certain embodiments, the alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region are alterations that reduce complement-dependent cytotoxicity (CDC), antibody-dependent cell-cytotoxicity (ADCC), antibody-dependent cell phagocytosis ADCP, or a combination thereof. In certain embodiments, the alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region are selected from the list consisting of the following according to EU numbering: (a) 297A, 297Q, 297G, or 297D, (b) 279F, 279K, or 279L, (c) 228P, (d) 235A, 235E, 235G, 235Q, 235R, or 235S, (e) 237A, 237E, 237K, 237N, or 237R, (f) 234A, 234V, or 234F, (g) 233P, (h) 328A, (i) 327Q or 327T, (j) 329A, 329G, 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 (ppp) any combination of (a)–(ppp). In certain embodiments, the alteration to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region comprises L234A, L235E, G237A, A330S and P331S according to EU numbering. In certain embodiments, the amino acid alteration to one or more amino acid residues that alters the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) shortens the serum half-life of the immunoconjugate. In certain embodiments, the alteration to one or more amino acid residues that alters the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to amino acid residues selected from the list consisting of the following according to EU numbering: 251, 252, 253, 254, 255, 288, 309, 310, 312, 385, 386, 388, 400, 415, 433, 435, 436, 439, 447 and combinations thereof. In certain embodiments, the alteration to one or more amino acid residues that alters the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to amino acid residues selected from the list consisting of the following according to EU numbering: 253, 254, 310, 435, 436 and combinations thereof. In certain embodiments,Alterations to one or more amino acid residues that alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are to amino acid residues selected from the list consisting of the following according to EU numbering: I253A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof. In certain embodiments, alterations to one or more amino acid residues that alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are to amino acid residues selected from the list consisting of the following according to EU numbering: I253A, S254A, H310A, H435Q, Y436A, and combinations thereof. In certain embodiments, alterations to one or more amino acid residues that alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are to amino acid residues selected from the list consisting of the following according to EU numbering: I253A, H310A, H435Q, and combinations thereof. In certain embodiments, the immunoconjugate has a serum half-life of less than 15 days. In certain embodiments, the immunoconjugate has a serum half-life of less than 10 days. In certain embodiments, the immunoconjugate has a serum half-life of less than 120 hours. In certain embodiments, the immunoconjugate has a serum half-life of less than 72 hours. In certain embodiments, the antigen-binding region is conjugated to the immunoglobulin heavy chain constant region via a linker amino acid sequence or the human IgG hinge region. In certain embodiments, the antigen-binding region is conjugated to the immunoglobulin heavy chain constant region via the human IgG hinge region.,

[0082] In certain embodiments, the chelator is conjugated to the antigen-binding region and / or the immunoglobulin heavy chain constant region in a ratio of 1:1 to 8:1. In certain embodiments, the chelator is conjugated to the antigen-binding region and / or the immunoglobulin heavy chain constant region in a ratio of 1:1 to 6:1. In certain embodiments, the chelator is conjugated to the antigen-binding region and / or the immunoglobulin heavy chain constant region in a ratio of 2:1 to 6:1. In certain embodiments, the immunoconjugate further comprises a radioisotope. In certain embodiments, the radioisotope is an α-emitter. In certain embodiments, the radioisotope is an α-emitter selected from the list consisting of: 225 Ac, radium-223 ( 223 Ra), radium-224 ( 224 Ra), thorium-227 ( 227 Th), lead-212 ( 212 Pb), bismuth-212 ( 212 Bi), and bismuth ( 213 Bi). In certain embodiments, the radioisotope is225 Ac. In certain embodiments, the radioisotope is a β-emitter. In certain embodiments, the radioisotope is a β-emitter selected from the following: 177 Lu, 90 Y, copper-67 ( 67 Cu), and samarium-153 ( 153 Sm). In certain embodiments, the molecular weight of the immunoconjugate is between 60 and 100 kDa. In certain embodiments, the molecular weight of the immunoconjugate is between 60 and 90 kDa. In certain embodiments, the molecular weight of the immunoconjugate is between 65 and 90 kDa. In certain embodiments, the molecular weight of the immunoconjugate is between 70 and 90 kDa. In certain embodiments, the immunoconjugate forms a dimer with another immunoconjugate. In certain embodiments, the immunoconjugate further comprises a pharmaceutically acceptable excipient or carrier. In certain embodiments, the immunoconjugate is formulated for intravenous administration.

[0083] Also described herein is a method for preparing the immunoconjugate, the method comprising loading the immunoconjugate with a radioisotope. In certain embodiments, the radioisotope is an α-emitter. In certain embodiments, the radioisotope is an α-emitter selected from the list consisting of: 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi, and 213 Bi. In certain embodiments, the radioisotope is 225 Ac. In certain embodiments, the radioisotope is a β-emitter. In certain embodiments, the radioisotope is a β-emitter selected from the following: 177 Lu, 90 Y, 67 Cu, and 153 Sm. In certain embodiments, the radioisotope is 177 Lu.

[0084] The present invention also describes a method for treating cancer or a tumor in an individual, the method comprising administering the immunoconjugate to the individual so as to treat the cancer or the tumor. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer or tumor is a solid cancer or tumor. In certain embodiments, the cancer or tumor includes lung cancer, breast cancer, ovarian cancer or neuroendocrine cancer. In certain embodiments, the method further comprises administering 0.5 μCi to 30.0 μCi / kg to the individual. In certain embodiments, the cancer or tumor expresses an antigen specifically bound by the immunoconjugate.

[0085] The present invention also describes an immunoconjugate for use in a method for treating cancer or a tumor in an individual. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer or tumor is a solid cancer or tumor. In certain embodiments, the cancer or tumor includes lung cancer, breast cancer, ovarian cancer or neuroendocrine cancer. In certain embodiments, 0.5 μCi to 30.0 μCi / kg is administered to the individual. In certain embodiments, the cancer or tumor expresses an antigen specifically bound by the immunoconjugate.

[0086] The present invention also describes a method for killing cancer cells in an individual, the method comprising administering the immunoconjugate to the individual so as to kill the cancer cells. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer cells include lung cancer cells, breast cancer cells, ovarian cancer cells or neuroendocrine cancer cells. In certain embodiments, the method comprises administering 0.1 μCi to 30.0 μCi / kg to the individual. In certain embodiments, the method comprises administering 10 mCi to 75 mCi per square meter of body surface area to the individual. In certain embodiments, the cancer cells express an antigen specifically bound by the immunoconjugate.

[0087] The present invention also describes the use of the immunoconjugate in a method for killing cancer cells in an individual. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer cells include lung cancer cells, breast cancer cells, ovarian cancer cells or neuroendocrine cancer cells. In certain embodiments, the method comprises administering 0.5 μCi to 30.0 μCi / kg to the individual. In certain embodiments, the cancer cells express an antigen specifically bound by the immunoconjugate.

[0088] The present invention also describes a method for delivering a radioisotope to cancer cells or tumor cells in an individual, the method comprising administering the immunoconjugate to the individual, thereby delivering the radioisotope to the cancer cells or the tumor cells. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer cells or the tumor cells include lung cancer cells, breast cancer cells, ovarian cancer cells or neuroendocrine cancer cells. In certain embodiments, the method comprises administering 0.5 μCi to 30.0 μCi / kg to the individual. In certain embodiments, the cancer cells or the tumor cells express an antigen specifically bound by the immunoconjugate.

[0089] The present invention also describes an immunoconjugate for delivering a radioisotope to cancer cells or tumor cells in an individual. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer cells or the tumor cells include lung cancer cells, breast cancer cells, ovarian cancer cells or neuroendocrine cancer cells. In certain embodiments, the cancer cells or the tumor cells express an antigen specifically bound by the immunoconjugate.

[0090] The present invention also describes a method for imaging a tumor in an individual, the method comprising administering the immunoconjugate to the individual. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer or tumor includes lung cancer, breast cancer, ovarian cancer or neuroendocrine cancer. In certain embodiments, the tumor expresses an antigen specifically bound by the immunoconjugate.

[0091] The present invention also describes an immunoconjugate for use in a method for imaging a tumor in an individual. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer or tumor includes lung cancer, breast cancer, ovarian cancer or neuroendocrine cancer. In certain embodiments, the tumor expresses an antigen specifically bound by the immunoconjugate.

[0092] The present invention also describes a nucleic acid encoding the immunoconjugate. In certain embodiments, an expression vector comprises the nucleic acid. In certain embodiments, a cell comprises the nucleic acid or the expression vector. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a CHO cell.

[0093] In some embodiments, the subject radioactive isotope delivery platform is large enough in molecular size (e.g., 60 kDa to 110 kDa) to substantially reduce off-target toxicity, particularly renal injury (e.g., from an α-emitting isotope cargo), and small enough in size to achieve increased tissue penetration compared to conventional IgG while maintaining target specificity and an increased probability of the first decay event in the target tissue. This size results in preferential elimination through the liver relative to the kidneys, sparing the kidneys from radiotoxicity.

[0094] In some embodiments, the subject radioactive isotope delivery platform can be used to safely and effectively target the delivery of α-emitters in vivo, in part, by reducing certain adverse effects caused by platforms with a half-life greater than 5 days and / or a molecular weight below 60 kDa.

[0095] In some embodiments, the subject radioactive isotope delivery platform can be used to safely and effectively target the delivery of α-emitters in vivo, in part, by exhibiting a reduced loss of targeting ability due to radiolysis compared to other possible delivery platforms.

[0096] In some embodiments, the subject radioactive isotope delivery platform can be used to safely and effectively target the delivery of α-emitters in vivo, in part, by exhibiting increased manufacturing stability at the temperatures required for certain radiolabeling processes (e.g., high-temperature chelation using certain chelating agents) compared to other possible delivery platforms that use antibody fragments.

[0097] In one embodiment, the present invention provides an immunoconjugate for the in vivo delivery of an α-emitting radioactive isotope. 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, gallium-68 ( 68 Ga) or cesium-134 ( 134 Ce)).

[0098] In one embodiment, the immunoconjugate comprises an antibody construct and a chelator and has a molecular weight between 60 and 110 kDa, preferably between 60 and 100 kDa, preferably between 60 and 90 kDa, preferably between 65 and 90 kDa, preferably between 70 and 90 kDa. The chelator is capable of chelating an α-emitting radioactive isotope such that the antibody construct is linked to the α-emitting radioactive isotope.

[0099] At least one of the variant constant regions in the immunoconjugate has at least one FcRn binding mutation. In a preferred embodiment, each of the two variant constant regions of the immunoconjugate has at least one FcRn binding mutation, and the FcRn binding mutations are the same or different.

[0100] In one embodiment, the chelator comprises DOTA or a DOTA derivative. In one embodiment, the chelator comprises DOTAGA. In one embodiment, the chelator comprises macropa or a macropa derivative. In one embodiment, the chelator comprises Py4Pa or a Py4Pa derivative. In one embodiment, the chelator comprises siderocalin or a siderocalin derivative.

[0101] In one embodiment, the chelator comprises a radioisotope chelating moiety and a functional group that permits covalent attachment to the antigen binding arm. In one embodiment, the functional group is directly attached to the radioisotope chelating moiety. In one embodiment, the chelator further comprises a linker between the functional group and the radioisotope chelating moiety.

[0102] In one embodiment, the radioisotope chelating moiety comprises DOTA or a DOTA derivative. In one embodiment, the radioisotope chelating moiety comprises DOTAGA. In one embodiment, the radioisotope chelating moiety comprises macropa or a macropa derivative. In one embodiment, the radioisotope chelating moiety comprises Py4Pa or a Py4Pa derivative.

[0103] In one embodiment, the invention provides a pharmaceutical composition comprising the radioactive immunoconjugate of the invention and a pharmaceutically acceptable carrier.

[0104] In one embodiment, the invention provides a method of delivering an α-emitting radioisotope to cancer cells in a patient, the method comprising administering to the patient the radioactive immunoconjugate or pharmaceutical composition of the invention. In one embodiment, the patient is a human patient.

[0105] In one embodiment, the invention provides a method of inhibiting the growth of cancer cells, the method comprising contacting the cancer cells with the radioactive immunoconjugate of the invention. In one embodiment, the cancer cells are in a patient. In one embodiment, the method involves administering to the patient the pharmaceutical composition of the invention. In one embodiment, the patient is a human patient.

[0106] In one embodiment, the present invention provides a method for killing cancer cells, the method comprising contacting the cancer cells with the radioimmunoconjugate of the present invention. In one embodiment, the cancer cells are in a patient. In one embodiment, the method involves administering to the patient a pharmaceutical composition of the present invention. In one embodiment, the patient is a human patient.

[0107] In one embodiment, the present invention provides a method for treating cancer in a patient in need thereof, the method comprising administering to the patient a radioimmunoconjugate or a pharmaceutical composition of the present invention. In one embodiment, the patient is a human patient.

[0108] In one embodiment, the present invention provides a targeted imaging complex, which comprises an immunoconjugate of the present invention and further comprises an imaging metal. In one aspect, the present invention provides a targeted imaging complex, which comprises an antibody construct of an immunoconjugate of the present invention and further comprises an imaging metal. In one embodiment, the imaging metal is a radioisotope. In one embodiment, the imaging metal is selected from the following: 111 In 89 Zr 64 Cu 68 Ga and 134 Ce. In one embodiment, the imaging metal is selected from 111 In 89 Zr 64 Cu 68 Ga and 134 Ce. In one embodiment, the imaging metal is 111 In. In one embodiment, the imaging metal is covalently bound to the immunoconjugate or the antibody construct. In one embodiment, the imaging metal is associated with a chelator of the immunoconjugate. In one embodiment, the present invention provides a method for determining the location of cancer cells in a patient, the method comprising administering to the patient the targeted imaging complex of the present invention. In one embodiment, the patient is a human patient.

[0109] In one embodiment, the present invention provides a kit for preparing a radiopharmaceutical of the present invention, which comprises an immunoconjugate of the present invention. In one embodiment, the present invention provides a kit comprising a radioimmunoconjugate of the present invention. In one embodiment, the present invention provides a kit for preparing a pharmaceutical composition of the present invention, which comprises an immunoconjugate of the present invention. In one embodiment, the present invention provides a kit for preparing a pharmaceutical composition of the present invention, which comprises a radioimmunoconjugate of the present invention. In one embodiment, the present invention provides a kit comprising a pharmaceutical composition of the present invention.

[0110] In some embodiments, the immunoconjugates or radioimmunoconjugates of the present invention comprise a dimerization domain or motif. In some additional embodiments, the dimerization domain or motif is in the hinge region and / or variant constant region.

[0111] In some embodiments, the half-life of the immunoconjugates, radioimmunoconjugates or pharmaceutical compositions of the present invention in human serum is less than 96 hours. In some additional embodiments, the half-life in human serum is less than 72 hours. In some additional embodiments, the half-life is less than 48, 36, 24 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.

[0112] 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 a radioimmunoconjugate.

[0113] In one aspect, the present invention provides a radioimmunoconjugate comprising the immunoconjugate of the present invention and further comprising an α-particle emitter and a β- and / or γ-particle emitter. In one aspect, the present invention provides a pharmaceutical composition comprising such a radioimmunoconjugate.

[0114] In some embodiments, the kits of the present invention, in addition to the immunoconjugates, radioimmunoconjugates or pharmaceutical compositions of the present invention, further comprise reagents or pharmaceutical devices.

[0115] In some embodiments, the kits of the present invention are immunoassay kits for specifically detecting an antigen in a biological sample, comprising: (a) an immunoconjugate, radioimmunoconjugate or targeting imaging complex and / or a composition thereof as described herein; and (b) instructions for detecting the immunoconjugate, radioimmunoconjugate or targeting imaging complex.

[0116] In another aspect, the present invention provides isolated nucleic acids encoding antigen-binding arms or components thereof as provided herein. In one aspect, the present invention provides isolated nucleic acids encoding the antigen-binding region of an immunoconjugate herein. In one aspect, the present invention provides isolated nucleic acids encoding the VHH polypeptide of an immunoconjugate herein. In one aspect, the present invention provides isolated nucleic acids encoding the hinge region of an immunoconjugate herein. In one aspect, the present invention provides isolated nucleic acids encoding the variant constant region of an immunoconjugate herein. In one aspect, the present invention provides isolated nucleic acids encoding the VHH polypeptide of an immunoconjugate herein and the hinge region of an immunoconjugate herein. In one aspect, the present invention provides isolated nucleic acids encoding the VHH polypeptide of an immunoconjugate herein, the hinge region of an immunoconjugate herein, and the variant constant region of an immunoconjugate herein.

[0117] In another aspect, the present invention provides vectors comprising the nucleic acids as provided herein. In some embodiments, the vector is an expression vector.

[0118] In another aspect, the present invention provides methods of using the immunoconjugates, radioimmunoconjugates, targeted imaging complexes, or pharmaceutical compositions of the present invention. In some embodiments, the present invention provides methods of treating a disease, disorder, or condition, the method comprising administering to a patient in need thereof a pharmaceutically effective amount of a radioimmunoconjugate or pharmaceutical composition herein.

[0119] In some embodiments, the methods of the present invention comprise the step of administering to a subject in need thereof any one of the radioimmunoconjugates or pharmaceutical compositions described herein. For some additional embodiments, the method is for inhibiting the growth and / or killing of cancer cells or tumors.

[0120] In some embodiments, there is provided the use of an immunoconjugate or radioimmunoconjugate described herein for the manufacture of a medicament for treating a disease, disorder, or condition of a subject such as, for example, cancer.

[0121] In another aspect, the present invention provides a method for preparing a radioimmunoconjugate or pharmaceutical composition of the present invention, the method comprising radio-labeling the immunoconjugate with a suitable isotope such as, for example, an α or β particle emitter.

[0122] These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and the appended claims. To form other embodiments of the present invention, the above elements of the present invention may be combined alone or removed freely without any description herein against such combination or removal. BRIEF DESCRIPTION OF THE DRAWINGS

[0123] Figure 1A and1B Show the binding of anti-HER2 and anti-DLL3 VHH-Fc constructs.

[0124] Figure 2A 、 2B and 2C show the binding of anti-HER2 and anti-DLL3 VHH-Fc constructs to cells expressing HER2 and / or DLL3.

[0125] Figure 3A and 3B show the internalization of anti-HER2 and anti-DLL3 VHH-Fc constructs in cells expressing HER2 and DLL3.

[0126] Figure 4 Show the self-interaction data of anti-HER2 and anti-DLL3 VHH-Fc constructs.

[0127] Figure 5 Show a schematic diagram of the chemical synthesis of the linker molecule.

[0128] Figure 6 Show a schematic diagram of the chemical synthesis of the linker molecule.

[0129] Figure 7A 、 7B and 7C show the immunoreactivity scores of different VHH-Fc constructs.

[0130] Figure 8 Show with 111 imaging with In-labeled VHH-Fc and 225 comparison of the biodistribution of Ac-labeled VHH-Fc.

[0131] Figure 9A 、 9B 、9C and 9D show the biodistribution of the labeled anti-HER2 VHH-Fc construct over time.

[0132] Figure 10A 、 10B and 10C show the tumor:non-tumor tissue ratio of the labeled anti-HER2 VHH-Fc construct.

[0133] Figure 11 Show the biodistribution of the labeled anti-HER2 VHH-Fc construct.

[0134] Figure 12 Show with 111 whole body clearance of In-labeled VHH-Fc (H101) and VHH-Fc variants (H105, H107 and H108).

[0135] Figure 13Show biodistribution over time of radiolabeled anti-DLL3 VHH-Fc constructs.

[0136] Figure 14 Show biodistribution of radiolabeled anti-DLL3 VHH-Fc constructs.

[0137] Figure 15A and 15B Show 225 Biodistribution of 111In-labeled anti-HER2 (15A) and anti-DLL3 (15B) VHH-Fc constructs.

[0138] Figure 16A 、 16B and 16C show results of toxicity studies with 225 111In-labeled anti-HER2 VHH-Fc constructs.

[0139] Figure 17 Show immunoreactivity scores of different anti-DDL3 VHH-Fc constructs loaded with 177 68Ga.

[0140] Figure 18 Show chemical structures of certain linker chelator agents described herein.

[0141] Figure 19A and 19B Show imaging experiments in untreated mice with 111 67Ga to measure whole-body clearance of radioactivity using different conjugates (see Example B-26). DETAILED DESCRIPTION

[0142] The present invention is described more fully hereinafter with reference to illustrative, 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 will be thorough and will convey the scope of the invention to those skilled in the art. For easier understanding of the invention, certain terms are defined below. Additional definitions may be found in the detailed description of the invention.

[0143] Specifically, in embodiments, the present invention addresses many challenges inherent in the targeted delivery of radioisotopes in vivo through the selection and specific assembly of specific immunoconjugates and radioimmunoconjugate components. The radioisotope delivery platform of the present invention provides a shorter half-life compared to traditional 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 large enough (e.g., 60 kDa to 110 kDa) to significantly reduce off-target toxicity, particularly kidney injury (e.g., from α- or β-emitting isotope cargo), and its size is small enough to achieve increased tissue penetration compared to traditional IgG while maintaining target specificity and increasing the probability of the first decay event in the target tissue. In some embodiments, the subject radioisotope delivery platform can be used to safely and effectively target the delivery of radioisotopes (such as α- or β-emitters) in vivo in part by reducing certain adverse effects caused by platforms with a half-life exceeding 5 days and / or a molecular weight below 60 kDa. In some embodiments, the subject radioisotope delivery platform can be used to safely and effectively target the delivery of radioisotopes (such as α- or β-emitters) in vivo in part by exhibiting a reduced loss of targeting ability due to radiolysis compared to other possible delivery platforms. In some embodiments, the subject radioisotope delivery platform can be used to safely and effectively target the delivery of radioisotopes (such as α- or β-emitters) in vivo in part by exhibiting increased manufacturing stability at the temperatures required for certain radiolabeling processes (e.g., high-temperature chelation using certain chelating agents) compared to other possible delivery platforms using antibody fragments.

[0144] Immunoconjugate

[0145] In one aspect, the present invention provides an immunoconjugate that specifically binds to a target antigen with high affinity. In some embodiments, the present invention provides an immunoconjugate that specifically binds to a cell surface antigen of a cancer cell. In some embodiments, the immunoconjugate comprises three, four, five, six or more CDRs or HVRs (Kabat). In some embodiments, the immunoconjugate binds to a specific antigen and / or epitope with an affinity characterized by KD ≤ 1 μM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM or < 0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M).

[0146] The immunoconjugates described herein can serve as platforms for the delivery of radioisotopes. Radioisotope delivery platforms are provided herein that have a relatively short half-life (e.g., less than one or two weeks, but more than two hours to eight hours).

[0147] In one embodiment, the immunoconjugates of the present disclosure comprise: a) an antigen-binding region; b) an immunoglobulin heavy chain constant region; and c) a chelating moiety or a radionuclide complex thereof. In one embodiment, the immunoconjugates of the present disclosure comprise: 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 and 110 kDa.

[0148] In one embodiment, the immunoconjugates of the present disclosure comprise: a) a VHH antigen-binding region; b) an immunoglobulin heavy chain constant region; and c) a chelating moiety or a radionuclide complex thereof. In one embodiment, the immunoconjugates of the present disclosure comprise: 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 and 110 kDa.

[0149] In one embodiment, the immunoconjugates of the present disclosure comprise: a) a VHH antigen-binding region; b) an immunoglobulin Fc region, together referred to as VHH-Fc; and c) a chelating moiety or a radionuclide complex thereof. In one embodiment, the immunoconjugates of the present disclosure comprise: 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 and 110 kDa.

[0150] In one embodiment, the immunoconjugates of the present disclosure comprise: a) a VHH antigen-binding region; b) a variant immunoglobulin Fc region; and c) a chelating moiety or a radionuclide complex thereof. In one embodiment, the immunoconjugates of the present disclosure comprise: 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 and 110 kDa. In certain embodiments, the variant immunoglobulin Fc region comprises one or more amino acid alterations that shorten the serum or plasma half-life of the immunoconjugate.

[0151] In some embodiments, the size of the radioisotope delivery platform is greater than about 60 kDa to avoid certain toxicities from α-emitting 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 enhance tumor penetration. In some embodiments, the size of the radioisotope delivery platform is between 60 kDa and 110 kDa due to its dimeric structure having two separate antigen-binding arms, each antigen-binding arm 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 one or more Fc effector functions.

[0152] In one embodiment, the antibody construct of the immunoconjugate consists of two antigen-binding arms covalently linked to each other (e.g., via a disulfide bond between the associated heavy-chain constant regions or immunoglobulin hinge region). Each of the antigen-binding arms independently consists of an antigen-binding region, a hinge region, and a variant constant region. Within each antigen-binding arm, the antigen-binding region of the arm is covalently linked to the hinge region of the arm, and the hinge region of the arm is covalently linked to the variant constant region of the arm such that the hinge region is interposed between the antigen-binding region and the variant constant region of the antigen-binding arm and thereby links them.

[0153] In a preferred embodiment, at least one of the two antigen-binding regions in the immunoconjugate consists of one or two heavy-chain only variable (VHH) polypeptides. In a preferred embodiment, at least one of the two antigen-binding regions consists of one VHH polypeptide. In a preferred embodiment, each of the two antigen-binding regions of the immunoconjugate consists of one VHH polypeptide, which may be the same or different.

[0154] In one embodiment, the antigen-binding regions of the immunoconjugate bind the same antigen. In one embodiment, the antigen-binding regions of the immunoconjugate bind different antigens. In one embodiment, the antigen-binding regions of the immunoconjugate are the same. In one embodiment, the antigen-binding regions of the immunoconjugate are different. In one embodiment, the antigen-binding region of each antigen-binding arm consists of one or two VHH polypeptides.

[0155] In one embodiment, the antigen-binding region of one antigen-binding arm consists of two VHH polypeptides and the antigen-binding region of the other antigen-binding arm does not contain a VHH polypeptide. In one embodiment, the two antigen-binding arms bind the same antigen. In one embodiment, the two antigen-binding arms bind different antigens. In one embodiment, the two VHH polypeptides are the same. In one embodiment, the two VHH polypeptides are different. In one embodiment, the immunoconjugate is bispecific.

[0156] In one embodiment, the antigen-binding region of one antigen-binding arm consists of a VHH polypeptide, and the antigen-binding region of the other antigen-binding arm consists of two VHH polypeptides. In one embodiment, the two antigen-binding arms bind the same antigen. In one embodiment, the two antigen-binding arms bind different antigens. In one embodiment, the 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.

[0157] 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 polypeptides bind the same antigen. In one embodiment, the VHH polypeptides bind different antigens. In one embodiment, the VHH polypeptides are identical. In one embodiment, the VHH polypeptides are different. In one embodiment, the immunoconjugate is bispecific.

[0158] Antigen-binding region

[0159] The antigen-binding region confers specificity to the immunoconjugate and may suitably comprise a small antigen-binding polypeptide. Such small antigen-binding polypeptides confer advantages such as reducing the overall size of the immunoconjugate molecule to achieve tumor penetration and labeling. The small antigen-binding polypeptide may lack certain regions that are dispensable for binding, such as the light chain constant region, the heavy chain constant region, the CH1 region, or the hinge region. In certain embodiments, the antigen-binding region may lack the light chain variable region. In certain embodiments, the molecular weight of the small antigen-binding region may be between 10 kDa and 40 kDa.

[0160] In some embodiments, 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 from about 10 kDa to about 15 kDa, from about 10 kDa to about 20 kDa, from about 10 kDa to about 25 kDa, from about 10 kDa to about 30 kDa, from about 10 kDa to about 35 kDa, from about 10 kDa to about 40 kDa, from about 15 kDa to about 20 kDa, from about 15 kDa to about 25 kDa, from about 15 kDa to about 30 kDa, from about 15 kDa to about 35 kDa, from about 15 kDa to about 40 kDa, from about 20 kDa to about 25 kDa, from about 20 kDa to about 30 kDa, from about 20 kDa to about 35 kDa, from about 20 kDa to about 40 kDa, from about 25 kDa to about 30 kDa, from about 25 kDa to about 35 kDa, from about 25 kDa to about 40 kDa, from about 30 kDa to about 35 kDa, from about 30 kDa to about 40 kDa, or from 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.

[0161] The antigen-binding region can comprise a VHH polypeptide, a scFv polypeptide, or a VNAR polypeptide. In certain embodiments, the antigen-binding region comprises a VHH polypeptide. In certain embodiments, the antigen-binding region comprises a ScFv polypeptide. In certain embodiments, the antigen-binding region comprises a VNAR polypeptide. In certain embodiments, the antigen-binding region is humanized.

[0162] The antigen region can include specificity for an antigen selected by a person skilled in the art to achieve a desired function, such as targeting a specific cancer, tumor, or cell type suitable for treatment with the immunoconjugate or radioimmunoconjugate described herein. As described herein, the antigen-binding region can be a fragment or form of an antibody known in the art. An intact antibody can be engineered to conform to the various small antigen-binding region forms (e.g., scFv) described herein. The antigen-binding region can specifically bind to a tumor antigen (e.g., an antigen specifically expressed or enriched in cancerous cells). In certain embodiments, the tumor antigen includes Her2, Trop2, CEA, NaPi2b, uPAR, CDCP1, MUC-1, MUC-16, CEACAM-5, MR-1, Fn14, MAGE-3, NY-ESO-1, EGFR, PDGFR, IGF1R, CSF-1R, PSMA, PSCA, STEAP-1, FAP, TEM8, 5T4, VEGFR, NRP1, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD39, CD44, CD47, CD52, CD70, CD71, CD74, CD79b, CD132, CD133, CD138, CD166, CD205, CD276, ROR1, ROR2, Glypican 3, Trail receptor 2 (DR5), PD-L1, mesothelin, Bombesin, EpCAM, DARPP, CSPG4, galectin-3, integrin αvβ1, integrin αvβ3, integrin αvβ5, integrin αvβ6, integrin α5β1, integrin α-3, integrin α-5, integrin β-6, Nectin-4, Wnt-activated inhibitor 1, DLL3, transferrin receptor, folate receptor α, tissue factor, BCMA, c-Met, LIV-1, AXL, AFP, ENPP3, CLDN6 / 9, DPEP3, RNF43, LRRC15, PTK7, P-cadherin, FLT3, EphA2, MTI-MMP, CXCR6, GD2, or Smoothened antigen (Smo). In certain embodiments, the tumor antigen includes human epidermal growth factor receptor 2 (HER2), delta-like ligand 3 (DLL3), folate receptor α (FOLR1), or Wnt-activated inhibitor 1 (WAIF1). In certain embodiments, the tumor antigen includes HER2. In certain embodiments, the tumor antigen includes DLL3. In certain embodiments, the tumor antigen includes FOLR1. In certain embodiments, the tumor antigen includes WAIF1. In certain embodiments, the tumor antigen includes TROP2. In certain embodiments, the tumor antigen includes EGFR. In certain embodiments, the tumor antigen includes PSA.In certain embodiments, the tumor antigen includes MUC-1. In certain embodiments, the tumor antigen includes CEA. In certain embodiments, the tumor antigen includes NY-ESO-1.

[0163] In certain embodiments, the antigen-binding region of the immunoconjugate comprises a sequence that has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to the sequence shown in SEQ ID NO:20 and binds HER2.

[0164] In certain 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.

[0165] In certain embodiments, the antigen-binding region of the immunoconjugate comprises a sequence that has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to the sequence shown in SEQ ID NO:30 and binds DLL3.

[0166] In certain 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.

[0167] In some embodiments, the immunoconjugates of the invention comprise synthetic engineered antibody derivatives such as, for example, proteins or polypeptides comprising autonomous V H domains (such as, for example, from camelids, murine, or human sources); single domain antibody domains (sdAb); heavy chain antibody domains derived from camelids (V H H fragments or V H domain fragments); heavy chain antibody domains derived from camelid V H H fragments or V H domain fragments; heavy chain antibody domains derived from cartilaginous fish; immunoglobulin new antigen receptors (IgNAR); V NAR fragments; single-chain variable (scFv) fragments; nanobodies; "camelized" scaffolds comprising V H domains; Fd fragments consisting of a heavy chain and a C H 1 domain; single-chain Fv-C H3 microantibodies; Fc antigen-binding domain (Fcab); scFv-Fc fusions; multimerized scFv fragments (diabodies, triabodies, tetra-bodies); disulfide-stabilized antibody variable (Fv) fragments (dsFv); disulfide-stabilized antigen-binding (Fab) fragments composed of V L , V H , C L and C H 1 domains; scFv with disulfide-stabilized heavy and light chains (sc-dsFv); bivalent nanobodies; bivalent microantibodies; bivalent F(ab’) 2 fragments (Fab dimers); bispecific tandem V H H fragments; bispecific tandem scFv fragments; bispecific nanobodies; bispecific microantibodies; and pairs of any of the foregoing genetic manipulations that retain complementarity and target antigen-binding function.

[0168] In some embodiments, the immunoconjugate is monovalent. In other embodiments, the immunoconjugate is multivalent, such as, for example, bivalent. In some additional embodiments, the immunoconjugate is bivalent and dimeric. In some additional embodiments, the bivalent immunoconjugate is homodimeric.

[0169] In one aspect, the invention provides antibody constructs (alone or in the context of the immunoconjugates, radioimmunoconjugates, or targeted imaging complexes of the invention), which comprise a VHH fragment that comprises a heavy-chain variable region that comprises three heavy-chain CDRs derived from a camelid and that binds an antigen with specificity and high affinity.

[0170] In some embodiments, the antibody construct, immunoconjugate, radioimmunoconjugate, or targeted imaging complex specifically binds at least one extracellular portion of an antigen expressed on the cell surface. In some embodiments, the immunoconjugate specifically binds at least one extracellular portion of an antigen expressed by a target cell (such as, for example, a tumor cell).

[0171] In some embodiments, the present disclosure provides an immunoconjugate that specifically binds an antigen. In some embodiments, the immunoconjugate comprises an antibody construct that comprises a heavy chain variable region (HVR-H) that comprises three CDRs: hCDR1, hCDR2, and hCDR3, such as, for example, those derived from camelid antibodies or IgNAR. In some embodiments, the immunoconjugate comprises: (a) a light chain variable region (HVR-L) that comprises three CDRs: lCDR1, lCDR2, and lCDR3, and (b) a heavy chain variable region (HVR-H) that comprises three CDRs: hCDR1, hCDR2, and hCDR3. In some embodiments, the antibody construct is chimeric or humanized.

[0172] In some embodiments, the immunoconjugate of the invention comprises an antibody construct that comprises an antigen-binding domain that is an antibody fragment, including but not limited to, for example, Fv, Fab, Fab’, scFv, HcAb fragment, VHH fragment, sdAb fragment, diabody, or F(ab')2 fragment. In some additional embodiments, the immunoconjugate of the invention comprises a multimer of two or more antibody fragments, such as, for example, a homodimer or heterodimer comprising two antibody fragments, each antibody fragment capable of binding the antigen with specificity and high affinity and each antibody fragment comprising a heavy chain variable region (HVR-H) that comprises three CDRs: hCDR1, hCDR2, and hCDR3.

[0173] Heavy chain constant region

[0174] The antigen-binding region of the immunoconjugates described herein can comprise an Fc or heavy chain constant region. The antigen-binding molecule can be conjugated to the Fc or heavy chain constant region directly, through a suitable linker, or through the IgG hinge region. Incorporating a heavy chain constant region or Fc region confers advantages such as allowing optimization and modulation of serum half-life, adding additional sites for conjugation of chelators or cytotoxic agents, and allowing purification of the immunoconjugate using standard processes and methods. Adding a heavy chain constant region also increases the size, thereby shifting catabolism and elimination of the immunoconjugate from the kidney to the liver. This can confer safety advantages, particularly for radioimmunoconjugates, as the kidney is more sensitive to radiation than the liver. Alterations that affect effector function or serum half-life can be made to residues present in the heavy chain constant region responsible for binding to the neonatal Fc receptor (FcRn). Binding to FcRn generally contributes to an increased half-life of molecules containing an immunoglobulin Fc, and thus reducing binding to FcRn can shorten the half-life of Fc-containing molecules. Reduction of FcRn binding can confer advantages such as shortening the half-life of the immunoconjugate and thus reducing subsequent toxicity attributed to the cytotoxic agent or radioisotope. In certain embodiments, the immunoglobulin constant region comprises or consists of an Fc region. In certain embodiments, the immunoglobulin heavy chain constant region comprises the CH2 domain of the immunoglobulin, the CH3 domain of the immunoglobulin, or the CH2 and CH3 domains of the immunoglobulin. In certain embodiments, the immunoglobulin heavy chain constant region comprises the CH2 and CH3 domains of the immunoglobulin. For treatment or imaging of a human individual, the immunoglobulin heavy chain constant region can be human, thereby preventing or reducing an endogenous immune response against the immunoconjugate. In certain embodiments, the immunoglobulin heavy chain constant region is a human immunoglobulin heavy chain constant region. In certain embodiments, the immunoglobulin heavy chain constant region is an IgA, IgG1, IgG2, IgG3, or IgG4 isotype. In certain embodiments, the immunoglobulin heavy chain constant region is an IgG1 isotype. In certain embodiments, the immunoglobulin heavy chain constant region is an IgG4 isotype.

[0175] The immunoglobulin heavy chain constant region can be a variant constant region that contains one or more amino acid residue alterations that confer additional utility and advantageous properties to the immunoconjugates described herein. In certain embodiments, the immunoglobulin heavy chain constant region contains 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 certain embodiments, the immunoglobulin heavy chain constant region contains alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region or decrease the binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region contains alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region and decrease the binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region contains alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region. In certain embodiments, the immunoglobulin heavy chain constant region contains alterations to one or more amino acid residues that decrease the binding of the immunoconjugate to the neonatal Fc receptor (FcRn).

[0176] Alterations in the constant region of the immunoconjugate heavy chain can reduce effector functions associated with the constant region of the heavy chain, such as the ability to fix complement, promote phagocytosis, or recruit other immune effector cells (e.g., NK cells) to the constant region of the heavy chain. In certain embodiments, the alteration of 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 cell phagocytosis ADCP, or a combination thereof. In certain embodiments, the alteration of one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region is selected from the list consisting of the following according to EU numbering: (a) 297A, 297Q, 297G, or 297D, (b) 279F, 279K, or 279L, (c) 228P, (d) 235A, 235E, 235G, 235Q, 235R, or 235S, (e) 237A, 237E, 237K, 237N, or 237R, (f) 234A, 234V, or 234F, (g) 233P, (h) 328A, (i) 327Q or 327T, (j) 329A, 329G, 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 (ppp) any combination of (a)–(ooo). In certain embodiments, the alteration of one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region comprises L234A, L235E, G237A, A330S and P331S according to EU numbering.,

[0177] Alterations in the heavy chain constant region of an immunoconjugate can shorten the serum half-life of the immunoconjugate. In certain embodiments, amino acid alterations that alter or reduce the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) shorten the serum half-life of the immunoconjugate. In certain embodiments, the alterations that alter or reduce the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are directed to amino acid residues selected from the list consisting of the following according to EU numbering: 251, 252, 253, 254, 255, 288, 309, 310, 312, 385, 386, 388, 400, 415, 433, 435, 436, 439, 447, and combinations thereof. In certain embodiments, the alterations that alter or reduce the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are directed to amino acid residues selected from the list consisting of the following according to EU numbering: 253, 254, 310, 435, 436, and combinations thereof. In certain embodiments, the alterations that alter or reduce the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are directed to amino acid residues selected from the list consisting of the following according to EU numbering: I253A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof. In certain embodiments, the alterations that alter or reduce the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are directed to amino acid residues selected from the list consisting of the following according to EU numbering: I253A, S254A, H310A, H435Q, Y436A, and combinations thereof. In certain embodiments, the alterations that alter or reduce the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are directed to amino acid residues selected from the list consisting of the following according to EU numbering: I253A, H310A, H435Q, and combinations thereof. In certain embodiments, the alterations that alter or reduce the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are directed to amino acid residues selected from the list consisting of the following according to EU numbering: H310A, H435Q, and combinations thereof.

[0178] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO:1. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:1. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO:1, wherein the heavy chain constant region comprises an I253A substitution according to EU numbering.

[0179] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:2. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:2. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:2, wherein the heavy chain constant region comprises an S254A substitution according to EU numbering.

[0180] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:3. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:3. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:3, wherein the heavy chain constant region comprises an H310A substitution according to EU numbering.

[0181] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:4. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:4. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:4, wherein the heavy chain constant region comprises an H435Q substitution according to EU numbering.

[0182] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:5. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:5. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:5, wherein the heavy chain constant region comprises a Y436A substitution according to EU numbering.

[0183] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO:6. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:6. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO:6, wherein the heavy chain constant region comprises the H310A / H435Q substitution according to EU numbering.

[0184] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO:7. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:7. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO:7, wherein the heavy chain constant region comprises the L234A, L235E, G237A, A330S, and P331S substitutions according to EU numbering.

[0185] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO:8. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:8, wherein the heavy chain constant region comprises the L234A, L235E, G237A, H310A, A330S, and P331S substitutions according to EU numbering.

[0186] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO:9. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:9. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:9, wherein the heavy chain constant region comprises the L234A, L235E, G237A, H435Q, A330S, and P331S substitutions according to EU numbering.

[0187] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:10. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:10 according to EU numbering.

[0188] In one embodiment, each of the two variant constant regions has at least one FcRn binding mutation. In one embodiment, each of the two variant constant regions has the same FcRn binding mutation. In one embodiment, each of the two variant constant regions has a different FcRn binding mutation.

[0189] In one embodiment, at least one of the variant constant regions in the immunoconjugate has at least one FcRn binding mutation. In a preferred embodiment, each of the two variant constant regions of the immunoconjugate has at least one FcRn binding mutation, which may be the same or different.

[0190] Alterations that affect FcRn binding can shorten the serum half-life of the immunoconjugate, thus allowing the skilled person to select a half-life suitable for a particular imaging or therapeutic goal. In certain embodiments, the serum half-life of the immunoconjugate is from about 12 hours to about 120 hours. In certain embodiments, the serum half-life of the immunoconjugate is from about 12 hours to about 24 hours, from about 12 hours to about 36 hours, from about 12 hours to about 48 hours, from about 12 hours to about 60 hours, from about 12 hours to about 72 hours, from about 12 hours to about 84 hours, from about 12 hours to about 96 hours, from about 12 hours to about 108 hours, from about 12 hours to about 120 hours, from about 24 hours to about 36 hours, from about 24 hours to about 48 hours, from about 24 hours to about 60 hours, from about 24 hours to about 72 hours, from about 24 hours to about 84 hours, from about 24 hours to about 96 hours, from about 24 hours to about 108 hours, from about 24 hours to about 120 hours, from about 36 hours to about 48 hours, from about 36 hours to about 60 hours, from about 36 hours to about 72 hours, from about 36 hours to about 84 hours, from about 36 hours to about 96 hours, from about 36 hours to about 108 hours, from about 36 hours to about 120 hours, from about 48 hours to about 60 hours, from about 48 hours to about 72 hours, from about 48 hours to about 84 hours, from about 48 hours to about 96 hours, from about 48 hours to about 108 hours, from about 48 hours to about 120 hours, from about 60 hours to about 72 hours, from about 60 hours to about 84 hours, from about 60 hours to about 96 hours, from about 60 hours to about 108 hours, from about 60 hours to about 120 hours, from about 72 hours to about 84 hours, from about 72 hours to about 96 hours, from about 72 hours to about 108 hours, from about 72 hours to about 120 hours, from about 84 hours to about 96 hours, from about 84 hours to about 108 hours, from about 84 hours to about 120 hours, from about 96 hours to about 108 hours, from about 96 hours to about 120 hours or from about 108 hours to about 120 hours. In certain 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 certain 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 certain 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.

[0191] In certain embodiments, the serum half-life of the immunoconjugate is from about 1 day to about 10 days. In certain embodiments, the serum half-life of the immunoconjugate is from about 1 day to about 2 days, from about 1 day to about 3 days, from about 1 day to about 4 days, from about 1 day to about 5 days, from about 1 day to about 6 days, from about 1 day to about 7 days, from about 1 day to about 8 days, from about 1 day to about 9 days, from about 1 day to about 10 days, from about 2 days to about 3 days, from about 2 days to about 4 days, from about 2 days to about 5 days, from about 2 days to about 6 days, from about 2 days to about 7 days, from about 2 days to about 8 days, from about 2 days to about 9 days, from about 2 days to about 10 days, from about 3 days to about 4 days, from about 3 days to about 5 days, from about 3 days to about 6 days, from about 3 days to about 7 days, from about 3 days to about 8 days, from about 3 days to about 9 days, from about 3 days to about 10 days, from about 4 days to about 5 days, from about 4 days to about 6 days, from about 4 days to about 7 days, from about 4 days to about 8 days, from about 4 days to about 9 days, from about 4 days to about 10 days, from about 5 days to about 6 days, from about 5 days to about 7 days, from about 5 days to about 8 days, from about 5 days to about 9 days, from about 5 days to about 10 days, from about 6 days to about 7 days, from about 6 days to about 8 days, from about 6 days to about 9 days, from about 6 days to about 10 days, from about 7 days to about 8 days, from about 7 days to about 9 days, from about 7 days to about 10 days, from about 8 days to about 9 days, from about 8 days to about 10 days, or from about 9 days to about 10 days. In certain embodiments, the serum half-life of the immunoconjugate is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days. In certain embodiments, the 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 certain 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.

[0192] In certain embodiments, the molecular weight of the heavy chain constant region is from about 10 kDa to about 25 kDa. In certain embodiments, the molecular weight of the heavy chain constant region is from about 10 kDa to about 15 kDa, from about 10 kDa to about 20 kDa, from about 10 kDa to about 25 kDa, from about 15 kDa to about 20 kDa, from about 15 kDa to about 25 kDa, or from about 20 kDa to about 25 kDa. In certain 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 certain embodiments, the molecular weight of the heavy chain constant region is at least about 10 kDa, about 15 kDa, or about 20 kDa. In certain embodiments, the molecular weight of the heavy chain constant region is at most about 15 kDa, about 20 kDa, or about 25 kDa.

[0193] In some embodiments, the immunoconjugates of the invention comprise a linker or hinge region, which is a polypeptide that links the antigen-binding region to the heavy chain constant region or variant constant region of the invention. Naturally occurring and synthetic hinge regions that link immunoglobulin components are well known in the art and can be used in the present invention. See, for example, US 8,067,548 and the references therein.

[0194] In one embodiment, the hinge regions of the immunoconjugate are the same. In one embodiment, the hinge regions of the immunoconjugate are different.

[0195] The antigen-binding region and the heavy chain constant region (with or without amino acid sequence changes) can be linked by a suitable hinge or linker sequence. In certain 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. Suitable IgG hinge regions include and encompass IgG1 or IgG4 hinge regions. In certain embodiments, the hinge region is an IgG1 hinge region. In certain embodiments, the hinge region is an IgG1 hinge region with a C220S substitution according to EU numbering. 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 September 1993, 90(17)7995-7999; Davis et al., “Abatacept binds to the Fc receptor CD64 but does not mediate complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity.” J Rheumatol. November 2007; 334(11):2204-10. Suitable hinges can also include non-IgG-based polypeptide linkers. The linker amino acid sequence can primarily comprise the following amino acid residues: Gly, Ser, Ala, or Thr. The length of the linker peptide should be sufficient to present the two molecules in the correct conformation relative to each other and thus link the two molecules in a manner that retains the desired activity. In one embodiment, the length of the linker is about 1 to 50 amino acids or a length of about 1 to 30 amino acids. In one embodiment, a linker with a length of 1 to 20 amino acids 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.Exemplarily, linkers for connecting antibody fragments or single-chain variable fragments can include AAEPKSS, AAEPKSSDKTHTCPPCP, GGGG or GGGGDKTHTCPPCP. Alternatively, a variety of non-proteinaceous polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyalkylene oxide or copolymers of polyethylene glycol and polypropylene glycol, can be used as linkers, i.e., can be used as linkers.

[0196] The total size of the immunoconjugate can be such that it facilitates tissue penetration, stability, and / or clearance. In certain embodiments, the molecular weight of the immunoconjugate is from about 60 kDa to about 120 kDa. In certain embodiments, the molecular weight of the immunoconjugate is from about 60 kDa to about 65 kDa, from about 60 kDa to about 70 kDa, from about 60 kDa to about 75 kDa, from about 60 kDa to about 80 kDa, from about 60 kDa to about 90 kDa, from about 60 kDa to about 100 kDa, from about 60 kDa to about 110 kDa, from about 60 kDa to about 120 kDa, from about 65 kDa to about 70 kDa, from about 65 kDa to about 75 kDa, from about 65 kDa to about 80 kDa, from about 65 kDa to about 90 kDa, from about 65 kDa to about 100 kDa, from about 65 kDa to about 110 kDa, from about 65 kDa to about 120 kDa, from about 70 kDa to about 75 kDa, from about 70 kDa to about 80 kDa, from about 70 kDa to about 90 kDa, from about 70 kDa to about 100 kDa, from about 70 kDa to about 110 kDa, from about 70 kDa to about 120 kDa, from about 75 kDa to about 80 kDa, from about 75 kDa to about 90 kDa, from about 75 kDa to about 100 kDa, from about 75 kDa to about 110 kDa, from about 75 kDa to about 120 kDa, from about 80 kDa to about 90 kDa, from about 80 kDa to about 100 kDa, from about 80 kDa to about 110 kDa, from about 80 kDa to about 120 kDa, from about 90 kDa to about 100 kDa, from about 90 kDa to about 110 kDa, from about 90 kDa to about 120 kDa, from about 100 kDa to about 110 kDa, from about 100 kDa to about 120 kDa, or from about 110 kDa to about 120 kDa. In certain 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 certain 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 certain 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.

[0197] In some embodiments, the molecular weight of the immunoconjugate is greater than 60, 70, 75, 80, 82, 83, 85, 86, 87, 88, or 89 kDa. In some embodiments, the molecular weight of the immunoconjugate is less than 110, 100, 95, 93, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, or 80 kDa. In some embodiments, the molecular weight of the immunoconjugate is greater than 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, or 79 kDa and less than 110, 100, 95, 93, 91, or 90 kDa.

[0198] The size of the immunoconjugates and / or heavy chain constant region variants described herein results in an increase in the safety or therapeutic index of the immunoconjugates included herein. This safety can be reflected in a decrease in radiation accumulation in radiosensitive major tissues such as the kidney and bone marrow and / or an increase in radiation accumulation in target tissues (i.e., tumor or cancerous tissue) or organs with greater radiation tolerance such as the liver.

[0199] In certain embodiments, the immunoconjugates of the present disclosure result in a total radiation exposure per treatment, as measured in grays (Gy). In certain embodiments, the kidney is exposed to 20 Gy or less per treatment. In certain embodiments, the kidney is exposed to 19 Gy or less per treatment. In certain embodiments, the kidney is exposed to 18 Gy or less per treatment. In certain embodiments, the kidney is exposed to 17 Gy or less per treatment. In certain embodiments, the kidney is exposed to 16 Gy or less per treatment. In certain embodiments, the kidney is exposed to 15 Gy or less per treatment. In certain embodiments, the kidney is exposed to 14 Gy or less per treatment. In certain embodiments, the kidney is exposed to 13 Gy or less per treatment. In certain embodiments, the kidney is exposed to 12 Gy or less per treatment. In certain embodiments, the kidney is exposed to 11 Gy or less per treatment. In certain embodiments, the kidney is exposed to 10 Gy or less per treatment. In certain embodiments, the kidney is exposed to 9 Gy or less per treatment. In certain embodiments, the kidney is exposed to 8 Gy or less per treatment. In certain embodiments, the kidney is exposed to 5 Gy or less per treatment.

[0200] In certain embodiments, the immunoconjugates of the present disclosure result in a total radiation exposure per treatment, as measured in grays (Gy). In certain embodiments, the bone marrow is exposed to 4 Gy or less per treatment. In certain embodiments, the bone marrow is exposed to 3 Gy or less per treatment. In certain embodiments, the bone marrow is exposed to 2 Gy or less per treatment. In certain embodiments, the bone marrow is exposed to 1.5 Gy or less per treatment. In certain embodiments, the bone marrow is exposed to 1.0 Gy or less per treatment. In certain embodiments, the bone marrow is exposed to 0.5 Gy or less per treatment.

[0201] In certain embodiments, when measured as a percentage of the injection dose per gram, the immunoconjugates of the present disclosure result in a higher amount of radiation in the tumor compared to the kidney. In certain embodiments, the ratio of the percentage of the injection dose per gram in the tumor to the percentage of the injection 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.

[0202] In certain embodiments, when measured as a percentage of the injection dose per gram, the immunoconjugates of the present disclosure result in a higher amount of radiation in the tumor compared to the blood. In certain embodiments, the ratio of the percentage of the injection dose per gram in the tumor to the percentage of the injection 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.

[0203] In certain embodiments, when measured as a percentage of the injection dose per gram, the immunoconjugates of the present disclosure result in a higher amount of radiation in the tumor compared to the bone marrow. In certain embodiments, the ratio of the percentage of the injection dose per gram in the tumor to the percentage of the injection 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.

[0204] In certain embodiments, when measured as the injection dose per gram, the immunoconjugates of the present disclosure result in a higher amount of radiation in the liver compared to the kidney. In certain embodiments, the ratio of the percentage of the injection dose per gram in the tumor to the percentage of the injection dose per gram in the bone marrow is greater than 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0205] In some embodiments, the present invention contemplates variants of the immunoconjugates of the invention that comprise an Fc region, where the variant has some but not all effector functions, making it a desirable candidate for applications where the in vivo half-life of the immunoconjugate 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 reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the immunoconjugate lacks FcγγR binding (and thus likely lacks ADCC activity), but retains FcRn binding ability. The major cell type mediating 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 the ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., 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-radiometric assay methods can be employed (see, e.g., ACTI TM Non-radiometric cytotoxicity assay (CellTechnology, Inc., Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assays (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be evaluated in vivo, for example in animal models such as those disclosed in Clynes et al., Proc Natl Acad Sci USA 95:652-656 (1998). A Clq binding assay can also be performed to confirm that the immunoconjugate does not bind Clq and thus lacks CDC activity (see, for example, Clq and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402). To evaluate complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, S.B. et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0206] Immunoconjugates with reduced effector function include those in which one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 are substituted (US 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of the amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (US 7,332,581).

[0207] Immunoconjugates can have altered effector function by including the following changes according to EU numbering: L234A, L235E, G237A, A330S, and P331S, which reduce Fc receptor binding. See, e.g., US 8,613,926 or Andersson C, Wenander et al., “Rapid-onset clinical and mechanistic effects of anti-C5aR treatment in the mouse collagen-induced arthritis model.” Clin Exp Immunol. July 2014;177(1):219-33.

[0208] Certain immunoconjugate variants with enhanced or diminished binding to FcR are described (see, e.g., US 6,737,056; WO 2004 / 056312; Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0209] In some embodiments, the changes are made in the Fc region, resulting in altered (i.e., enhanced or reduced) Clq binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in US 6,194,551; WO 1999 / 051642; Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0210] Antibodies with extended half-life and enhanced binding to the neonatal Fc receptor (FcRn), which is responsible for transferring maternal IgG to the fetus, are described in US2005 / 0014934 (Guyer et al., J. Immunol. 117:587 (1976); Kim et al., J. Immunol. 24:249 (1994)). These antibodies contain an Fc region with one or more substitutions therein that enhance the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues 434 or 435, e.g., substitutions of 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 relate to other examples of Fc region variants.

[0211] 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 of 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.

[0212] As will be recognized by those of ordinary skill in the art, certain teachings herein apply to the antibody constructs, targeting 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 encompassed by the present invention.

[0213] Chelating agent

[0214] As described herein, in some embodiments, a chelator (i.e., R 1 ) is conjugated to a tumor targeting moiety (e.g., a polypeptide comprising an antigen binding region and an immunoglobulin heavy chain constant region described herein). The chelating moiety allows the tumor targeting moiety to be loaded with a suitable radioisotope, such as a β-emitter or an α-emitter. The chelating agent can be conjugated to the antigen binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof. Such conjugation can be effected appropriately by covalent attachment to one or more amino acids of the immunoconjugate, antigen binding region, heavy chain constant region, immunoglobulin Fc region, or any combination thereof.

[0215] In one embodiment, the chelator of the immunoconjugate is covalently linked to the antigen binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof. In one embodiment, the chelator is directly covalently linked to the antigen binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof (e.g., without using a spacer, stretcher, or linker). In one embodiment, the chelator is covalently linked to the antigen binding arm through a linker that is covalently linked to the chelator and covalently linked to the antigen binding arm. In one embodiment, the linker is hydrophilic (e.g., a PEG chain). In one embodiment, the linker is hydrophobic (e.g., an alkyl or olefin chain). The chelating agent can be linked or conjugated to the immunoconjugate as described by Sadiki, A. et al., "Site-specific conjugation of native antibody." Antibody Therapeutics 2020, 3, 271-284.

[0216] In some embodiments, the immunoconjugate is formed by attaching a chelator-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 the framework region with a chelator-linker. In other embodiments, this residue can be functionalized with a different reactive functional group and then reacted with the chelator-linker in a second step to yield the immunoconjugate. In some embodiments, this reactive functional group is a thioester group.

[0217] In some embodiments, a non-natural cysteine residue is engineered into the framework of the antibody as a site for thiol-directed conjugation to yield the immunoconjugate. In some embodiments, other non-natural amino acids or amino acid sequences are engineered into the framework to serve as attachment sites for the chelator-linker or a second reactive group, to which the chelator-linker will conjugate to yield the immunoconjugate.

[0218] In some embodiments, a non-natural amino acid containing a crosslinking group is engineered into the framework for attachment of the chelator-linker. In some embodiments, this non-natural amino acid contains an azide.

[0219] In some embodiments, the chelator-linker is attached to a glutamine residue through the action of transglutaminase. In other embodiments, a second reactive group is attached through transglutaminase, and the chelator-linker is added to the transglutaminase to yield the immunoconjugate.

[0220] In some embodiments, one or more N-glycans are modified with a reactive functional group through the action of a glycosidase, and then the chelator-linker is conjugated to the site to attach the chelator-linker. In some embodiments, the glycan is modified through the action of β-galactosidase. In some embodiments, the glycan is modified with a glycoside containing an azide for attachment of a suitably functionalized chelator-linker.

[0221] In one embodiment, the immunoconjugate comprises more than one chelator, which are the same or different.

[0222] In one embodiment, an immunoconjugate having more than one chelator has more than one chelator attached to the same antigen-binding arm.

[0223] In one embodiment, an immunoconjugate having more than one chelator and fewer than eleven chelators has more than two chelators, more than three chelators, more than four chelators, more than five chelators, more than six chelators, more than seven chelators, more than eight chelators, or more than nine chelators. In one embodiment, the chelators are the same. In one embodiment, each antigen-binding arm is directly or indirectly linked to more than one chelator.

[0224] In one embodiment, the chelator comprises a radioisotope chelating moiety and a functional group that permits covalent attachment to the antigen-binding arm. In one embodiment, the functional group is directly attached to the radioisotope chelating moiety. In one embodiment, the chelator further comprises a linker between the functional group and the radioisotope chelating moiety.

[0225] In one embodiment, the radioisotope chelating moiety comprises DOTA or a DOTA derivative. In one embodiment, the radioisotope chelating moiety comprises DOTAGA. In one embodiment, the radioisotope chelating moiety comprises macropa or a macropa derivative. In one embodiment, the radioisotope chelating moiety comprises Py4Pa or a Py4Pa derivative.

[0226] In a preferred embodiment, the chelator of the immunoconjugate is not attached to the antigen-binding region in the antigen-binding arm of the immunoconjugate.

[0227] In one embodiment, the chelator of the immunoconjugate is non-covalently associated with the antigen-binding arm. In a preferred embodiment, the chelating agent is not associated with the antigen-binding region in the antigen-binding arm of the immunoconjugate.

[0228] In one embodiment, the chelator comprises DOTA or a DOTA derivative. In one embodiment, the chelator comprises DOTAGA. In one embodiment, the chelator comprises macropa or a macropa derivative. In one embodiment, the chelator comprises Py4Pa or a Py4Pa derivative. In one embodiment, the chelator comprises transferrin or a transferrin derivative.

[0229] In certain embodiments, the chelator is a radioisotope chelator. In certain embodiments, the radioisotope chelator is selected from the list consisting of: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), α-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTAGA), or (Py4Pa). In certain embodiments, the radioisotope chelator is DOTA. In certain embodiments, the radioisotope chelator is DOTAGA. In certain embodiments, the radioisotope chelator is Py4Pa. In certain embodiments, the radioisotope, wherein the radioisotope chelator is directly conjugated to the antigen-binding region and / or the constant region of the immunoglobulin heavy chain. In certain embodiments, the radioisotope chelator is conjugated to the antigen-binding region or the constant region of the immunoglobulin heavy chain via a linker. In certain embodiments, the linker is selected from: 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzoxycarbonyl (PAB), and those obtained by conjugation with the following linker reagents: N-succinimidyl 4-(2-pyridylthio)valerate (SPP) that forms the linker moiety 4-mercaptopentanoic acid, N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl 4-(2-pyridyldithio)butyrate (SPDB), N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB), polyethylene glycol (PEG), polyethylene glycol polymer (PEGn), and S-2-(4-isothiocyanobenzyl) (SCN). In certain embodiments, the linker is selected from: polyethylene glycol (PEG), polyethylene glycol polymer (PEG), and S-2-(4-isothiocyanobenzyl) (SCN). In certain embodiments, the linker is PEG5. In certain embodiments, the linker is SCN. In certain embodiments, the radioisotope chelator is a linker-chelate factor selected from the list consisting of: TFP-Ad-PEG5-DOTAGA, p-SCN-Bn-DOTA, p-SCN-Ph-Et-Py4Pa, and TFP-Ad-PEG5-Ac-Py4Pa.

[0230] In some embodiments, the chelating agent is conjugated to a polypeptide (i.e., an antigen-binding region and / or an immunoglobulin heavy chain constant region) at a predetermined ratio of the chelating agent. In certain embodiments, the radioisotope chelator 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 certain embodiments, the radioisotope chelator 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 certain embodiments, the radioisotope chelator is coupled to the antigen-binding region and / or the immunoglobulin heavy chain constant region at a ratio of 2:1 to 6:1.

[0231] For example, a bifunctional chelating agent is used to conjugate a radioisotope to the radioisotope delivery platform of the invention to produce an immunoconjugate of the invention. (See, e.g., Scheinberg D, McDevitt M, Curr Radiopharm 4:306-20 (2011)). Examples of bifunctional chelating agents 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.

[0232] Examples of bifunctional chelating agents are 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), and related analogs thereof. Such chelating agents are suitable for coordinating metal ions such as α- and β-emitting radionuclides.

[0233] In some embodiments, the chelating agent of the immunoconjugate or radioimmunoconjugate of the present invention is selected from bifunctional chelating agents, DOTA, DO3A-NHS, DOTAGA-NHS, DOTAGA-anhydride DOTAGA-TFP, p-SCN-Bn-DOTA, p-SCN-Bn-DTPA, p-SCN-Bn-CHX-A”-DTPA, p-SCN-Bn-TCMC, macropa-NCS (Thiele NA et al., Angew. Chem. Int. Ed. 56:1 (2017)), crown (Yang H et al., Chem. Eur. J. 26:11435 (2020)), P-SCN-Ph-Et-Py4Pa (Li L et al., Bioconjugate Chem. ASAP (2020)), 3,2-HOPO (Wickstroem K et al., Int. J. Rad. Oncol. Biol. Phys. 105:410 (2019)) (for reviews of these and other bifunctional chelating agents, see, for example, Price EW and Orvig C Chem. Soc. Rev., 2014, 43:260 (2014) and Brechbiel MW Q. J. Nucl. Med. Mol. Imaging 52:166 (2008)).

[0234] In some embodiments, the chelating agent of the immunoconjugate or radioimmunoconjugate of the present invention is selected from bifunctional chelating agents, DOTA, DO3A-NHS, DOTAGA-NHS, DOTAGA-anhydride DOTAGA-TFP, p-SCN-Bn-DOTA, p-SCN-Bn-DTPA, p-SCN-Bn-CHX-A”-DTPA, p-SCN-Bn-TCMC, macropa-NCS (Thiele NA et al., Angew. Chem. Int. Ed. 56:1 (2017)), crown (Yang H et al., Chem. Eur. J. 26:11435 (2020)), P-SCN-Ph-Et-Py4Pa (Li L et al., Bioconjugate Chem. ASAP (2020)), 3,2-HOPO (Wickstroem K et al., Int. J. Rad. Oncol. Biol. Phys. 105:410 (2019)) (for reviews of these and other bifunctional chelating agents, see, for example, Price EW and Orvig C Chem. Soc. Rev., 2014, 43:260 (2014) and Brechbiel MW Q. J. Nucl. Med. Mol. Imaging 52:166 (2008)).

[0235] For 225 Ac immunoconjugates, a variety of acyclic and cyclic ligands known in the art are 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)).

[0236] In certain embodiments, 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. January 2022; 63(1):5-13.

[0237] In certain embodiments, chelating agents suitable for α-emitter chelation are selected from the list consisting of: DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; DO3A, 1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane; DOTAGA, α-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; DOTAGA anhydride, (2,2',2”-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid; Py4Pa, 6,6',6”,6”'-(((pyridine-2,6-diylbis(methylene))bis(azanetriyl))-tetrakis(methylene))tetrapicolinic acid; Py4Pa-NCS, 6,6'-((((4-isothiocyanatopyridine-2,6-diyl)bis(methylene))bis((carboxymethyl)azanediyl))-bis(methylene))dipicolinic acid; Crown, 2,2',2”,2”'-(1,10-dioxo-4,7,13,16-tetraazacyclooctadecane-4,7,13,16-tetrayl)tetraacetic acid; Macropa, 6,6'-((1,4,10,13-tetraoxo-7,16-diaza-cyclooctadecane-7,16-diyl)bis(methylene))-dipicolinic acid; Macropa-NCS, 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxo-7,16-diazacyclooctadecane-7-yl)methyl)-4-isothiocyanatopicolinic acid; HEHA, 1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid; CHXoctapa, 6,6'-[(1R,2R)-1,2-cyclohexanediylbis[[(carboxymethyl)imino]methylene]]bis[2-pyridinecarboxylic acid]; Bispa, 3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylic acid, 7-[(6-carboxy-2-pyridinyl)methyl]-9-hydroxy-3-methyl-2,4-di-2-pyridinyl-, 1,5-dimethyl ester; Noneunpa, 6,6'-(((oxy-bis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))-dipicolinic acid; and combinations thereof.

[0238] In certain embodiments, the chelating agent is a chelating agent suitable for chelating a β or γ emitter. In certain embodiments, chelating agents suitable for chelating a β or γ emitter are selected from the list consisting of: 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-tetraazacyclo-dodecane-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(acetamido-methylenephosphonic acid); CB-TE2A, 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid; NOTA, 1,4,7-triazacyclononane-1,4,7-triacetic acid; NOTP, 1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid); TETPA, 1,4,8,11-tetraazacyclo-tetradecane-1,4,8,11-tetrapropionic acid; TETA, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid; PEPA, 1,4,7,10,13-pentaazacyclopentadecane-N,N',N”,N”',N””-pentaacetic acid; H4Octapa, N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid; H2Dedpa, 1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane; H6phospa, N,N'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane; TTHA, triethylenetetraamine-N,N,N',N”,N”',N”'-hexaacetic acid; DO2P, tetraazacyclododecane dimethane-phosphonic acid; HP-DO3A, hydroxypropyltetraazacyclododecane triacetic acid; EDTA, ethylenediaminetetraacetic acid; DTPA, diethylenetriaminepentaacetic acid; DTPA-BMA, diethylenetriaminepentaacetic acid-bisdimethylamide; HOPO, octadentate hydroxypyridone; 3,2,3-LI(HOPO), N,N'-(butane-1,4-diyl)bis(1-hydroxy-N-(3-(1-hydroxy-6-oxo-1,6-dihydro-pyridin-2-carboxamido)propyl)-6-oxo-1,6-dihydropyridin-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)amino)-propyl)azanediyl)bis(ethane-2,1-diyl))bis(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamide); Neunpa, 6,6'-(((azanediylbis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))-bis(methylene))dipicolinic acid; Neunpa-NCS, 6,6'-(((((4-isothiocyanatophenethyl)azanediyl)-bis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid; Octapa, 6,6'-((ethane-1,2-diylbis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid; Octox, 2,2'-(ethane-1,2-diylbis(((8-hydroxyquinolin-2-yl)methyl)azanediyl))diacetic acid; PyPa, 6,6'-(((pyridine-2,6-diylbis(methylene))bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid; porphyrin, 21,22,23,24-tetraazapentacyclo[16.2.1.13,6.1.18,11.113,16]tetracosa-1,3,5,7,9,11(23),12,14,16,18(21),19-undecene; desferrioxamine, 30-amino-3,14,25-trihydroxy-3,9,14,20,25-pentaazatricosan-2,10,13,21,24-pentone; DFO*, N1-[5-(acetylhydroxyamino)-pentyl]-N26-(5-aminopentyl)-N26,5,16-trihydroxy-4,12,15,23-tetraoxo-5,11,16,22-tetraazatetracosanediamide; and combinations thereof.;

[0239] In some embodiments, R 1 comprises a chelating moiety selected from the list consisting of: DOTA, DO3A, DO3Apic, DOTAGA, DOTAGA anhydride, Py4Pa, Py4Pa-NCS, Crown, Macropa, Macropa-NCS, HEHA, CHXoctapa, Bispa, and Noneunpa or a radionuclide complex thereof.

[0240] In some embodiments, R 1comprises a chelating moiety selected from the list consisting of: DOTMA, DOTPA, DO3Apic, DO3AM-acetic acid, DOTP, DOTMP, DOTA-4AMP, CB-TE2A, NOTA, NOTP, TETPA, TETA, PEPA, H4Octapa, H2Dedpa, DO2P, EDTA, DTPA-BMA, 3,2,3-LI(HOPO), 3,2-HOPO, Neunpa, Neunpa-NCS, Octapa, PyPa, porphyrin, and desferrioxamine or its radionuclide complex.

[0241] In some embodiments, R 1 is a chelating moiety selected from the list consisting of:

[0242] 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA);

[0243] 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A);

[0244] 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A);

[0245] α,α',α”,α”'-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTMA);

[0246] 1,4,7,10-tetra(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM); 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid (DOTPA);

[0247] 2,2',2”-(10-(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid;

[0248] 10-((6-carboxypyridin-2-yl)methyl)-1,4,7,10-tetra-azacyclododecane-1,4,7-triacetic acid (DO3Apic);

[0249] 6,6'-(((pyridine-2,6-diylbis(methylene))bis((carboxymethyl)azanediyl))-bis(methylene))dipicolinic acid (H 4 pypa);

[0250] 6,6',6”,6”'-(((pyridine-2,6-diylbis(methylene))bis(azanetriyl))tetra(methylene))-tetrapicolinic acid (H 4py4pa); and

[0251] 3,6,9,12 - tetra(carboxymethyl)-3,6,9,12 - tetraazatetradecanedioic acid (TTHA);

[0252] or a radionuclide complex thereof.

[0253] In some embodiments, R 1 comprises a chelating moiety having one of the following structures:

[0254]

[0255]

[0256]

[0257]

[0258]

[0259] In some embodiments, R 1 comprises DOTA and is attached to a tumor targeting moiety at the indicated position:

[0260] In some embodiments, R 1 comprises DOTA and is attached to a tumor targeting moiety at the indicated position:

[0261] In some embodiments, R 1 comprises DOTA and is attached to a tumor targeting moiety at the indicated position:

[0262] It should be understood that the immunoconjugates described herein comprise one or more R 1 moieties. In some embodiments, the immunoconjugates described herein comprise one or more than one R 1 moiety, wherein each R 1 moiety is the same.

[0263] Radionuclide

[0264] In some embodiments, the radionuclide in the immunoconjugates described herein is an Auger electron-emitting radionuclide. In some embodiments, the radionuclide is an α-emitting radionuclide. In some embodiments, the radionuclide is a β-emitting radionuclide. In some embodiments, the radionuclide is a γ-emitting radionuclide. In some embodiments, the type of radionuclide used in a non-peptide targeting therapeutic compound can be customized for the specific type of cancer, the type of targeting moiety (e.g., non-peptide ligand), etc. A radionuclide that undergoes α decay emits an α particle (a helium ion with a +2 charge) from its atomic nucleus. Due to α decay, the daughter nuclide has 2 fewer protons and 2 fewer neutrons than the parent nuclide. This means that in α decay, the number of protons decreases by 2 and the number of nucleons decreases by 4. A radionuclide that undergoes β decay emits a β particle (an electron) from its atomic nucleus. During β decay, one of the neutrons turns into a proton and an electron. The proton remains in the atomic nucleus, and the electron is emitted as a β particle. This means that in β decay, the atomic nucleus loses a neutron but gains a proton. In γ decay, an atomic nucleus in an excited state (higher energy state) emits a γ-ray photon to become a lower energy state. During γ decay, the number of protons and the number of nucleons do not change. The emission of γ rays is usually accompanied by the emission of α particles and β particles.

[0265] Auger electrons (AEs) are very low energy electrons emitted by radionuclides (e.g., 111 In, gallium-67 ( 67 Ga), technetium-99m ( 99m Tc), platinum-195m ( 195m Pt), iodine-125 ( 125 I) and iodine-123 ( 123 I)) that decay by electron capture (EC). The energy deposition occurs over nano-micron distances, resulting in high linear energy transfer, which is effective in causing lethal damage to cancer cells. Therefore, radiotherapeutic agents that emit AEs have great potential for treating cancer.

[0266] β particles are electrons emitted from the atomic nucleus. They typically have a relatively long range in tissue (1 - 5 mm), and they are the most commonly used.

[0267] An α-particle is a helium nucleus (two protons and two neutrons) emitted from the nucleus of a radioactive atom. Depending on its emission energy, it can travel 50 - 100 μm in tissue. They are positively charged and are on the order of magnitude larger than electrons. The amount of energy deposited per unit path length of an α-particle (referred to as "linear energy transfer") is approximately 400 times that of an electron. This results in much greater damage along its path than that caused by electrons. α-particle tracks result in a large number of complex and mostly irreparable DNA double-strand breaks. The absorbed dose required to achieve cytotoxicity is related to the number of α-particles passing through the cell nucleus. Using this as a measure, cytotoxicity can be achieved within the range of 1 to 20 α-particles passing through the cell nucleus. The combination of the resulting high potency with the short range of α-particles (which reduces normal organ toxicity) has led to a great deal of interest in the development of α-particle emitters. Commonly used α-particle emitters include 212 Bi, 212 Pb, 213 Bi, 225 Ac, 223 Ra and 229 Th.

[0268] In some embodiments, the radionuclide is a diagnostic or therapeutic radionuclide.

[0269] Representative radionuclides

[0270] Isotope <![CDATA[t 1 / 2 (h)]]> Decay mode <![CDATA 66 Ga]]> 9.5 β+(56%), EC(44%) <![CDATA 67 Ga]]> 78.2 EC(100%) <![CDATA 68 Ga]]> 1.1 β+(90%), EC(10%) <![CDATA 111 In]]> 67.2 EC(100%) <![CDATA 114m In]]> 49.5d EC(100%) <![CDATA 114 In (daughter)]]> 73s β-(100％) <![CDATA 177 Lu]]> 159.4 β-(100％) <![CDATA 89 Zr]]> 78.5 β+(23%), EC(77%) <![CDATA 212 Bi]]> 1.1 α(36％)，β-(64％) <![CDATA 213 Bi]]> 0.76 α(2.2％)，β-(97.8％) <![CDATA 212 Pb (the daughter is 212 Bi)]]> 10.6 β-(100％) <![CDATA 225 Ac]]> 240 α(100％)

[0271] 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 embodiments, the radionuclide is an Auger electron-emitting radionuclide, which is 111 In, 67 Ga, 68 Ga or 99m Tc.

[0272] In some embodiments, the radionuclide is an α-emitting radionuclide. In some embodiments, the radionuclide is an α-emitting radionuclide, which is 225 Ac, 213 Bi, 223 Ra or 212 Pb. In some embodiments, the radionuclide is an α-emitting radionuclide, which is 225 Ac. In some embodiments, the radionuclide is a β-emitting radionuclide. In some embodiments, the radionuclide is a β-emitting radionuclide, which is90 Y, 177 Lu, rhenium-186 ( 186 Re), rhenium-188 ( 188 Re), 64 Cu, 67 Cu, 153 Sm, 89 Sr, gold-198 ( 198 Au), erbium-169 ( 169 Er), dysprosium-165 ( 165 Dy), 99m Tc, 89 Zr or manganese-52 ( 52 Mn). In some embodiments, the radionuclide is a β-emitting radionuclide, which is 90 Y, 177 Lu, 99m Tc or 89 Zr.

[0273] In some embodiments, the radionuclide is a γ-emitting radionuclide. In some embodiments, the radionuclide is a γ-emitting radionuclide, which is 60 Co cobalt-60 ( 60 Co), palladium-103 ( 103 Pd), cesium-137 ( 137 Cs), ytterbium-169 ( 169 Yb), iridium-192 ( 192 Ir), 212 B1, 213 Bi or 226 Ra.

[0274] Linker

[0275] In some embodiments, a linker is used to connect the tumor targeting moiety and the radionuclide chelating factor moiety R 1 . In some embodiments, L is a hydrophobic linker. In some other embodiments, L is a hydrophilic linker. In some embodiments, the linker is flexible. In some embodiments, the linker is rigid. In some embodiments, the linker is linear. In other embodiments, the linker is branched. In some embodiments, the branched linker allows attachment to more than one R 1Partial conjugation. In some embodiments, the branched linker allows conjugation with a cell-penetrating peptide (CPP) 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 moieties, one or more rigid moieties, or combinations thereof.

[0276] The linker length can be observed based on the number of linear atoms between the radionuclide chelating factor moiety R 1 and the tumor targeting moiety, where the cyclic moiety is counted by taking the shortest path around the ring. In some embodiments, the linear stretch of the linker is between 1 and 100 atoms, between 1 - 50 atoms, between 1 - 40 atoms, between 1 - 30 atoms, in other embodiments 1 - 20 atoms, in still other embodiments 1 - 15 atoms, in still other embodiments 1 - 10 atoms, and in still other embodiments 1 - 5 atoms. In some embodiments, the linker length is in a range with a lower limit selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and an 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 embodiments, the linker length is in a range with a lower limit selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and an 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.

[0277] Linker considerations include effects on the physical or pharmacokinetic properties of the resulting radioimmunoconjugate, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable and programmed degradation), rigidity, flexibility, immunogenicity, modulation of antibody binding, etc.

[0278] The optimal linker should connect the carrier mAb to the radiochelating factor without compromising either function, provide a stable connection in circulation, and degrade under specific conditions. Stability in circulation has proven to be a particularly important factor in the successful use of this strategy.

[0279] In malignant tumors, the accessibility of tumor cells can be targeted by specific antibodies. Antigens expressed on tumor cells but not on normal healthy cells allow for the selective targeting of tumor cells while sparing non-tumor cells. Monoclonal antibodies (mAbs) labeled with short-lived radionuclides that emit short-range, high-linear energy transfer particles represent an attractive approach for treating tumors. These short-range particles may be able to kill individual cells while sparing bystanders. High-energy α-emitters used in radioimmunotherapy include 211 At (astatine-211), 212 Bi, 213 Bi, and 225 Ac. The latter isotope is an in vivo isotope generator because it has a long (10-day) half-life and decays via the α-emission of three short-lived atoms, each of which produces an α-particle.

[0280] In some cases, a limitation to the general use of 225 Ac radioimmunotherapy is the excessive irradiation of normal tissues by the radioimmunoconjugate. Except for antibodies targeting leukemia, most (>99%) of the injected antibody dose remains circulating in the blood or is not associated with the target tumor. Prolonged circulation times of non-targeted antibodies (typically days to weeks) result in radiation exposure of normal organs that catabolize or retain proteins and peptides (primarily the liver and kidneys).

[0281] In the case of in vivo decay of α-emitters, another major potential obstacle to their safe use is the sequential release of three unchelated α-daughter atoms following the initial decay of the parent radionuclide. Uptake into organs such as the kidney or liver can cause toxicity. An attractive approach to overcoming the long circulation times of untargeted radioimmunoconjugates is to develop linkers that allow for the conditional release of the chelated radioactive payload. Introduction of a cleavage site in the linkage between the radio-chelate and the mAb means allowing the low molecular weight radio-chelate to be released from its carrier mAb and its subsequent rapid clearance after the radio-conjugate has accumulated in the metabolic organs. The chelated radioactive metal is then rapidly cleared from the body via the kidneys, thus 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, Vriesendorp HM. Effects of linker chemistry on the pharmacokinetics of radioimmunoconjugates. Q J Nucl Med. 1998;42:250-261). The optimal linker should attach the carrier mAb to the radio-chelation factor without compromising either function, provide a stable linkage in circulation, and degrade under specific conditions. Stability in circulation has proven to be a particularly important factor in the successful use of this strategy.

[0282] Peptide linkers that are generally more stable than esters and disulfides in serum have been the most successful in the design of radioimmunoconjugates and antibody-drug conjugates (ADCs). The most widely studied peptide linkers are sensitive to cathepsins. More precisely, ADCs incorporating the dipeptide valine-citrulline have been shown to enter target cells and migrate to lysosomes, where they specifically release the drug 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., Preclinical evaluation of cathepsin-degradable peptide linkers for radioimmunoconjugates. Clin Cancer Res. 2003;9:3865S-3872S; DeNardo GL et al. Comparison of 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA)-peptide-ChL6, a novel immunoconjugate with 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-labile radioactive conjugates (e.g., glycylglycylglycyl-L-p-isothiocyanatophenylalanine-) has shown a reduced liver dose and a slightly increased tumor dose was observed (DeNardo GL et al., Clin Cancer Res. 2003; 9:3865S-3872S; DeNardo GL et al., Clin Cancer Res. 1998; 4:2483-2490).

[0283] 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 of the amino acids of the linker are non-natural amino acids.

[0284] In some embodiments, the linker may comprise flexible and / or rigid regions. Exemplary flexible linker regions include those comprising Gly and Ser residues ("GS" linker), glycine residues, alkylene chains, PEG chains, etc. Exemplary rigid linker regions include those comprising sequences that form α-helices, proline-rich sequences, and regions rich in double and / or triple bonds.

[0285] In some embodiments, the linker comprises a peptidyl linker. In some embodiments, the length of the peptidyl linker can be between 3-20 amino acids, such as a repeating sequence of a single amino acid residue (e.g., polyglycine) or a combination of amino acid residues to produce a peptide linker that confers favorable pharmacokinetics.

[0286] In some embodiments, the linker contains peptide bonds. The peptide bonds contain 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. The cellular uptake of oligomeric-D-arginine sequences is known to be as good as or better than that of oligomeric-L-arginine.

[0287] In some embodiments, the linker is cleavable. In some embodiments, the linker is designed to cleave in the presence of specific conditions or in a specific environment, such conditions or environments being proximal to such target cells, tissues, or regions. Cleavable linkers rely on the inherent property of the cytoplasmic compartment of cells for the selective release of cytotoxic drugs. Such linkers mainly include chemically cleavable linkers that are responsive to low pH (acid-labile linkers) or a reducing environment (disulfide linkers), and enzymatically cleavable linkers that are sensitive to the action of certain lysosomal enzymes (peptide linkers or β-glucuronide linkers).

[0288] 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, a pH-sensitive linker can be hydrolyzable under acidic conditions. For example, the linker can be an acid-labile linker (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitate, orthoester, acetal, ketal, etc.) that is hydrolyzable in lysosomes. Such linkers can be relatively stable at neutral pH conditions (such as in blood), but are unstable at pH values below 7.0 (such as pH 6.5 - 4.5, the approximate pH of lysosomes and / or endosomes).

[0289] 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). A β-glucuronide linker can be readily cleaved by the abundant lysosomal enzyme β-glucuronidase, which facilitates the easy and selective release of the active drug. In other embodiments, the linker is non-cleavable.

[0290] In some embodiments, the linker component comprises amino acid units. In one such embodiment, the amino acid units allow protease cleavage of the linker, thereby facilitating the release of the drug from the immunoconjugate upon exposure to intracellular proteases (such as lysosomal enzymes). Exemplary amino acid units include but are not limited to dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys), or N-methyl-valine-citrulline (Me-val-cit). These dipeptide linkers exhibit good stability in serum, but can be recognized and rapidly hydrolyzed by certain lysosomal proteases (such as cathepsin B) after internalization. Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly).

[0291] The amino acid units can contain naturally occurring amino acid residues as well as minor and non-naturally occurring amino acid analogs (such as citrulline). The amino acid units can be designed and optimized for selective enzymatic cleavage by a specific enzyme (e.g., tumor-associated proteases, cathepsins B, C, and D, or plasmin protease).

[0292] In some embodiments, the linker is cleaved by a protease, a matrix metalloproteinase, a serine protease, or a combination thereof. In some embodiments, the linker is cleaved by a reducing agent. In some embodiments, the linker is cleaved by an oxidizing agent or oxidative stress.

[0293] In some embodiments, the linker is cleaved by MMP. The hydrolytic activity of matrix metalloproteinases (MMPs) is associated with the invasive migration of metastatic tumor cells. In some embodiments, the linker comprises the amino acid sequences PLG-C(Me)-AG, PLGLAG that are cleaved by the metalloproteinases MMP-2, MMP-9, or MMP-7 (MMPs involved in cancer and inflammation).

[0294] In some embodiments, the linker is cleaved by a proteolytic enzyme or a reducing environment, such as can be found near cancer cells. Such an environment or such enzymes are generally not found near normal cells.

[0295] In some embodiments, the linker is cleaved by serine proteases (including but not limited to thrombin and cathepsin). In some embodiments, the linker is cleaved by cathepsin K, cathepsin S, cathepsin D, cathepsin E, cathepsin W, cathepsin F, cathepsin A, cathepsin C, cathepsin H, cathepsin Z, or any combination thereof. In some embodiments, the linker is cleaved by cathepsin K and / or cathepsin S.

[0296] In some embodiments, the linker is cleaved in a necrotic environment. Necrosis typically results in the release of enzymes or other cellular contents that can be used to trigger linker cleavage. In some embodiments, cleavage of the linker occurs by a necrotic enzyme (e.g., by calpain).

[0297] In some embodiments, the linker comprises one or more disulfide bonds.

[0298] Alternatively, the linker can be a non-peptidyl linker. Typical examples of these types of linkers are those based on straight-chain or branched-chain hydrocarbons or polyethylene glycols of different lengths. These can incorporate other groups to affect solubility, rigidity, isoelectric point, such as aromatic or non-aromatic rings, halogens, ketones, aldehydes, esters, sulfonyl groups, phosphate ester groups, etc.

[0299] In some embodiments, the linker moiety is "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 after 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 spacers sensitive to sequence-specific enzymatic cleavage are also contemplated. For example, enzymatic cleavage of a glycine-glycine spacer sequence unit by a tumor cell-associated protease will 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 then undergoes a separate hydrolysis step in the tumor cell to cleave the glycine-glycine spacer sequence unit from the chelator moiety.

[0300] A "self-immolative" spacer sequence unit allows for the release of the drug moiety without a separate hydrolysis step. In certain embodiments, the spacer sequence unit of the linker contains a p-aminobenzyl unit. In one such embodiment, p-aminobenzyl alcohol is attached to an amino acid unit via an amide bond, and a carbamate, methylcarbamate, or carbonate is formed between the benzyl alcohol and the cytotoxic agent (see, e.g., Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-103). In one embodiment, the spacer sequence unit is p-aminobenzyloxycarbonyl (PAB). In certain embodiments, the phenylene moiety of the p-aminobenzyl unit is substituted with Qm, where Q is halogen, nitro, cyano, -OH, -OR 5 , -CO 2 H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C 1 -C 6 alkyl, where the substituted C 1 -C 6 alkyl is -OH, -CO 2 H, -NHR 5 , -C(=O)NHR 5 and -NHC(=O)R 5is substituted; and m is an integer from 0 to 4 (see, e.g., Yam B. Poudel et al., ACS Medicinal Chemistry Letters 2020 11(11), 2190-2194). Examples of the suicide spacer units also include, but are not limited to, aromatic compounds electronically similar to p-aminobenzyl alcohol (see, e.g., US2005 / 0256030 A1), such as 2-aminoimidazole-5-methanol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and o- or p-aminobenzyl acetals. Spacers that cyclize after amide bond hydrolysis can be used, such as substituted and unsubstituted 4-aminobutyramides (Rodrigues et al., Chemistry Biology, 1995, 2, 223); appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., J. Amer. Chem. Soc., 1972, 94:5815); and 2-aminophenylpropionamides (Amsberry et al., J. Org. Chem., 1990, 55:5867). Elimination of amine-containing drugs substituted at the α-position of glycine (Kingsbury et al., J. Med. Chem., 1984, 27:1447) is also an example of a suicide spacer available in ADCs.

[0301] In some embodiments, the immunoconjugate comprises a linker, such as, for example, a dendritic linker, to covalently attach more than one drug moiety to the antibody via a branched multifunctional linker moiety (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-5; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-8). The dendritic linker can increase the drug-to-antibody molar ratio, i.e., the loading, which is related to the potency of the ADC. Thus, when the cysteine-engineered antibody bears only one reactive cysteine thiol group, multiple drug moieties can be attached via the dendritic linker.

[0302] In some embodiments, the linker connecting the tumor targeting moiety to the chelating moiety (R 1 ) or its radionuclide complex comprises L as described herein; v is 1, 2, 3, or 4; L c is -C(=O)-, -phenyl-C(=O)-, -NHC(=S)-, -X-phenyl-NHC(=S)-, -S-, -C(=O)CH 2 -, X is absent, -O-, -S-, -S(=O)-, -S(=O) 2- , -NR a -, -C(=O)-, -NR a C(=O)-, -C(=O)NR a -, -C(=O)O-, -OC(=O)-, -OC(=O)NR a -, -NR a C(=O)NR a -, -NR a C(=S)NR a -, -NR a -, -C(=O)O-; each R a is independently selected from hydrogen and C 1 -C 4 alkyl group.

[0303] In some embodiments, L c is -C(=O)-. In some embodiments, L c is -phenyl-C(=O)-. In some embodiments, L c is -NHC(=S)-. In some embodiments, L c is -X-phenyl-NHC(=S)-. In some embodiments, X is absent or -NHC(=S)NH-. In some embodiments, L c is -NHC(=S)NH-. In some embodiments, L c is -S-, -C(=O)CH 2 -, In some embodiments, L c is -C(=O)CH 2 -. In some embodiments, L c is

[0304] In some embodiments, X 1 is -O-, -S-, -S(=O)-, -S(=O) 2- , -NR a -, -C(=O)-, -NR a C(=O)-, -C(=O)NR a -, -(C 1 -C 6 alkylene)-X 2 - or -(C 4 -C 20 polyethylene glycol)-X 2 -; X 2 is absent, -C(=O)-, -C(=O)NR a-or -C(=O)X 4 -; X 4 is -NR a -, -NR a S(=O) 2 -, -NR a S(=O) 2 NR a - or one or more independently selected natural or unnatural amino acids, wherein any free amine of the amino acid is optionally and independently substituted by R 5 or -C(=O)R 5 and wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 Alternative.

[0305] In some embodiments, X 1 is absent, -O-, -S-, -S(=O)-, -S(=O) 2- -, -NR a -, -C(=O)-, -NR a C(=O)- or -C(=O)NR a -. In some embodiments, X 1 is -O-. In some embodiments, X 1 is absent, -S-. In some embodiments, X 1 is -NR a -. In some embodiments, X 1 is -C(=O)-. In some embodiments, X 1 is -C(=O)NR a -.

[0306] In some embodiments, X 1 is absent, -O-, -S-, -NR a -, -C(=O)-, -NR a C(=O)- or -C(=O)NR a -.

[0307] In some embodiments, X 1 is -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2CH 2 CH 2 -、 -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -、 -CH 2 -X 2 -、 -CH 2 CH 2 -X 2 -、 -CH 2 CH 2 CH 2 -X 2 -、 -CH 2 CH 2 CH 2 CH 2 -X 2 -、 -CH 2 CH 2 CH 2 CH 2 CH 2 -X 2 -、 -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -X 2 -。 In some embodiments, X 1 is -CH 2 -、 -CH 2 CH 2 -、 -CH 2 -X 2 - or -CH 2 CH 2 -X 2 -。 In some embodiments, X 1 is -CH 2 CH 2 -。 In some embodiments, X 1 is -CH 2 CH 2 -X 2 -。 In some embodiments, X 2 is -C(=O)-、 -C(=O)NR a - or -C(=O)X 4 -。 In some embodiments, X 2 is -C(=O)-。 In some embodiments, X 2 is -C(=O)NR a -。 In some embodiments, X2 is -C(=O)X 4 -.

[0308] In some embodiments, X 1 is -CH 2 CH 2 - or -CH 2 CH 2 -X 2 -; X 2 is -C(=O)X 4 -; X 4 is -NH-, -N(CH 3 )-, -N(CH 2 CH 3 )-, -NHS(=O) 2 -, -N(CH 3 )S(=O) 2 -, -N(CH 2 CH 3 )S(=O) 2 -, lysine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, serine, tyrosine, valine, citrulline, methionine-valine-lysine, glycine-phenylalanine-glycine-glycine, tyrosine-arginine-valine, arginine-valine or a combination thereof; wherein any free amine (-NH 2 ) of the amino acid is optionally independently substituted by R 5 or -C(=O)R 5 and any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 . In some embodiments, X 4 is -NH-, -N(CH 3 )-, -N(CH 2 CH 3 )-, -NHS(=O) 2 -, -N(CH 3 )S(=O) 2 - or -N(CH 2 CH 3 )S(=O) 2 -. In some embodiments, X 4 is -NH-, -N(CH 3 )- or -N(CH 2 CH 3 )-. In some embodiments, X 4 is -NHS(=O) 2 -, -N(CH 3 )S(=O) 2 - or -N(CH2 CH 3 )S(=O) 2 -. In some embodiments, X 4 is lysine, glutamine, glutamate, glycine, leucine, lysine, methionine, phenylalanine, serine, tyrosine, valine, citrulline, methionine-valine-lysine, glycine-phenylalanine-glycine-glycine, tyrosine-arginine-valine, arginine-valine, or a combination thereof; wherein any free amine (-NH 2 ) of the amino acid is optionally independently substituted by R 5 or -C(=O)R 5 and wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 .

[0309] In some embodiments, L is -L 1 -, -L 2 -, -L 3 -, -L 4 -, -L 5 -, -L 1 -L 2 -L 3 -L 4 -L 5 -, or a combination thereof;

[0310] L 1 is absent, unsubstituted, or substituted C 1 -C 10 alkylene, unsubstituted or substituted C 1 -C 10 heteroalkylene, unsubstituted or substituted C 2 -C 20 alkenylene, unsubstituted or substituted C 2 -C 20 alkynylene, C 4 -C 20 polyethylene glycol, -(X 3 CH 2 CH 2 ) t -, unsubstituted or substituted cycloalkylene, unsubstituted or substituted heterocycloalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene;

[0311] Each X 3 is independently selected from O and NR 4 ;

[0312] Each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; L2 is an unsubstituted or substituted C 1 -C 10 alkylene, an unsubstituted or substituted C 1 -C 10 heteroalkylene, -C(=O)-(an unsubstituted or substituted C 1 -C 10 alkylene), -C(=O)NR 4 -(an unsubstituted or substituted C 1 -C 10 alkylene)-, -NR 4 C(=O)-(an unsubstituted or substituted C 1 -C 10 alkylene)-, -C(=O)-(CH 2 CH 2 X 3 ) m -(CH 2 ) P -, -C(=O)NR 4 -(CH 2 CH 2 X 3 ) m -(CH 2 ) p -, -NR 4 C(=O)-(CH 2 CH 2 X 3 ) m -(CH 2 ) p - or -(CH 2 CH 2 X 3 ) m -(CH 2 ) p ;

[0313] Each R 4 is independently selected from hydrogen and C 1 -C 6 alkyl;

[0314] Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0315] Each p is independently 0, 1 or 2;

[0316] L 3is absent or is one or more independently selected groups chosen from: natural or unnatural amino acids and optionally amino (unsubstituted or substituted benzyl) carbamate; wherein any free amine (-NH 2 ) of the amino acid is optionally independently substituted by R 5 or -C(=O)R 5 ; wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 ; and wherein the substituted benzyl is substituted by 1 or 2 groups selected from halogen, -OH, -OR 5 , -CO 2 H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C 1 -C 6 alkyl; wherein the substituted C 1 -C 6 alkyl is substituted by -OH, -CO 2 H, -NHR 5 , -C(=O)NHR 5 and -NHC(=O)R 5 ;

[0317] L 4 is absent, unsubstituted or substituted C 1 -C 10 alkylene, -C(=O)-(unsubstituted or substituted C 1 -C 10 alkylene), -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 10 alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 10 alkylene)-, -C(=O)-(CH 2 CH 2 X 3 ) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 X 3 ) n -(CH 2 ) q -, -NR 4 C(=O)-(CH 2 CH2 X 3 ) n -(CH 2 ) q -、-(CH 2 CH 2 X 3 ) n -(CH 2 ) q - or -(X 3 CH 2 CH 2 ) n -;

[0318] Each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0319] Each q is independently 0, 1 or 2;

[0320] L 5 is absent, -C(=O)-(CH 2 ) n -, -C(=O)NR 4 -(CH 2 ) n -, -NR 4 C(=O)-(CH 2 ) n -, -C(=O)-(CH 2 CH 2 X 3 ) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 X 3 ) n -(CH 2 ) q -, -NR 4 C(=O)-(CH 2 CH 2 X 3 ) n -(CH 2 ) q -, -(CH 2 CH 2 X 3 ) n -(CH 2 ) q -, -C(=O)-(X 3 CH 2 CH 2 )n - or - (X 3 CH 2 CH 2 ) n -;

[0321] Each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0322] Each q is independently 0, 1 or 2;

[0323] Each R a is independently selected from hydrogen and C 1 -C 4 alkyl;

[0324] Each R b is independently selected from hydrogen and C 1 -C 4 alkyl;

[0325] wherein the heteroalkylene is one in which a carbon atom is replaced by -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(=N-R 5 )NR a - or -NR a C(=O)O-substituted alkylene;

[0326] wherein when any one of -L 1 -, -L 2 -, -L 3 -, -L 4 - and -L 5 - is substituted, then -L 1 -, -L 2 -, -L 3 -, -L 4 - and -L 5 - is substituted by 1, 2, 3 or 4 substituents selected from halogen, -OH, -OR 5 , -CO 2 H, -NHR5 、 -C(=O)NHR 5 、 -NHC(=O)R 5 and a substituted C 1 -C 6 alkyl group, wherein the substituted C 1 -C 6 alkyl is substituted by -OH, -CO 2 H, -NHR 5 、 -C(=O)NHR 5 and -NHC(=O)R 5 ;

[0327] Each R 5 is independently selected from C 1 -C 10 alkyl, C 4 -C 30 polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene.

[0328] In some embodiments, L is -L 1 -, -L 2 -, -L 3 -, -L 4 -, -L 5 -, -L 1 -L 2 -L 3 -L 4 -L 5 - or a combination thereof.

[0329] In some embodiments, L is -L 1 -. In some embodiments, L is -L 2 -. In some embodiments, L is -L 3 -. In some embodiments, L is -L 4 -. In some embodiments, L is -L 5 -. In some embodiments, L is -L 1 -L 2 -. In some embodiments, L is -L 1 -L 3 -. In some embodiments, L is -L 1 -L 4 -. In some embodiments, L is -L 1 -L 5 -. In some embodiments, L is -L 2 -L 3 -. In some embodiments, L is -L 2 -L 4- In some embodiments, L is -L 2 -L 5 - In some embodiments, L is -L 3 -L 4 - In some embodiments, L is -L 3 -L 5 - In some embodiments, L is -L 4 -L 5 - In some embodiments, L is -L 1 -L 2 -L 3 - In some embodiments, L is -L 1 -L 2 -L 4 - In some embodiments, L is -L 1 -L 2 -L 5 - In some embodiments, L is -L 1 -L 4 -L 5 - In some embodiments, L is -L 1 -L 2 -L 3 -L 4 - In some embodiments, L is -L 2 -L 3 -L 4 -L 5 - In some embodiments, L is -L 1 -L 2 -L 4 -L 5 - In some embodiments, L is -L 1 -L 2 -L 3 -L 4 -L 5 -。

[0330] In some embodiments, the heteroalkylene is an alkylene in which one carbon atom is replaced by -O-, -S-, -S(=O)-, -S(=O) 2 -,-S(=O)(=NH)-,-S(=O)(=NR 5 )-,-NR 5 -,-P(=O)OH-,-NHC(=O)-,-C(=O)NH-,-OC(=O)NH-,-NHC(=N-CN)NH- or -NHC(=N-R 5 )NH-.

[0331] In some embodiments, the heteroalkylene is an alkylene in which one carbon atom is replaced by -S(=O)(=NH)-, -S(=O)(=NR 5 )-, -P(=O)OH-, -NHC(=N-CN)NH- or -NHC(=N-R 5 )NH-.

[0332] In some embodiments, L 1 is an unsubstituted or substituted C 1 -C 20 alkylene, an unsubstituted or substituted C 1 -C 20 heteroalkylene, a C 4 -C 20 polyethylene glycol, an unsubstituted or substituted C 3 -C 8 cycloalkylene, an unsubstituted or substituted monocyclic C 3 -C 8 heterocycloalkylene, an unsubstituted or substituted phenylene, an unsubstituted or substituted monocyclic heteroarylene.

[0333] In some embodiments, L 1 is an unsubstituted or substituted C 1 -C 6 alkylene, an unsubstituted or substituted C 1 -C 10 heteroalkylene, a C 4 -C 20 polyethylene glycol, an unsubstituted or substituted cyclohexylene or an unsubstituted or substituted phenylene.

[0334] In some embodiments, L 2 is absent, -C(=O)NR 4 -(an unsubstituted or substituted C 1 -C 10 alkylene)-, -NR 4 C(=O)-(an unsubstituted or substituted C 1 -C 10 alkylene)-, -C(=O)-(CH 2 CH 2 O) m -(CH 2 ) P -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) P -, -NR 4C(=O)-(CH 2 CH 2 O) n -(CH 2 ) P - or -(CH 2 CH 2 O) n -(CH 2 ) P -; each m is independently 1, 2, 3, 4, 5 or 6; each p is independently 1 or 2.

[0335] In some embodiments, L 3 is absent or is one or more independently selected groups from the following: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, tyrosine, valine, and (unsubstituted or substituted benzyl) carbamate; wherein any free amine of the amino acid is optionally substituted by R 5 or -C(=O)(R 5 ) and any free carboxylic acid of the amino acid is optionally replaced by -C(=O)NH(R 5 ).

[0336] In some embodiments, L 3 is lysine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, serine, tyrosine, valine, citrulline, methionine-valine-lysine, glycine-phenylalanine-glycine-glycine, tyrosine-arginine-valine, arginine-valine, valine-citrulline-(p-aminobenzyl carbamate) or a combination thereof; wherein any free amine (-NH 2 ) of the amino acid is optionally substituted by -C(=O)(unsubstituted or substituted C 1 -C 20 alkylene) or -C(=O)-C 4 -C 20 polyethylene glycol; wherein any free carboxylic acid (-CO 2 H) of any amino acid is optionally replaced by -C(=O)NH-(2,4,6-trimethyl-3-bromophenyl), -C(=O)NH-(unsubstituted or substituted C 1 -C 20 alkylene) or -C(=O)NH-(C 4 -C 20 polyethylene glycol).

[0337] In some embodiments, L 3 is:

[0338]

[0339] In some embodiments, L 4 is absent, -C(=O)-(unsubstituted or substituted C 1 -C 6 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 6 alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 6 alkylene)-, -C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q -, -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q - or -(CH 2 CH 2 O) n -(CH 2 ) q -; each n is independently 1, 2, 3, 4, 5 or 6; each q is independently 1 or 2.

[0340] In some embodiments, L 5 is absent, -C(=O)-(CH 2 ) n -, -C(=O)NR 4 -(CH 2 ) n -, -NR 4 C(=O)-(CH 2 ) n -, -C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 O)n -(CH 2 ) q -、-NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -、-(CH 2 CH 2 O) n -(CH 2 ) q -、-C(=O)-(OCH 2 CH 2 ) n -or-(OCH 2 CH 2 ) n -; each p is independently 0, 1 or 2.

[0341] In some embodiments, L 5 Does not exist, -NR 4 C(=O)-(CH 2 ) n -、-C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -、-C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q -or-NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -; each q is independently 1 or 2.

[0342] In some embodiments,

[0343] L 1 is unsubstituted or substituted C 1 -C 6 Alkylene, unsubstituted or substituted C 1 -C 10 Heteroalkylene, C 4 -C 20 Polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene;

[0344] L2 is absent, -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 10 -alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 10 -alkylene)-, -C(=O)-(CH 2 CH 2 O) m -(CH 2 ) P -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) P -, -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) P - or -(CH 2 CH 2 O) n -(CH 2 ) P -; each m is independently 1, 2, 3, 4, 5 or 6; each p is independently 1 or 2;

[0345] L 4 is absent, -C(=O)-(unsubstituted or substituted C 1 -C 6 -alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 6 -alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 6 -alkylene)-, -C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q -, -NR 4 C(=O)-(CH2 CH 2 O) n -(CH 2 ) q - or -(CH 2 CH 2 O) n -(CH 2 ) q -;

[0346] L 5 is -NR 4 C(=O)-(CH 2 ) n -, -C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q - or -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -; each n is independently 1, 2, 3, 4, 5 or 6; each q is independently 1 or 2.

[0347] In some embodiments,

[0348] L 1 is unsubstituted or substituted C 1 -C 6 alkylene, unsubstituted or substituted C 1 -C 10 heteroalkylene, C 4 -C 20 polyethylene glycol, unsubstituted or substituted cyclohexylene or unsubstituted or substituted phenylene;

[0349] L 2 is absent, -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 10 alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 10 alkylene)-, -C(=O)-(CH 2 CH2 O) m -(CH 2 ) P -、-C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) P -、-NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) P -or-(CH 2 CH 2 O) n -(CH 2 ) P -; each m is independently 1, 2, 3, 4, 5 or 6; each p is independently 1 or 2;

[0350] L 4 does not exist; L 5 Does not exist, -NR 4 C(=O)-(CH 2 ) n -、-C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -、-C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q -or-NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -; each n is independently 1, 2, 3, 4, 5 or 6; each q is independently 1 or 2.

[0351] In some embodiments,

[0352] L 1 is unsubstituted or substituted C 1 -C 6 Alkylene, unsubstituted or substituted C 1 -C 10 Heteroalkylene, C 4 -C 20Polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene;

[0353] L 2 is absent, -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 10 -alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 10 -alkylene)-, -C(=O)-(CH 2 CH 2 O) m -(CH 2 ) P -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) P -, -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) P - or -(CH 2 CH 2 O) n -(CH 2 ) P -; each m is independently 1, 2, 3, 4, 5 or 6; each p is independently 1 or 2; L 3 is one or more independently selected groups 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 substituted by R 5 or -C(=O)(R 5 ) and any free carboxylic acid of the amino acid is optionally replaced by -C(=O)NH(R 5 ) each R 5 is independently unsubstituted or substituted C 1 -C 10 -alkylene, C 4 -C 20 - polyethylene glycol or unsubstituted or substituted phenyl, wherein the substituted phenyl is substituted by 1, 2, 3, 4 or 5 independently selected from F, Cl, Br, I, -CH 3 and CF3 substituted by a group;

[0354] L 4 is absent; L 5 is -NR 4 C(=O)-(CH 2 ) n -, -C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q -, or -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2.

[0355] In some embodiments, -X 1 -L- is:

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362] In some embodiments, is:

[0363]

[0364]

[0365]

[0366] In some embodiments, is:

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373] In addition, a conjugate moiety (R 2 ) is used to facilitate attachment to the tumor targeting moiety. In some embodiments, R 2 is a moiety capable of reacting with the amine (-NH 3 ) or thiol (-SH) of the tumor targeting moiety R 2 . In some embodiments, R 2 is a moiety capable of reacting with the amine (-NH 3 ) of the side chain of the lysine residue of the tumor targeting moiety R 2 .

[0374] In some embodiments, R 2 is a moiety capable of reacting with the amine (-NH 3 ) of the tumor targeting moiety R 2 and includes tetrafluorophenyl ester, pentafluorophenyl ester, dinitrophenyl ester, succinimidyl ester, sulfosuccinimidyl ester, or isothiocyanate.

[0375] In some embodiments, R 2 is a moiety capable of reacting with the amine (-NH 3 ) of the tumor targeting moiety R 2 and includes:

[0376]

[0377] X is absent, -O-, -S-, -S(=O)-, -S(=O) 2- , -NR a -, -C(=O)-, -NR a C(=O)-, -C(=O)NR a -, -C(=O)O-, -OC(=O)-, -OC(=O)NR a -, -NR a C(=O)NR a -, -NR a C(=S)NR a -, -NR a C(=O)O-; each R aIndependently selected from hydrogen and C 1 -C 4 alkyl group.

[0378] In some embodiments, R 2 is a moiety capable of reacting with the amine (-NH 3 ) of the tumor targeting moiety R 2 and includes:

[0379]

[0380] X is absent or -NR a C(=S)NR a -; each R a is independently selected from hydrogen and C 1 -C 4 alkyl group. In some embodiments, X is absent or -NHC(=S)NH-.

[0381] In some embodiments, R 2 is a moiety capable of reacting with the amine (-NH 3 ) of the tumor targeting moiety R 2 and includes: X is absent or -NHC(=S)NH-.

[0382] In some embodiments, R 2 is a moiety capable of reacting with the amine (-NH 3 ) of the tumor targeting moiety R 2 and includes:

[0383] In some embodiments, R 2 is a moiety capable of reacting with the amine (-NH 3 ) of the tumor targeting moiety R 2 and includes: X is absent or -NHC(=S)NH-.

[0384] Alternatively, or additionally, an isothiocyanate linker (such as p-SCN-Bn-DOTA) can be used, which involves lysine residues in the immunoconjugates of the present invention.

[0385] In some embodiments, R 2 is a moiety capable of reacting with the thiol (-SH) of the side chain of the cysteine residue of the tumor targeting moiety R 3 and includes: maleimide group, haloacetamide group, haloacetyl group, haloacetic acid group, pyridylthio group, vinylcarbonyl group, aziridinyl group, disulfide group, acetylene group, hydroxysuccinimide group or thiol group.

[0386] In some embodiments, R 2 is a moiety capable of reacting with the thiol (-SH) of the tumor targeting moiety R 3 and comprises

[0387] m is 0, 1, 2, 3, 4 or 5.

[0388] In some embodiments, R 2 is a moiety capable of reacting with the thiol (-SH) of the tumor targeting moiety R 3 and comprises

[0389] In some embodiments, the linker can be conjugated to the antibody via a cysteine bridging functional group such as or DBM (dibromomaleimide). These linkers can serve to re-stabilize the intra-chain disulfide bond 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 re-bridging extension sequence elements are shown below (where the wavy line indicates the site of covalent attachment to the immunoconjugate):

[0390]

[0391] For additional details regarding linkers and their use in the compounds described herein, see: Wu, A.M.; Senter, P.D. Nat.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, J.R.; Owen, S.C. AAPS J. 2015, 17(2):339 - 351; Jun Lu et al., Int J Mol Sci. April 2016; 17(4):561; regarding such linker disclosures, each of the said documents is incorporated herein by reference.

[0392] Exemplary immunoconjugate

[0393] In some embodiments, compounds of formula (I) or pharmaceutically acceptable salts thereof are described herein:

[0394]

[0395] Wherein:

[0396] R 1 is a chelating moiety or a radionuclide complex thereof;

[0397] X 1 is -O-, -S-, -S(=O)-, -S(=O) 2- -, -NR a -, -C(=O)-, -NR a C(=O)-, -

[0398] C(=O)NR a -, -C(=O)O-, -OC(=O)-, -OC(=O)NR a -, -NR a C(=O)NR a -,

[0399] -NR a C(=S)NR a -, -NR a C(=O)O-, -(unsubstituted or substituted C 1 -C 6 -alkylene)-X 2 -, -(unsubstituted or substituted C 2 -C 10 -alkenylene)-X 2 -, -(unsubstituted or substituted C 2 -C 10 -alkynylene)-X 2 -, or -(C 4 -C 20 -polyethylene glycol)-X 2 -;

[0400] X 2 is absent, -C(=O)-, -NR a C(=O)-, -C(=O)NR a -, or -C(=O)X 4 -;

[0401] Each R a is independently selected from hydrogen and C 1 -C 4 -alkyl;

[0402] X 4 is -NR a -, -NR a S(=O) 2 -, -NRa S(=O) 2 NR a - or one or more independently selected natural or unnatural amino acids, wherein any free amine of the amino acid is optionally and independently substituted by R 5 or -C(=O)R 5 and wherein any free carboxylic acid of any amino acid is optionally substituted by -C(=O)NH-R 5 substituted;

[0403] Each R 5 is independently selected from C 1 -C 10 alkyl, C 4 -C 30 polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene;

[0404] L is an optional linker;

[0405] R 2 is a moiety capable of reacting with the amine (-NH 3 ) or thiol (-SH) of the tumor targeting moiety R 2 ); and

[0406] v is 1, 2, 3, or 4.

[0407] In some embodiments, R 2 is a moiety capable of reacting with the amine (-NH 3 ) of the tumor targeting moiety R 2 and includes tetrafluorophenyl ester, pentafluorophenyl ester, dinitrophenyl ester, succinimide ester, sulfosuccinimide ester, or isothiocyanate.

[0408] In some embodiments, R 2 is a moiety capable of reacting with the amine (-NH 3 ) of the tumor targeting moiety R 2 and includes:

[0409]

[0410] X is absent, -O-, -S-, -S(=O)-, -S(=O) 2- , -NR a -, -C(=O)-, -NR a C(=O)-, -C(=O)NR a -, -C(=O)O-, -OC(=O)-, -OC(=O)NR a -, -NR a C(=O)NR a -, -NRa C(=S)NR a -, -NR a C(=O)O-; and each R a is independently selected from hydrogen and C 1 -C 4 alkyl.

[0411] In some embodiments, immunoconjugates of formula (II), formula (III) or formula (IV) or pharmaceutically acceptable salts thereof are described herein:

[0412]

[0413] wherein:

[0414] R 1 is a chelating moiety or a radionuclide complex thereof;

[0415] X 1 is -O-, -S-, -S(=O)-, -S(=O) 2- , -NR a -, -C(=O)-, -NR a C(=O)-, -

[0416] C(=O)NR a -, -C(=O)O-, -OC(=O)-, -OC(=O)NR a -, -NR a C(=O)NR a (-),

[0417] -NR a C(=S)NR a -, -NR a C(=O)O-, -(unsubstituted or substituted C 1 -C 6 alkylene)-X 2 -, -(unsubstituted or substituted C 2 -C 10 alkenylene)-X 2 -, -(unsubstituted or substituted C 2 -C 10 alkynylene)-X 2 - or -(C 4 -C 20 polyethylene glycol)-X 2 (-);

[0418] X 2 is absent, -C(=O)-, -NR a C(=O)-, -C(=O)NR a - or -C(=O)X4 -;

[0419] Each R a is independently selected from hydrogen and C 1 -C 4 alkyl;

[0420] X 4 is -NR a -, -NR a S(=O) 2 -, -NR a S(=O) 2 NR a -, or one or more independently selected natural or unnatural amino acids, wherein any free amine of the amino acid is optionally and independently substituted by R 5 or -C(=O)R 5 and wherein any free carboxylic acid of any amino acid is optionally substituted by -C(=O)NH-R 5 ;

[0421] Each R 5 is independently selected from C 1 -C 10 alkyl, C 4 -C 30 polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene;

[0422] L is an optional linker;

[0423] -NH-R 3 is a tumor targeting moiety; and

[0424] v is 1, 2, 3 or 4.

[0425] In some embodiments, R 2 is a moiety capable of reacting with the thiol (-SH) of the tumor targeting moiety R 3 and includes a maleimide group, a haloacetamide group, a haloacetyl group, a haloacetic acid group, a pyridylthio group, a vinylcarbonyl group, an aziridinyl group, a disulfide group, an acetylene group, a hydroxysuccinimide group or a thiol group.

[0426] In some embodiments, R 2 is a moiety capable of reacting with the thiol (-SH) of the tumor targeting moiety R 3 and includes

[0427] m is 0, 1, 2, 3, 4 or 5.

[0428] In some embodiments, there are described herein immunoconjugates of formula (V), formula (VI), formula (VII) or formula (VIII) or pharmaceutically acceptable salts thereof:

[0429]

[0430] Wherein:

[0431] R 1 is a chelating moiety or a radionuclide complex thereof;

[0432] X 1 is -O-, -S-, -S(=O)-, -S(=O) 2- 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-, -(unsubstituted or substituted C 1 -C 6 alkylene)-X 2 -, -(unsubstituted or substituted C 2 -C 10 alkenylene)-X 2 -, -(unsubstituted or substituted C 2 -C 10 alkynylene)-X 2 - or -(C 4 -C 20 polyethylene glycol)-X 2 -;

[0433] X 2 is absent, -C(=O)-, -NR a C(=O)-, -C(=O)NR a - or -C(=O)X 4 -;

[0434] Each R a is independently selected from hydrogen and C 1 -C 4 alkyl;

[0435] X 4 is -NR a -, -NR a S(=O) 2-, -NR a S(=O) 2 NR a - or one or more independently selected natural or non-natural amino acids, wherein any free amine of the amino acid is optionally independently substituted by R 5 or -C(=O)R 5 and wherein any free carboxylic acid of any amino acid is optionally substituted by -C(=O)NH-R 5 substituted;

[0436] Each R 5 is independently selected from C 1 -C 10 alkyl, C 4 -C 30 polyethylene glycol and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene;

[0437] L is an optional linker; -S-R 3 is a tumor targeting moiety; v is 1, 2, 3 or 4.

[0438] In some embodiments, R 2 is a moiety capable of reacting with the amine (-NH 3 ) of the lysine of the tumor targeting moiety R 2 .

[0439] In some embodiments, the immunoconjugate of formula (II) has the structure of formula (IIa) or a pharmaceutically acceptable salt thereof:

[0440]

[0441] wherein: -NHCH 2 CH 2 CH 2 CH 2 - is the side chain of the lysine residue of the tumor targeting moiety R 3 .

[0442] In some embodiments, the immunoconjugate of formula (III) has the structure of formula (IIIa) or a pharmaceutically acceptable salt thereof:

[0443]

[0444] wherein: -NHCH 2 CH 2 CH 2 CH 2 - is the side chain of the lysine residue of the tumor targeting moiety R 3 .

[0445] In some embodiments, the immunoconjugate of formula (IV) has the structure of formula (IVa) or a pharmaceutically acceptable salt thereof:

[0446]

[0447] wherein: -NHCH 2 CH 2 CH 2 CH 2 - is the side chain of a lysine residue of the tumor targeting moiety R 3

[0448] In some embodiments, R 2 is a moiety capable of reacting with the thiol (-SH) of a cysteine of the tumor targeting moiety R 3

[0449] In some embodiments, the immunoconjugate of formula (V) has the structure of formula (Va) or a pharmaceutically acceptable salt thereof:

[0450]

[0451] wherein: -SCH 2 - is the thiol (-SH) of the side chain of a cysteine residue of the tumor targeting moiety R 3

[0452] In some embodiments, the immunoconjugate of formula (VI) has the structure of formula (VIa) or a pharmaceutically acceptable salt thereof:

[0453]

[0454] wherein: -SCH 2 - is the thiol (-SH) of the side chain of a cysteine residue of the tumor targeting moiety R 3

[0455] In some embodiments, the immunoconjugate of formula (VII) has the structure of formula (VIIa) or a pharmaceutically acceptable salt thereof:

[0456]

[0457] wherein: -SCH 2 - is the thiol (-SH) of the side chain of a cysteine residue of the tumor targeting moiety R 3

[0458] In some embodiments, the immunoconjugate of formula (VIII) has the structure of formula (VIIIa) or a pharmaceutically acceptable salt thereof:

[0459] ​​​​​

[0460] Wherein: -SCH 2 - is the thiol (-SH) of the side chain of a cysteine residue of the tumor targeting moiety R 3 of.

[0461] In some embodiments, R 1 is a chelating moiety selected from: 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'-(((pyridine-2,6-diylbis(methylene))bis((carboxymethyl)azanediyl))-bis(methylene))dipicolinic acid (H 4 pypa); 6,6',6”,6”'-(((pyridine-2,6-diylbis(methylene))bis(azanetriyl))tetra(methylene))-tetrapicolinic acid (H 4 py4pa); 10-((6-carboxypyridin-2-yl)methyl)-1,4,7,10-tetra-azacyclododecane-1,4,7-triacetic acid (DO3Apic); 3,6,9,12-tetra(carboxymethyl)-3,6,9,12-tetraazatetradecanedioic acid (TTHA); or a radionuclide complex thereof.

[0462] In some embodiments, R 1 is a chelating moiety selected from:

[0463]

[0464] or a radionuclide complex thereof.

[0465] In some embodiments, R 1 is a chelating moiety selected from: or a radionuclide complex thereof. In some embodiments, R 1 is a chelating moiety selected from:

[0466] or a radionuclide complex thereof.

[0467] In some embodiments, X 1 is -O-, -S-, -S(=O)-, -S(=O) 2- , -NR a -, -C(=O)-, -NR a C(=O)-, -C(=O)NR a -, -(C 1 -C 6 -alkylene)-X 2 - or -(C 4 -C 20 -polyethylene glycol)-X 2 -; X 2 is absent, -C(=O)-, -C(=O)NR a - or -C(=O)X 4 -; X 4 is -NR a -, -NR a S(=O) 2 -, -NR a S(=O) 2 NR a - or one or more independently selected natural or unnatural amino acids, wherein any free amine of the amino acid is optionally independently substituted by R 5 or -C(=O)R 5 and wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 .

[0468] In some embodiments, X 1 is absent, -O-, -S-, -S(=O)-, -S(=O) 2- , -NR a (=O)-, -C(=O)-, -NR a C(=O)- or -C(=O)NR a . In some embodiments, X 1 is absent, -O-, -S-, -NR a (=O)-, -C(=O)-, -NR a C(=O)- or -C(=O)NR a .

[0469] In some embodiments, X 1 is -CH 2 (=O)-, -CH 2 CH 2 (=O)-, -CH 2 CH 2 CH 2 (=O)-, -CH 2 CH2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 -X 2 -, -CH 2 CH 2 -X 2 -, -CH 2 CH 2 CH 2 -X 2 -, -CH 2 CH 2 CH 2 CH 2 -X2 4 -, -CH 2 CH 2 CH 2 CH 2 CH 2 -X 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 -CH 2 -X 2 -.

[0470] In some embodiments, X 2 is -C(=O)NR a - or -C(=O)X 4 -. In some embodiments, X 2 is -C(=O)NR a -. In some embodiments, X 2 is -C(=O)X 4 -. In some embodiments, X 4 is -NR a - or -NR a S(=O) 2 -.

[0471] In some embodiments, X 1 is -CH 2 CH 2 - or -CH2 CH 2 -C(=O)X 2 -; X 4 is -NH-, -N(CH 3 )-, -N(CH 2 CH 3 )-, -NHS(=O) 2 -, -N(CH 3 )S(=O) 2 -, -N(CH 2 CH 3 )S(=O) 2 -, lysine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, serine, tyrosine, valine, citrulline, methionine - valine - lysine, glycine - phenylalanine - glycine - glycine, tyrosine - arginine - valine, arginine - valine or a combination thereof; wherein any free amine (-NH 2 ) of the amino acid is optionally independently substituted by R 5 or –C(=O)R 5 and any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 .

[0472] In some embodiments, L is -L 1 -, -L 2 -, -L 3 -, -L 4 -, -L 5 -, -L 1 -L 2 -L 3 -L 4 -L 5 - or a combination thereof;

[0473] L 1 is absent, unsubstituted or substituted C 1 -C 10 alkylene, unsubstituted or substituted C 1 -C 10 heteroalkylene, unsubstituted or substituted C 2 -C 20 alkenylene, unsubstituted or substituted C 2 -C 20 alkynylene, C 4 -C 20 polyethylene glycol, -(X 3 CH 2 CH 2 ) t- An unsubstituted or substituted cycloalkylene, an unsubstituted or substituted hetero cycloalkylene, an unsubstituted or substituted arylene, an unsubstituted or substituted heteroarylene;

[0474] Each X 3 is independently selected from O and NR 4 ;

[0475] Each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0476] L 2 is absent, an unsubstituted or substituted C 1 -C 10 alkylene, an unsubstituted or substituted C 1 -C 10 heteroalkylene, -C(=O)-(an unsubstituted or substituted C 1 -C 10 alkylene), -C(=O)NR 4 -(an unsubstituted or substituted C 1 -C 10 alkylene)-, -NR 4 C(=O)-(an unsubstituted or substituted C 1 -C 10 alkylene)-, -C(=O)-(CH 2 CH 2 X 3 ) m -(CH 2 ) P -, -C(=O)NR 4 -(CH 2 CH 2 X 3 ) m -(CH 2 ) p -, -NR 4 C(=O)-(CH 2 CH 2 X 3 ) m -(CH 2 ) p - or -(CH 2 CH 2 X 3 ) m -(CH 2 ) p ;

[0477] Each R 4 is independently selected from hydrogen and C 1 -C 6 alkyl;

[0478] 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;

[0479] L 3 is absent or is one or more independently selected groups from: natural or unnatural amino acids and optionally amino (unsubstituted or substituted benzyl) carbamates; wherein any free amine (-NH 2 ) of the amino acid is optionally independently substituted by R 5 or -C(=O)R 5 , and any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 , and the substituted benzyl is substituted by 1 or 2 groups selected from halogen, -OH, -OR 5 , -CO 2 H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C 1 -C 6 alkyl, wherein the substituted C 1 -C 6 alkyl is substituted by -OH, -CO 2 H, -NHR 5 , -C(=O)NHR 5 and -NHC(=O)R 5 ;

[0480] L 4 is absent, unsubstituted or substituted C 1 -C 10 alkylene, -C(=O)-(unsubstituted or substituted C 1 -C 10 alkylene), -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 10 alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 10 alkylene)-, -C(=O)-(CH 2 CH 2 X 3 ) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH2 X 3 ) n -(CH 2 ) q -、-NR 4 C(=O)-(CH 2 CH 2 X 3 ) n -(CH 2 ) q -、-(CH 2 CH 2 X 3 ) n -(CH 2 ) q -or-(X 3 CH 2 CH 2 ) n -;

[0481] 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;

[0482] L 5 does not exist, -C(=O)-(CH 2 ) n -、-C(=O)NR 4 -(CH 2 ) n -、-NR 4 C(=O)-(CH 2 ) n -、-C(=O)-(CH 2 CH 2 X 3 ) n -(CH 2 ) q -、-C(=O)NR 4 -(CH 2 CH 2 X 3 ) n -(CH 2 ) q -、-NR 4 C(=O)-(CH 2 CH 2 X 3 ) n -(CH 2 ) q -、-(CH 2 CH 2 X 3 ) n -(CH2 ) q -, -C(=O)-(X 3 CH 2 CH 2 ) n - or -(X 3 CH 2 CH 2 ) n -;

[0483] 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;

[0484] Each R a is independently selected from hydrogen and C 1 -C 4 alkyl;

[0485] Each R b is independently selected from hydrogen and C 1 -C 4 alkyl;

[0486] wherein the heteroalkylene is an alkylene in which one carbon atom is replaced by -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(=N-R 5 )NR a - or -NR a C(=O)O-substituted alkylene;

[0487] where when any one of -L 1 -, -L 2 -, -L 3 -, -L 4 - and -L 5 is substituted, then -L 1 -, -L 2 -, -L 3 -, -L 4 - and -L 5- substituted by one, two, three or four groups selected from halogen, -OH, -OR 5 , -CO 2 H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C 1 -C 6 alkyl group, wherein the substituted C 1 -C 6 alkyl is substituted by -OH, -CO 2 H, -NHR 5 , -C(=O)NHR 5 and -NHC(=O)R 5 ;

[0488] Each R 5 is independently selected from C 1 -C 10 alkyl, C 4 -C 30 polyethylene glycol and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene.

[0489] In some embodiments, the heteroalkylene is an alkylene in which one carbon atom is replaced by -O-, -S-, -S(=O)-, -S(=O) 2 -, -S(=O)(=NH)-, -S(=O)(=NR 5 ), -, -NR 5 -, -P(=O)OH-, -NHC(=O)-, -C(=O)NH-, -OC(=O)NH-, -NHC(=N-CN)NH- or -NHC(=N-R 5 )NH-. In some embodiments, the heteroalkylene is an alkylene in which one carbon atom is replaced by -S(=O)(=NH)-, -S(=O)(=NR 5 ), -, -P(=O)OH-, -NHC(=N-CN)NH- or -NHC(=N-R 5 )NH-. In some embodiments, the heteroalkylene is an alkylene in which one carbon atom is replaced by -P(=O)OH-. In some embodiments, the heteroalkylene is an alkylene in which one carbon atom is replaced by -NHC(=N-CN)NH- or -NHC(=N-R 5 )NH-.

[0490] In some embodiments, L 1 is unsubstituted or substituted C 1 -C 20 alkylene, unsubstituted or substituted C 1-C 20 heteroalkylene, C 4 -C 20 polyethylene glycol, unsubstituted or substituted C 3 -C 8 cycloalkylene, unsubstituted or substituted monocyclic C 3 -C 8 heterocycloalkylene, unsubstituted or substituted phenylene, unsubstituted or substituted monocyclic heteroarylene.

[0491] In some embodiments, L 1 is unsubstituted or substituted C 1 -C 6 alkylene, unsubstituted or substituted C 1 -C 10 heteroalkylene, C 4 -C 20 polyethylene glycol, unsubstituted or substituted cyclohexylene or unsubstituted or substituted phenylene.

[0492] In some embodiments, L 2 is absent, -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 10 alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 10 alkylene)-, -C(=O)-(CH 2 CH 2 O) m -(CH 2 ) P -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) P -, -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) P - or -(CH 2 CH 2 O) n -(CH 2 ) P -; each m is independently 1, 2, 3, 4, 5 or 6; each p is independently 1 or 2. In some embodiments, L 2 is absent.

[0493] In some embodiments, L 3 is absent or is one or more independently selected groups chosen from: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, tyrosine, valine, and (unsubstituted or substituted benzyl) carbamate; wherein any free amine of the amino acid is optionally substituted by R 5 or -C(=O)(R 5 ) and any free carboxylic acid of the amino acid is optionally replaced by -C(=O)NH(R 5 ). In some embodiments, L 3 is absent.

[0494] In some embodiments, L 3 is lysine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, serine, tyrosine, valine, citrulline, methionine-valine-lysine, glycine-phenylalanine-glycine-glycine, tyrosine-arginine-valine, arginine-valine, valine-citrulline-(p-aminobenzyl carbamate), or a combination thereof; wherein any free amine (-NH 2 ) of the amino acid is optionally substituted by -C(=O)(unsubstituted or substituted C 1 -C 20 alkylene) or -C(=O)-C 4 -C 20 polyethylene glycol; wherein any free carboxylic acid (-CO 2 H) of any amino acid is optionally replaced by -C(=O)NH-(2,4,6-trimethyl-3-bromophenyl), -C(=O)NH-(unsubstituted or substituted C 1 -C 20 alkylene) or -C(=O)NH-(C 4 -C 20 polyethylene glycol).

[0495] In some embodiments, L 4 is absent, -C(=O)-(unsubstituted or substituted C 1 -C 6 alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 6 alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 6 alkylene)-, -C(=O)-(CH2 CH 2 O) n -(CH 2 ) q -、-C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q -、-NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q - or -(CH 2 CH 2 O) n -(CH 2 ) q -; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2. In some embodiments, L 4 is absent.

[0496] In some embodiments, L 5 is absent, -C(=O)-(CH 2 ) n -, -C(=O)NR 4 -(CH 2 ) n -, -NR 4 C(=O)-(CH 2 ) n -, -C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q -, -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -, -(CH 2 CH 2 O) n -(CH 2 ) q -, -C(=O)-(OCH2 CH 2 ) n - or -(OCH 2 CH 2 ) n -; each p is independently 0, 1 or 2. In some embodiments, L 5 is absent.

[0497] In some embodiments, L 5 is absent, -NR 4 C(=O)-(CH 2 ) n -, -C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q - or -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -; each q is independently 1 or 2.

[0498] In some embodiments, L 1 is unsubstituted or substituted C 1 -C 6 alkylene, unsubstituted or substituted C 1 -C 10 heteroalkylene, C 4 -C 20 polyethylene glycol, unsubstituted or substituted cyclohexylene or unsubstituted or substituted phenylene; L 2 is absent, -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 10 alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 10 alkylene)-, -C(=O)-(CH 2 CH 2 O) m -(CH 2 ) P -, -C(=O)NR 4-(CH 2 CH 2 O) n -(CH 2 ) P -、-NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) P -or-(CH 2 CH 2 O) n -(CH 2 ) P -; each m is independently 1, 2, 3, 4, 5 or 6; each p is independently 1 or 2; L 4 is absent, -C(=O)-(unsubstituted or substituted C 1 -C 6 Alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 6 Alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 6 alkylene)-, -C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -、-C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q -、-NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -or-(CH 2 CH 2 O) n -(CH 2 ) q -;L 5 Yes - NR 4 C(=O)-(CH 2 ) n -、-C(=O)-(CH 2 CH 2 O) n -(CH2 ) q -,-C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q -or-NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -; each n is independently 1, 2, 3, 4, 5 or 6; each q is independently 1 or 2.

[0499] In some embodiments, L 1 is unsubstituted or substituted C 1 -C 6 alkylene, unsubstituted or substituted C 1 -C 10 heteroalkylene, C 4 -C 20 polyethylene glycol, unsubstituted or substituted cyclohexylene or unsubstituted or substituted phenylene; L 2 is absent, -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 10 alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 10 alkylene)-, -C(=O)-(CH 2 CH 2 O) m -(CH 2 ) P -,-C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) P -,-NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) P -or-(CH 2 CH 2 O) n -(CH 2 ) P-; each m is independently 1, 2, 3, 4, 5 or 6; each p is independently 1 or 2; L 4 is absent; L 5 is absent, -NR 4 C(=O)-(CH 2 ) n -, -C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q -, or -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -; each n is independently 1, 2, 3, 4, 5 or 6; each q is independently 1 or 2.

[0500] In some embodiments, L 1 is unsubstituted or substituted C 1 -C 6 alkylene, unsubstituted or substituted C 1 -C 10 heteroalkylene, C 4 -C 20 polyethylene glycol, unsubstituted or substituted cyclohexylene or unsubstituted or substituted phenylene; L 2 is absent, -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 10 alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 10 alkylene)-, -C(=O)-(CH 2 CH 2 O) m -(CH 2 ) P -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) P -, -NR 4C(=O)-(CH 2 CH 2 O) n -(CH 2 ) P - or -(CH 2 CH 2 O) n -(CH 2 ) P -; each m is independently 1, 2, 3, 4, 5 or 6; each p is independently 1 or 2; L 3 is one or more independently selected groups 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 substituted by R 5 or -C(=O)(R 5 ) and any free carboxylic acid of the amino acid is optionally replaced by -C(=O)NH(R 5 ); each R 5 is independently unsubstituted or substituted C 1 -C 10 alkylene, C 4 -C 20 polyethylene glycol or unsubstituted or substituted phenyl, wherein the substituted phenyl is substituted by 1, 2, 3, 4 or 5 groups independently selected from F, Cl, Br, I, -CH 3 and CF 3 ; L 4 is absent; L 5 is -NR 4 C(=O)-(CH 2 ) n -, -C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q - or -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q-; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2.

[0501] In some embodiments, each m is independently 3, 4, 5, or 6. In some embodiments, each m is independently 4, 5, or 6. In some embodiments, each p is independently 1. In some embodiments, each p is independently 2.

[0502] In some embodiments, each n is independently 3, 4, 5, or 6. In some embodiments, each n is independently 4, 5, or 6. In some embodiments, each q is independently 1. In some embodiments, each q is independently 2.

[0503] In some embodiments, each m is independently 1, 2, 3, 4, 5, or 6; each p is independently 2; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2. In some embodiments, each m is independently 1, 2, 3, 4, 5, or 6; each p is independently 2; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 2.

[0504] In some embodiments, each X 3 is independently O. In some embodiments, each X 3 is independently NR 4 . In some embodiments, each R 4 is hydrogen. In some embodiments, each R 4 is C 1 -C 6 alkyl.

[0505] In some embodiments, each R 5 is independently selected from C 1 -C 10 alkyl, C 4 -C 30 polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene. In some embodiments, each R 5 is independently selected from C 1 -C 10 alkyl or C 4 -C 30 polyethylene glycol. In some embodiments, each R 5 is independently selected from unsubstituted or substituted arylene or unsubstituted or substituted heteroarylene.

[0506] In some embodiments, each R a is independently selected from hydrogen and C 1 -C 4 alkyl. In some embodiments, each Ra Independently is hydrogen. In some embodiments, each R a Independently is C 1 -C 4 alkyl.

[0507] In some embodiments, each R b Independently is selected from hydrogen and C 1 -C 4 alkyl. In some embodiments, each R b Independently is hydrogen. In some embodiments, each R b Independently is C 1 -C 4 alkyl.

[0508] Exemplary compounds for preparing the immunoconjugates described herein include the compounds depicted in Table A.

[0509] Table A

[0510]

[0511]

[0512]

[0513]

[0514] Radioimmunoconjugate

[0515] In one embodiment, the present invention provides an immunoconjugate. In one embodiment, when so labeled, linked, or loaded with an α-emitter, the immunoconjugate is capable of delivering the α-emitter in vivo. In one embodiment, when so labeled, linked, 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, linked, or loaded, the immunoconjugate is capable of delivering an imaging metal (e.g., 111 In, 89 Zr, 64 Cu, 68 Ga or 13 4Ce).

[0516] The immunoconjugates of the present disclosure can be loaded with radioisotopes to achieve a therapeutic or diagnostic effect. In certain embodiments, the chelating agent may also contain a radioisotope. In certain embodiments, the radioisotope is an α-emitter. In certain embodiments, the radioisotope is an α-emitter selected from the list consisting of: 225 Ac, 223 Ra, 224Ra, 227 Th, 212 Pb, 212 Bi and 213 Bi. In certain embodiments, the radioisotope is 225 Ac. In certain embodiments, the radioisotope is a β-emitter. In certain embodiments, the radioisotope is a β-emitter selected from the following: 177 Lu, 90 Y, 67 Cu and 153 Sm.

[0517] Also described herein is a method for preparing a radioimmunoconjugate, the method comprising loading or conjugating the immunoconjugate of the present disclosure to a radioisotope. In certain embodiments, the radioisotope is an α-emitter. In certain embodiments, the radioisotope is an α-emitter selected from the list consisting of: 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi and 213 Bi. In certain embodiments, the radioisotope is 225 Ac. In certain embodiments, the radioisotope is a β-emitter. In certain embodiments, the radioisotope is a β-emitter selected from the following: 177 Lu, 90 Y, 67 Cu and 153 Sm.

[0518] In one aspect, the present invention provides a radioimmunoconjugate comprising the immunoconjugate of the present invention and an α-emitting radioisotope. In one embodiment, the α-emitting radioisotope of the radioimmunoconjugate is selected from the group comprising: 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi and 213 Bi. In one embodiment, the α-emitting radioisotope of the radioimmunoconjugate is selected from the group consisting of: 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi and 213 Bi. In one embodiment, the α-emitting radioisotope of the radioimmunoconjugate is 225Ac. In one embodiment, the α-emitting radioisotope of the radioimmunoconjugate is 223 Ra. In one embodiment, the α-emitting radioisotope of the radioimmunoconjugate is 224 Ra. In one embodiment, the α-emitting radioisotope of the radioimmunoconjugate is 227 Th. In one embodiment, the α-emitting radioisotope of the radioimmunoconjugate is 212 Pb. In one embodiment, the α-emitting radioisotope of the radioimmunoconjugate is 212 Bi. In one embodiment, the α-emitting radioisotope of the radioimmunoconjugate is 213 Bi.

[0519] In some embodiments, the immunoconjugates of the invention are combined with a radioisotope to provide the radioimmunoconjugates of the invention. In some embodiments, the radioisotope is 225 Ac, 86 Y, 90 Y, 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 213 Bi, 213 Po, 212 Bi, 223 Ra, 224 Ra, 227 Th, 149 Tb, 68 Ga, 64 Cu, 67 Cu, 89 Zr, 137 Cs, 212 Pb or 103 Pd. In some embodiments, the radioisotope is an α-emitter, such as, for example 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi and 213 Bi. In some embodiments, the radioisotope is a β-particle emitter, such as, for example 177 Lu, 90 Y, 67 Cu and 153Sm. In some embodiments, the radioisotope is both an α-particle emitter and a β- and / or γ-particle emitter. In some embodiments, the radioisotope is both a β-particle emitter and a γ-particle and / or photon emitter. In some embodiments, the radioimmunoconjugate is labeled, linked, or loaded with an α-emitter and a β-emitter and thus contains them. In some embodiments, such as, for example, selected from 68 Ga, 64 Cu, 89 Zr, 111 In, and 134 Ce for use in radioimaging.

[0520] The immunoconjugates and radioimmunoconjugates of the present invention may comprise other cargo or payloads in addition to the radioisotope, including a variety of 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 cytotoxins to target cells. Non-limiting examples of cytotoxic agents include aziridines, cisplatins, tetrazines, procarbazine, hexamethylmelamine, vinca 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.

[0521] 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.

[0522] In some embodiments, the radioimmunoconjugates of the present invention comprise radioisotopes 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.

[0523] In some embodiments, the radioisotope is an alpha-emitting radioisotope, which comprises 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi or 213 Bi.

[0524] In some embodiments, the radioisotope is an alpha-emitting radioisotope selected from the group consisting of: 225 Ac, 223 Ra, 224 Ra, 227Th 212 Pb 212 Bi and 213 Bi

[0525] Additional embodiments of the immunoconjugates, antigen-binding regions, and heavy chain variable regions are described below:

[0526] In some embodiments, the immunoconjugate comprises a dimerization domain or motif. In some additional embodiments, the dimerization domain or motif is in the variant constant region, linker, or hinge region.

[0527] One of ordinary skill in the art can engineer the multimeric immunoconjugates of the invention using methods and approaches known in the art. For example, engineered cysteine residues can form covalent bonds to stabilize the spontaneously assembled multimeric structure (see, e.g., Glockshuber R et al., Biochemistry 29:1362-7 (1990)). For example, cysteine residues can be introduced at specific positions to generate disulfide bond-stabilized structures such as Cys-diabodies, scFv' multimers, VHH multimers, VNAR multimers, and IgNAR multimers, such as, 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)).

[0528] Alternatively, polypeptide domains that self-associate or multimerize with each other can be used to link two or more polypeptide chains together (see, e.g., US 6,329,507). For example, the addition of a carboxyl-terminal multimerization domain has been used to construct multivalent proteins comprising immunoglobulin domains, such as, for example, scFv, autonomous V H domain, V H H, V NARand IgNAR. Examples of self-associating domains known to the person skilled in the art include: immunoglobulin constant domains (such as knobs-into-holes; electrostatic steering and IgG / IgA chain exchange); immunoglobulin Fab chains (e.g., (Fab-scFv) 2 and (Fab'scFv) 2 ); immunoglobulin Fc domains (e.g., (sc diabody-Fc) 2 , (scFv-Fc) 2 and scFv-Fc-scFv); immunoglobulin CHX domains; immunoglobulin CH1-3 regions; immunoglobulin CH3 domains (e.g., (sc diabody-CH3) 2, LD microantibodies and Flex-microantibodies); immunoglobulin CH4 domain; CHCL domain; amphipathic helix bundle (e.g., scFv-HLX); helix-turn-helix domain (e.g., scFv-dHlx); coiled-coil structure including leucine zipper and cartilage oligomeric matrix protein (e.g., scZIP); combination of the cAMP-dependent protein kinase (PKA) dimerization and docking domain (DDD) and the A-kinase anchor protein (AKAP) anchor domain (AD) (also known as "dock-and-lock" or "DNL"); streptavidin; Shiga-like toxin (verotoxin) B polymerization domain; tetramerization region from p53; and barnase-barstar interaction domain (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 Mol Cell Biol 5:959-70 (2004);Zhang J et al., J Mol Biol 335:49-56(2004); Baillie G et al., FEBS Letters 579:3264-70(2005); Rossi E et al., Proc Natl Acad Sci USA 103:6841-6(2006); Simmons D et al., J Immunol Methods 315:171-84(2006); Braren I et al., Biotechnol Appl Biochem 47:205-14(2007); Chang C et al., Clin Cancer Res 13:5586-91s(2007); Liu M et al., Biochem J 406:237-46(2007); Zhang J et al., Protein Expr Purif 65:77-82(2009); Bell A et al., Cancer Lett 289:81-90(2010); Iqbal U et al., Br J Pharmacol 160:1016-28(2010); Asano R et al., FEBS J 280:4816-26(2013); Gil D, Schrum A, Adv Biosci Biotechnol 4:73-84(2013)).;

[0529] Those skilled in the art can engineer the multimeric immunoconjugates of the present invention using various scFv-based polypeptide interactions known in the art, such as, for example, scFv-based dimers, trimers, tetrameric complexes, etc. For example, the length of the linker in the scFv can affect the spontaneous assembly of non-covalent, multimeric, multivalent structures. Generally, linkers of twelve amino acids or less, including the absence of any linker, promote the multimerization of scFv-containing polypeptides or protein into higher molecular weight species via intermolecular domain exchange that is more favorable than intrastrand domain pairing (see, for example, Dolezal O et al., Protein Eng 16:47-56 (2003)). However, scFvs that have no linker at all or have a linker of an exemplary length of 15 amino acid residues can multimerize (Whitlow M et al., Protein Eng 6:989-95 (1993); Desplancq D et al., Protein Eng 7:1027-33 (1994); Whitlow M et al., Protein Eng 7, 1017-26 (1994); Alfthan K et al., Protein Eng 8:725-31 (1995)). Those skilled in the art can identify multimeric structures produced and / or purified using techniques known in the art and / or described herein.

[0530] In some embodiments, amino acid sequence variants of the immunoconjugates described herein are contemplated. For example, it may be desirable to improve the binding affinity, stability, and / or other biological properties of the immunoconjugates of the present invention (e.g., alter the half-life or therapeutic window, reduce immunogenicity or increase 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, the fusion of immunoglobulin domains or polypeptide sequences; the substitution of hinge, linker, and / or chelator factor components; the substitution of radioisotopes. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the immunoconjugate. Any combination of fusions, deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics, such as a level of binding affinity for a particular antigen binding, a particular K D level and / or a particular Koff level.

[0531] The present invention provides antigen-binding antibody fragments and CDR sets. For example, such fragments can be truncated at the N-terminus or C-terminus, or can lack internal residues, when compared to a full-length native antibody (e.g., full-length camelid VHH IgG2 or IgG3). Certain fragments may lack amino acid residues or domains that are not essential for the desired biological activity of the antibody or for reducing the overall size of the immunoconjugates of the present invention.

[0532] 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 moiety or a readily detectable moiety. In some additional embodiments, the linkage comprises a proteinaceous fusion. In some additional embodiments, the heterologous moiety is a cytotoxic agent. In some embodiments, a carboxy-terminal lysine residue is added to provide a site-specific attachment site. Amino acid sequence insertions include N-terminal and / or C-terminal fusions of polypeptides ranging in length from one residue to polypeptides containing one hundred or more residues, as well as in-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include immunoconjugates having an N-terminal methionyl residue. Other insertion variants of the immunoconjugate include fusions 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.

[0533] The nucleic acid encoding the immunoconjugates of the present invention can be modified to produce chimeric or fusion immunoconjugate polypeptides, e.g., by substituting the human heavy and light chain constant domains (substituting homologous murine sequences with CH and CL (U.S. Patent No. 4,816,567; and Morrison et al., Proc Natl Acad Sci USA 81:6851 (1984))) or by fusing all or a portion of the immunoglobulin-encoding sequence with the encoding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide). The non-immunoglobulin polypeptide sequence can replace the constant domain of the immunoconjugate, or they replace the variable domain of one of the antigen-binding sites of the immunoconjugate to produce a chimeric bivalent immunoconjugate containing one antigen-binding site specific for one antigen and another antigen-binding site specific for a different antigen.

[0534] Variations of antibody constructs that serve as antigen-binding domains in the inventions described herein can be generated using, for example, any techniques and guidance for conservative and non-conservative mutations set forth in, for example, U.S. Patent No. 5,364,934. The variations can be substitutions, deletions, or insertions of one or more codons encoding the immunoconjugate or polypeptide, which result 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 for any other amino acid in one or more domains of the immunoconjugate. Guidance for determining which amino acid residue can be inserted, substituted, or deleted without adversely affecting the desired activity can be found by comparing the sequence of the immunoconjugate to the sequences of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. The amino acid substitution can be the result of replacing one amino acid with another having a similar structure and / or chemical property, such as replacing leucine with serine, i.e., a conservative amino acid substitution. The insertion or deletion can optionally be in the range of about 1 to 5 amino acids. Permissible variations can be determined by systematically making amino acid insertions, deletions, or substitutions in the sequence and testing the activity exhibited by the full-length or mature native sequence of the resulting variant.

[0535] In specific embodiments, conservative substitutions of interest are shown in Tables B and C, including under the heading of preferred substitutions. If such substitutions result in a change in biological activity, more substantial changes are introduced (designated as exemplary substitutions in Table C, or further described below with reference to amino acid classes), and the products are screened.

[0536] Table C

[0537]

[0538] Substantial modifications of the function or immunological identity of the immunoconjugates of the invention are accomplished by selecting substitutions that differ significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the substituted region, e.g., a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the bulk of the side chain packing. Based on common side-chain properties, naturally occurring residues are divided into the following groups:

[0539] (1) Hydrophobic: norleucine, met, ala, val, leu, ile;

[0540] (2) Neutral hydrophilic: cys, ser, thr;

[0541] (3) Acidic: asp, glu;

[0542] (4) Basic: asn, gln, his, lys, arg;

[0543] (5) Residues affecting chain orientation: gly, pro; and

[0544] (6) Aromatic: trp, tyr, phe.

[0545] Non-conservative substitutions would require the exchange of a member of one of these categories for another. Residues of such substitutions can also be introduced at conservative substitution sites, or more preferably, at the remaining (non-conservative) sites.

[0546] Mutations can be made using methods known in the art, such as, for example, oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis can be performed on cloned DNA (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction-selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London Ser A, 317:415 (1986)), or other known techniques to generate DNA molecules encoding variants of the immunoconjugates of the present invention.

[0547] In some embodiments, immunoconjugate variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include immunoglobulin variable domain HVRs and FRs and regions within immunoglobulin constant domains. Amino acid substitutions can be introduced into the immunoconjugates of interest, and the products screened for the desired activities, e.g., enhanced / retained antigen binding, reduced / retained immunogenicity, enhanced / retained antibody-dependent cell 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 immunoconjugates of interest, and the products screened for reduced or eliminated activities, e.g., ADCC, CDC, target inhibition, and / or ADCP.

[0548] One type of substituted variant involves substituting one or more hypervariable region residues of a parental antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting variants selected for further study will have a modification (e.g., an improvement) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parental antibody and / or will substantially retain certain biological properties of the parental antibody. Illustrative substituted variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0549] Alterations (e.g., substitutions) can be made in the HVRs, for example, to improve immunoconjugate affinity. Such alterations can be made in HVR "hotspots" (i.e., residues encoded by codons that are mutated at high frequency during somatic maturation) (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or in the SDR (a-CDR), and the binding affinity of the resulting variant VH or VL is tested. For example, Hoogenboom et al. describe affinity maturation by constructing a secondary library and reselecting from the secondary library in Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify any antibody variants having the desired affinity. Another method of introducing diversity involves HVR-directed methods in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine-scan mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly often targeted.

[0550] In some embodiments, substitutions, insertions, or deletions can occur within one or more HVRs, provided that such alterations do not substantially reduce the ability of the immunoconjugate to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not significantly reduce binding affinity can be made in the HVRs. Such alterations can be outside of HVR "hotspots" or the SDR. In some embodiments of the variant VH and VL sequences provided above, each HVR is unaltered or contains no more than one, two, or three amino acid substitutions.

[0551] A method for identifying residues or regions of an antibody that can be targeted by mutagenesis is called "alanine-scanning mutagenesis", as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or a group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Additional substitutions can be introduced at the amino acid positions to demonstrate functional sensitivity to the initial substitution.

[0552] Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points 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 whether they possess the desired properties.

[0553] In some embodiments, the immunoconjugates of the invention comprise an antibody construct (used herein as the antigen-binding region) that comprises a humanized immunoglobulin domain.

[0554] A humanized form of a non-human (e.g., camelid, murine, or rabbit) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as an Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequence of an antibody) that contains a minimal sequence derived from a non-human immunoglobulin. A humanized antibody includes a human immunoglobulin (recipient antibody) in which residues from the recipient complementarity-determining regions (CDRs) are replaced with residues from the CDRs of a non-human species (such as a camelid, mouse, rat, or rabbit) (donor antibody) having the desired specificity, affinity, and capacity. In some instances, the Fv framework residues of the human immunoglobulin are replaced with the corresponding non-human residues. A humanized antibody may also contain residues that are not found in either the recipient antibody or the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all of the CDR regions correspond to those of the non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. A humanized antibody may also comprise at least a portion of the immunoglobulin constant region (Fc), usually at least a portion of the constant region of a human immunoglobulin (Jones et al., Nature, 321:522-5 (1986); Riechmann et al., Nature, 332:323-9 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-6 (1992)).

[0555] Methods for humanizing non-human antibodies are well known in the art. Generally, humanized antibodies have one or more amino acid residues introduced from non-human sources. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from "import" variable domains. Humanization can generally follow the methods 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)), and is carried out by replacing the corresponding sequences of a human antibody with rodent CDRs or CDR sequences. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which substantially less than the entire human variable domain is replaced by the corresponding sequences from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from similar sites in a rodent antibody.

[0556] According to another method, antigen binding can be restored during humanization of an antibody by selecting repaired hypervariable regions (see, for example, U.S. Application Serial No. 11 / 061,841, filed February 18, 2005). The method includes incorporating non-human hypervariable regions onto a receptor framework and further introducing one or more amino acid substitutions in one or more hypervariable regions without modifying the receptor framework sequence. Alternatively, the introduction of one or more amino acid substitutions can be accompanied by modifications in the receptor framework sequence.

[0557] Generally, any cysteine residue not involved in maintaining the correct conformation of the immunoconjugates of the present invention can also be replaced with serine to increase 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 increase their stability (especially in cases where the antibody is an antibody fragment, such as an Fv fragment or a VHH fragment).

[0558] In some embodiments, it may be desirable to generate cysteine-engineered immunoconjugates in which one or more residues of the immunoconjugate are replaced with cysteine residues. In some embodiments, the replaced residues occur at accessible sites of the immunoconjugate. By replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the immunoconjugate and can be used to conjugate the immunoconjugate to other moieties, such as a drug moiety or a linker-drug moiety. In some embodiments, any one or more of the following residues may be replaced with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies can be generated as described, for example, in US 7,521,541.

[0559] One of ordinary skill in the art will understand that amino acid changes can alter post-translational processes of the immunoconjugate, such as changing the number or location of glycosylation sites or altering membrane anchoring characteristics.

[0560] In some embodiments, the immunoconjugates provided herein are altered to increase or decrease the degree to which the immunoconjugate is glycosylated and / or to alter the glycosylation pattern. "Altering the native glycosylation pattern" for purposes herein is intended to mean the deletion of one or more carbohydrate moieties found in the parental immunoconjugate of the invention (by removing potential glycosylation sites or by chemical and / or enzymatic means to remove glycosylation), and / or the addition of one or more glycosylation sites not present in the native sequence immunoconjugate of the invention. In addition, the phrase includes qualitative changes in the glycosylation of the native protein, involving changes in the nature and proportions of the various carbohydrate moieties present.

[0561] Glycosylation of antibodies and other polypeptides is generally N-linked or O-linked. N-linked refers to the attachment of a 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 other than proline) are recognition sequences for the enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxy amino acid, most commonly serine or threonine, but also 5-hydroxyproline or 5-hydroxylysine.

[0562] Addition or deletion of glycosylation sites to an immunoconjugate can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed. Addition of glycosylation sites to the immunoconjugates of the invention can be readily achieved by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration can also be effected by adding or substituting one or more serine or threonine residues to the sequence of the original immunoconjugate of the invention (for O-linked glycosylation sites). The amino acid sequence of the immunoconjugates of the invention can optionally be altered by changes at the DNA level, particularly by mutating the DNA encoding the immunoconjugate of the invention at preselected bases such that codons are generated that will translate into the desired amino acids.

[0563] In the case where the immunoconjugate comprises an Fc region, the carbohydrate attached thereto can be altered. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides attached to Asn297 of the Fc region CH2 domain via N-linkage (see, e.g., Wright et al., TIBTECH 15:26-32 (1997)). The oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the “backbone” of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the immunoconjugates of the invention can be modified to produce immunoconjugate variants with certain improved properties.

[0564] Another means of increasing the number of carbohydrate moieties on the immunoconjugates of the invention is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, for example, in WO87 / 05330 published Sep. 11, 1987 and Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0565] Removal of carbohydrate moieties present on the immunoconjugates of the invention can be accomplished by chemical or enzymatic means or by mutational substitution of codons encoding amino acid residues that serve as glycosylation targets. Chemical deglycosylation techniques are known in the art and are described, for example, by Hakimuddin et al., Arch. Biochem. Biophys., 259:52 (1987) and Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by using a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).

[0566] In some embodiments, immunoconjugate variants are provided that have a carbohydrate structure lacking fucose (directly or indirectly) attached to the Fc region. For example, the amount of fucose in such immunoconjugates can be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose at Asn297 within the sugar chain relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, 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 numbering of Fc region residues); however, due to minor sequence variations in antibodies, 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, e.g., US2003 / 0157108; US2004 / 0093621). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO2000 / 61739; WO 2001 / 29246; US2003 / 01 15614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 01 10282; US2004 / 0109865; WO 2003 / 0851 19; 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 defucosylated antibodies include the protein fucosylation-deficient Lec13 CHO cell line (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US 2003 / 0157108; WO 2004 / 056312, Adams et al., especially in Example 11) and knockout cell lines, such as the α-1,6-fucosyltransferase gene FUT8 knockout CHO cell (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).

[0567] Immunoconjugate variants also have bisected oligosaccharides, e.g., in which the biantennary oligosaccharide attached to the antibody Fc region 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, US2005 / 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.

[0568] Immunoconjugate derivatives and other modifications

[0569] Covalent modifications of the immunoconjugates of the present invention are included within the scope of the present invention. One type of covalent modification involves reacting a targeted amino acid residue of the immunoconjugate of the present invention with an organic derivatizing agent capable of reacting with a selected side chain or N-terminal or C-terminal residue of the immunoconjugate. 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; homobifunctional imidates, including disuccinimidyl esters, such as 3,3'-dithiobis(succinimidyl propionate); bismaleimides, such as bis-N-maleimidyl-1,8-octane; and agents such as methyl-3-[(p-azidophenyl)dithio]propionimidate.

[0570] Other modifications include: deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively; hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of serine or threonine residues; methylation of the α-amino groups of the side chains of lysine, arginine, and histidine (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)); acetylation of the N-terminal amine; and amidation of any C-terminal carboxyl group.

[0571] In some embodiments, the immunoconjugates provided herein can be further modified to contain additional non-proteinaceous moieties known and readily available in the art. Moieties suitable for derivatization of immunoconjugates include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to: polyethylene glycol (PEG), copolymers of ethylene glycol / propanediol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, poly(propylene oxide / ethylene oxide) copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacture 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 can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymer 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, whether the immunoconjugate derivative will be used in therapy under defined conditions, etc.

[0572] The PEG-derivatized immunoconjugates of the invention can contain a linker containing one or more -CH 2 CH 2 O- and can be used to alter the biodistribution and pharmacokinetics of the immunoconjugate. PEG can be prepared in polymeric form or as discrete oligomers. The bifunctionalized forms of these polymers can link the immunoconjugate to a chelator and / or provide additional size and / or solubility to the overall molecule. In some embodiments, the PEG-derivatized immunoconjugates exhibit reduced immunogenicity compared to their underivatized parent molecules. Methods for producing the immunoconjugates of the present invention

[0573] The present invention provides a composition comprising one or more of the immunoconjugates as in any of the above embodiments or as described herein. In another aspect, the present invention provides an isolated nucleic acid encoding a radioisotope delivery platform as described herein. Also provided herein are nucleic acids encoding the protein components of the immunoconjugates of the present invention, expression vectors comprising the foregoing nucleic acids, and host cells comprising the foregoing expression vectors.

[0574] In another aspect, the present invention provides a host cell comprising the nucleic acid and / or vector as provided herein. In some embodiments, the host cells of the present invention are isolated or purified. In some embodiments, the host cells of the present invention are in cell culture medium. The nucleic acids, expression vectors, and host cells of the present invention can be used to produce a composition 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 prokaryotic. In some embodiments, the host cell is Escherichia coli.

[0575] Illustrative techniques for producing the immunoconjugates and radioimmunoconjugates of the present invention for use in the methods according to the present invention are described below. In some embodiments, the present invention provides a method for preparing an immunoconjugate of the present invention, the method comprising culturing a host cell 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 α- or β-particle emitter).

[0576] Generation and identification of antigen-binding domains, immunoconjugates, and nucleic acids

[0577] Antigen-binding domains that can be used as antigen-binding regions herein can be identified in antibodies, which are monoclonal antibodies and / or polyclonal antibodies. DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures. Once isolated, the DNA can be placed into an expression vector and then transfected into a host cell (such as an Escherichia coli cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not otherwise produce antibody protein) to obtain synthesis of the monoclonal antibody in the recombinant host cell (see, for example, Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Plückthun, Immunol Revs. 130:151-188 (1992)).

[0578] In some embodiments, the antigen-binding domain or fragment thereof of the immunoconjugates of the invention is isolated by screening a phage library 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 the desired target antigen or epitope. Clones expressing Fv fragments, scFv or VHH capable of binding the desired antigen are adsorbed to the antigen and are thus separated from non-binding clones in the library. The binding clones are then eluted from the antigen and can be further enriched by additional antigen adsorption / elution cycles.

[0579] In some embodiments, the antibody or antibody fragment thereof is isolated from an antibody phage library generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J Mol Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies using phage libraries, respectively. Subsequent publications describe the generation of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), and combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc Acids Res. 21:2265-2266 (1993)). The variable domains can be functionally displayed on the phage, either 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).

[0580] VH and VL gene libraries can be cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, and then their antigen-binding clones can be searched as described by Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). The initial library for screening can be constructed from non-immunized sources to provide high-affinity antibodies against antigens (see, for example, Griffiths et al., EMBO J, 12:725-734 (1993)). Another example is an initial library constructed synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences encoding highly variable CDR3 regions and performing rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

[0581] Screening of the library can be accomplished by various techniques known in the art. For example, the target antigen can be used to coat the wells of an adsorption plate, expressed on host cells attached to the adsorption plate or used for cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning a display library. Selection of antibodies with slow dissociation kinetics (and strong binding affinity) can be facilitated by using long washes and monovalent phage display (as described by Bass et al., Proteins, 8:309-314 (1990) and in WO 1992 / 09690) and low antigen coating density (as described by Marks et al., Biotechnol., 10:779-783 (1992)).

[0582] Techniques for screening cDNA libraries are well known in the art. The library can be screened with probes (such as oligonucleotides of at least about 20-80 bases) designed to identify genes or proteins encoded thereby. Standard procedures (such as those described by Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) can be used to screen cDNA or genomic libraries with the selected probe. An alternative means of isolating genes encoding the immunoconjugates of the present invention is to use PCR methods (Sambrook et al. (as above); Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)).

[0583] DNA encoding the immunoconjugates of the present invention can be obtained from a cDNA library prepared from tissues believed to have the mRNA of the immunoconjugates of the present invention and expressing it at a detectable level. Thus, the DNA of the human immunoconjugates of the present invention can be conveniently obtained from a cDNA library prepared from human tissues. The gene encoding the immunoconjugates of the present invention can also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis). For some embodiments, the desired polynucleotide sequence encoding an antibody can be isolated from antibody-producing cells such as hybridoma cells and sequenced.

[0584] Sequences identified in such library screening methods can be compared and aligned with other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence 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 antibody clones using the variable domain and / or CDR sequences from the phage clones of interest and the appropriate constant region (Fc) sequences as described in Kabat et al., 1991 (as above).

[0585] Immunoconjugate production; host cells and expression vectors of the present invention

[0586] The following description mainly relates to the production of the antibody constructs of the present invention by culturing cells transformed or transfected with a vector containing the nucleic acid encoding the immunoconjugates of the present invention. Of course, alternative methods well known in the art are envisioned for preparing the antibody constructs of the present invention. For example, the appropriate amino acid sequence or a portion thereof can be produced by direct peptide synthesis using solid-phase techniques (e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J, Am. Chem. Soc., 85:2149-54 (1963)). In vitro protein synthesis can be performed using manual techniques or by automation. For example, an Applied Biosystems peptide synthesizer (Foster City, CA) can be used to complete automated synthesis using the manufacturer's instructions. The individual parts of the immunoconjugates of the present invention can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired immunoconjugates of the present invention.

[0587] Antibody constructs can be produced using recombinant methods and compositions, e.g., as described in US 4,816,567. In one embodiment, an isolated nucleic acid encoding an antibody as described herein is provided. Such nucleic acids can encode an amino acid sequence comprising an antibody VH and / or an amino acid sequence comprising a VL (e.g., an 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., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VH. In some other embodiments, the host cell comprises: (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL and a nucleic acid encoding an amino acid sequence comprising an antibody VH, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL and a second vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VH. In one embodiment, the host cell is a eukaryotic cell, e.g., a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., YO, NSO, Sp20 cells). In one embodiment, a method of preparing an immunoconjugate of the invention is provided, wherein the method comprises culturing a host cell comprising 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 the host cell culture medium).

[0588] For recombinant production of the immunoconjugates of the 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 a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically hybridize to genes encoding the antibody heavy and / or light chains). Nucleic acid molecules encoding the amino acid sequences of the immunoconjugates of the invention (including sequence variants) can be prepared by a variety of methods known to those of skill in the art. These methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of earlier prepared variants or non-variant forms of the antibody constructs.

[0589] Manipulation of host cells for immunoconjugate production

[0590] The host cells are transfected or transformed with the expression or cloning vectors described herein to effect the production of the conjugates of the invention and are cultured in a conventional nutrient medium modified as appropriate to induce the promoter, select for transformants, or amplify the gene encoding the desired sequences. The culture conditions, such as the medium, temperature, pH, etc., can be selected by the skilled person without undue experimentation. In general, the principles, protocols, and actual techniques for maximizing the productivity 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).

[0591] Host cells suitable for the cloning or expression of nucleic acids and vectors encoding immunoconjugates include the prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For antibody fragment and polypeptide expression in bacteria, see, e.g., US 5,648,237; US 5,789,199; US 5,840,523; and Charlton, Methods in Molecular Biology, Vol. 248 (edited by B.K.C. Lo, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describe the expression of antibody fragments in Escherichia coli. After expression, the immunoconjugate can be isolated from the bacterial cell paste in the form of a soluble fraction and can be further purified.

[0592] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding immunoconjugates, including fungal and yeast strains in which the glycosylation pathway has been "humanized", resulting in the production of antibodies with a partially or fully human glycosylation pattern (see, e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004); Li et al., Nat. Biotech. 24:210-215 (2006)).

[0593] 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. A number of baculovirus strains have been identified that are suitable for use in conjunction with insect cells, particularly for the transfection of Spodoptera frugiperda cells. Plant cell cultures are also used as hosts (see, e.g., US 5,959,177; US 6,040,498; US 6,420,548; US 7,125,978; and US 6,417,429).

[0594] 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 by SV40 (COS-7); human embryonic kidney line, 293 or 293 cells, as described, e.g., in Graham et al., J Gen Viral. 36:59 (1977); baby hamster kidney cells (BHK); mouse Sertoli cells, TM4 cells, as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980); monkey kidney cells (CV 1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MOCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep02); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., 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 immunoconjugate production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0595] Methods for transfection of eukaryotic cells and transformation of prokaryotic cells are known to those skilled in the art, which means introducing DNA into a host in an extrachromosomal form or through chromosomal integrants such that the DNA is replicable, such as CaCl2, CaPO4, liposome-mediated, polyethylene glycol / DMSO, and electroporation. Depending on the host cell used, transformation is carried out using standard techniques suitable for such cells. Calcium treatment with calcium chloride or electroporation as described by Sambrook et al. (as above) is commonly used for prokaryotes. Agrobacterium tumefaciens infection is used to transform certain plant cells, as described by Shaw et al., Gene, 23:315 (1983) and WO 89 / 05859 published on June 29, 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) can be employed. General aspects of transfection of mammalian cell host systems have been described in U.S. Patent No. 4,399,216. Transformation into yeast is usually carried out according to the methods of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al., Proc Natl Acad Sci USA 76:3829 (1979). However, other methods for introducing DNA into cells can also be used, such as by nuclear microinjection, electroporation, fusion of bacterial protoplasts with intact cells, or polycations, such as polybrene, 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).

[0596] Prokaryotic host cells

[0597] Suitable prokaryotes include, but are not limited to, archaebacteria and eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia coli. Various strains of E. coli are publicly available, such as strain K12 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., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens) and Shigella, as well as Bacillus (such as B. subtilis and B. licheniformis) (e.g., B. licheniformis 41P published in DD 266,710 published on April 12, 1989), Pseudomonas (such as P. aeruginosa), Rhizobia, Vitreoscilla, Paracoccus and Streptomyces. These examples are illustrative and not restrictive. The E. coli strain W3110 is a preferred host or parental host because it is a common host strain for the fermentation of recombinant DNA products. Preferably, the host cell secretes a minimal amount of proteolytic enzymes.For example, the strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) can be modified to effect a gene mutation of a gene encoding a protein endogenous to the host, examples of such hosts including: Escherichia coli strain W3110 1A2, which has the complete genotype tonA; Escherichia coli strain W3110 9E4, which has the complete genotype tonA ptr3; Escherichia coli strain W3110 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169degP ompT kanr; Escherichia coli strain W3110 37D6, which has the complete genotype tonA ptr3 phoA E15(argF-lac)169degP ompT rbs7 ilvG kanr; Escherichia coli strain W3110 40B4, which is strain 37D6 having a non-kanamycin resistant degP deletion mutation; Escherichia coli strain W3110 33D3, which has the genotype W3110ΔfhuA(ΔtonA)ptr3 lac Iq lacL8ΔompTΔ(nmpc-fepE)degP41 kanR (U.S. Patent No. 5,639,635); and an Escherichia coli strain having a mutant periplasmic protease as disclosed in U.S. 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 restrictive. Methods for constructing derivatives of any of the above-described bacteria having a defined genotype are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to consider the replicability of the replicon in the bacterial cell to select an appropriate bacterium. For example, when using well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410 to provide the replicon, Escherichia coli, Serratia, or Salmonella species may be suitable as hosts. Generally, the host cell should secrete a minimal amount of proteolytic enzymes, and it may be desirable to incorporate additional protease inhibitors into the cell culture. Alternatively, in vitro cloning methods, such as PCR or other nucleic acid polymerase reactions, are suitable.

[0598] Full-length antibodies, antibody fragments, and antibody fusion proteins can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. Full-length antibodies have a longer half-life in circulation. Production in E. coli is faster and more cost-effective. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. 5,648,237, U.S. 5,789,199, and U.S. 5,840,523, which describe translation initiation regions (TIRs) and signal sequences for optimizing expression and secretion. After expression, the immunoconjugate can be isolated from the E. coli cell paste in the soluble fraction and purified, depending on the isotype, by, for example, protein A or G columns. Final purification can be carried out similarly to the process for purifying antibodies expressed, for example, in CHO cells.

[0599] Eukaryotic host cells

[0600] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding the immunoconjugates of the present invention. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include: Schizosaccharomyces pombe (Beach and Nurse, Nature, 290:140 (1981); EP 139,383 published on May 2, 1985); Kluyveromyces hosts (U.S. Patent No. 4,943,529; Fleer et al., Bio / Technology, 9:968-75 (1991)), such as, for example, Kluyveromyces lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 (1983)), Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces wickeramii (ATCC 24,178), Kluyveromyces waltii (ATCC 56,500), Kluyveromyces drosophilarum (ATCC 36,906; Van den Berg et al., Bio / Technology, 8:135 (1990)), Kluyveromyces thermotolerans, 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., Proc Natl Acad Sci USA 76:5259-5263 (1979)); Schwanniomyces, such as Schwanniomyces occidentalis (EP 394,538 published October 31, 1990); and filamentous fungi such as, for example, Neurospora, Penicillium, Tolypocladium (WO 91 / 00357 published January 10, 1991) 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)). Methylotropic yeast is suitable herein and includes, but is not limited to, yeast capable of growing on methanol, selected from Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are examples of such yeast can be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

[0601] 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. Many baculovirus strains and variants, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori, have been identified. A variety of virus strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as the viruses herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.

[0602] However, there is the greatest interest in vertebrate cells, and the proliferation of vertebrate cells in culture (tissue culture) has become routine. Examples of useful mammalian host cell lines are: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc Natl Acad Sci USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma line (Hep G2).

[0603] The host cells are transformed with the expression or cloning vectors described above to effect production of the conjugates of the invention and are cultured in an appropriate modified conventional nutrient medium to induce the promoter, select transformants or amplify the gene encoding the desired sequence.

[0604] Selection and use of replicable vectors

[0605] To recombinantly produce the radioisotope delivery platform of the invention, the nucleic acid encoding it (e.g., cDNA or genomic DNA) is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or for expression. The DNA encoding the immunoconjugate can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of the antibody). There are many vectors available. The choice of vector depends in part on the host cell to be used. Generally, suitable host cells are of prokaryotic or eukaryotic (usually mammalian) origin.

[0606] The vector can be in the form of, for example, a plasmid, cosmid, viral particle or phage. Appropriate nucleic acid sequences can be inserted into the vector by a number of 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, promoter and transcription termination sequences. Construction of suitable vectors containing one or more of these components employs standard ligation techniques known to those skilled in the art.

[0607] The immunoconjugates of the present invention can be produced not only by direct recombination, but also as a fusion polypeptide with a heterologous polypeptide, which can be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. Generally, the signal sequence can be a component of the vector, or it can be part of the DNA encoding the immunoconjugate 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 the heat-stable enterotoxin II leader sequence. For yeast secretion, the signal sequence can be, for example, the yeast invertase leader sequence, the α-factor leader sequence (including Saccharomyces and Kluyveromyces α-factor leader sequences, the latter described in U.S. Patent No. 5,010,182) or the acid phosphatase leader sequence, the Candida albicans glucoamylase 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 direct the secretion of proteins, such as signal sequences of secreted polypeptides from the same or related species and viral secretion leader sequences.

[0608] Culturing host cells for producing a radioisotope delivery platform

[0609] Host cells for producing the immunoconjugates of the present invention can be cultured in a variety of media and culture conditions.

[0610] Prokaryotic host cell cultures

[0611] Prokaryotic cells for producing the polypeptides of the present invention are grown in media known in the art and suitable for culturing the selected host cells. Examples of suitable media include Luria broth (LB) plus necessary nutrient supplements. In some embodiments, the medium also contains a selection agent selected based on the construction of the expression vector to selectively permit the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium for the growth of cells expressing the ampicillin resistance gene.

[0612] In addition to carbon, nitrogen, and inorganic phosphate sources, any necessary supplements can be included at appropriate concentrations, introduced individually or as a mixture with another supplement or medium (such as a complex nitrogen source). Optionally, the medium can contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cysteamine, thioglycolate, dithiothreitol, and dithioerythritol.

[0613] The prokaryotic host cells are cultured at a suitable temperature. For example, for E. coli growth, 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 about 30°C. The pH of the medium can be any pH in the range from about 5 to about 9, depending primarily on the host organism. For E. coli, the pH is preferably from about 6.8 to about 7.4, more preferably about 7.0.

[0614] If an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activation of the promoter. In one aspect of the present invention, the PhoA promoter is used to control transcription of the polypeptide. Thus, the transformed host cells are cultured in a phosphate-limited medium for induction. In some embodiments, the phosphate-limited medium is C.R.A.P medium (see, for example, Simmons et al., J. Immunol. Methods (2002), 263:133-47). Depending on the vector construct employed, a variety of other inducers can be used, as known in the art.

[0615] In one embodiment, the expressed polypeptides of the present invention are secreted into the periplasm of the host cells and recovered from the periplasm. Protein recovery generally involves disrupting the microorganisms, typically by means such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transported into the medium and isolated therein. The cells can be removed from the culture, and the culture supernatant can be filtered and concentrated for further purification of the resulting protein. Commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blotting can be used to further separate and identify the expressed polypeptides.

[0616] In one aspect of the present invention, immunoconjugates are produced in large quantities by a fermentation method. Different large-scale fed-batch fermentation procedures can be used for the production of recombinant proteins. Large-scale fermentation has a capacity of at least 1000 liters, preferably about 1,000 to 100,000 liters. These fermenters use agitator impellers to distribute oxygen and nutrients, especially glucose (a preferred carbon source / energy source). Small-scale fermentation generally refers to fermentation in a fermenter with a volume capacity not exceeding approximately 100 liters, and the range can be from about 1 liter to about 100 liters.

[0617] In the fermentation process, induction of protein expression is typically initiated after the cells have grown to the desired density (e.g., an OD550 of about 180 - 220) under suitable conditions (at this stage the cells are in the early stationary phase). Depending on the vector construct employed, a variety of inducers can be used, as are known in the art and described above. The cells can be allowed to grow for a short period before induction. The cells are typically induced for about 12 - 50 hours, but longer or shorter induction times can be used.

[0618] To increase the yield and quality of the polypeptides of the present invention, various fermentation conditions can be modified. For example, to improve the correct assembly and folding of the secreted immunoconjugate polypeptides, host prokaryotic cells can be co-transformed with a vector that additionally overexpresses chaperone proteins such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (a peptidyl-prolyl cis,trans isomerase with chaperone activity). It has been demonstrated that chaperone proteins facilitate the correct folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al., (1999) J Bio Chem 274:19601 - 5; U.S. Patent No. 6,083,715; U.S. Patent No. 6,027,888; 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.

[0619] To minimize proteolysis of the heterologous proteins expressed, particularly those sensitive to proteolysis, certain host strains lacking proteolytic enzymes can be used in the present invention. For example, host cell strains can be modified to achieve one or more gene mutations in genes encoding known bacterial proteases such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Some Escherichia coli protease-deficient strains are available and described, for example, in Joly et al., (1998), supra; U.S. Patent No. 5,264,365; U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0620] In one embodiment, an Escherichia coli strain lacking proteolytic enzymes and transformed with a plasmid overexpressing one or more chaperone proteins is used as a host cell in the expression system of the present invention.

[0621] Eukaryotic host cell cultures

[0622] Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM, Sigma) are suitable for culturing host cells. In addition, any of the 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; WO 87 / 00195; or U.S. Patent No. 30,985 can be used as a medium for host cells. Any of these media can be supplemented, as needed, with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN TM drug), trace elements (defined as inorganic compounds that are typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Appropriate concentrations of any other necessary supplements known to those skilled in the art can also be included. Culture conditions, such as temperature, pH, etc., are those previously used with the host cells selected for expression and will be apparent to those of ordinary skill in the art.

[0623] Purification of immunoglobulin-derived structures of the present invention

[0624] The immunoconjugates of the invention can be recovered from the 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 cleavage. Cells used for expressing the immunoconjugates of the invention can be disrupted by various physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysing agents.

[0625] It may be necessary to purify the immunoconjugates of the invention from recombinant cell proteins or polypeptides. The following procedures are examples of suitable purification procedures: fractionation on an ion-exchange column; ethanol precipitation; reverse-phase HPLC; chromatography on silica or cation-exchange resins such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A agarose columns to remove contaminants such as IgG; and metal chelate columns to bind the immunoconjugates of the invention in epitope-tagged form. A variety of 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 purification steps chosen will depend, for example, on the nature of the production method used and the particular immunoconjugate of the invention produced.

[0626] When recombinant techniques are used, immunoconjugates can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the immunoconjugate is produced intracellularly, as a first step, particulate debris, host cells, or lysed fragments are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-7 (1992) describe procedures for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 min. Cell debris can be removed by centrifugation. When the immunoconjugate is secreted into the culture medium, the supernatant from such an expression system is usually first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor (such as PMSF) can be included in any of the foregoing steps to inhibit proteolysis, and an antibiotic can be included to prevent the growth of adventitious contaminants.

[0627] Immunoconjugate compositions prepared from cells 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 class 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 murine isotypes and human γ3 (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to which the affinity ligand is attached is usually agarose, but other matrices can also be used. A mechanically stable matrix (such as controlled pore glass or poly(styrene-divinyl)benzene) can achieve a faster flow rate and shorter processing time compared to what is achieved using agarose. When the immunoconjugate contains a CH3 domain, Bakerbond ABX TM resin (J.T. Baker, 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, reverse-phase HPLC, silica chromatography, heparin SEPHAROSE TM chromatography, anion or cation exchange resin (such as a polyaspartic acid column) chromatography, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0628] After any initial purification step, low pH hydrophobic interaction chromatography can be used to subject a mixture comprising the immunoconjugate of interest and contaminants to an elution buffer having a pH between about 2.5 - 4.5 and typically at a low salt concentration (e.g., about 0 - 0.25 M salt).

[0629] Immunoconjugates (including antibody-drug conjugates (ADCs))

[0630] In another aspect of the invention, an immunoconjugate according to any of the above embodiments or of the invention described herein is conjugated to a heterologous moiety or agent, such as, for example, those described below and including any additional foreign materials as described herein.

[0631] In one embodiment, the invention provides an immunoconjugate comprising an antibody construct of the invention conjugated to one or more therapeutic agents or radioisotopes.

[0632] In some embodiments, the immunoconjugate comprises an antibody construct as described herein conjugated to a radioactive atom to form a radio conjugate. As described herein, a variety of radioisotopes can be used to produce the radio conjugates of the invention.

[0633] Conjugates of immunoconjugates or antibody constructs can be prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as hexamethylenediamine dimethyl ester H), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazido compounds (such as bis(p-azidobenzoyl)hexanediamine), diazo derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bifunctional fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described by Vitetta et al., Science 238:1098 (1987). Carbon 14-labeled 1-isothiocyanatobenzyl-3-methyl diethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radio nucleotides to antibodies (see, for example, WO 1994 / 11026). The linker can be a "cleavable linker" which facilitates the release of the cytotoxic drug within the cell. For example, acid-labile linkers, protease-sensitive linkers, photo-labile 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).

[0634] The immunoconjugates or ADCs of the present disclosure specifically contemplate, but are not limited to, such conjugates prepared with crosslinkers, including but not limited to BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., available from Pierce Biotechnology, Inc., Rockford, IL., U.S.).

[0635] As will be recognized by those of ordinary skill in the art, certain of the methods described above can also be used to prepare radioimmunoconjugates and targeted imaging complexes (although the text only mentions immunoconjugates or antibody constructs), and such preparation methods are also encompassed within the present invention.

[0636] Immunoconjugation using chelating factors and / or linkers

[0637] Methods for attaching radioisotopes to immunoconjugates or antibody constructs (i.e., "labeling" antibodies with radioisotopes) are well known to those skilled in the art. Some of these methods are described, for example, in WO 2017 / 155937.

[0638] Bifunctional chelating agents such as, for example, DOTA, DTPA, and related analogs are suitable for coordinating metal ions, such as alpha and beta emitting radionuclides. For example, these chelating molecules can be linked to the targeting molecule by forming a new amide bond between an amine on the antibody construct (e.g., the functional group of a lysine residue) and a carboxylate ester on DOTA / DTPA. In the case of peptide synthesis, the characterization and purification of linker addition can be part of the overall synthesis of the antibody platform or immunoconjugate for radioisotope conjugation.

[0639] For some embodiments, the method of generating an immunoconjugate involves a click chemistry step described by Poty, S et al., Chem Commun. (Camb) 54:2599 (2018).

[0640] For some embodiments, the peptide can be biosynthetic or can be synthesized by chemical amino acid synthesis using suitable amino acid precursors (involving, for example, replacing hydrogen with fluorine 19). In some embodiments, a radiolabel can be incorporated into the peptide. In some embodiments, the radiolabel can be attached to the peptide. The IODOGEN method (Fraker et al., (1978) Biochem Biophys Res Commun. 80:49 - 57) can be used to incorporate iodine - 123. Other methods are described in detail in “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989).

[0641] Characterization of the immunoconjugates of the present invention

[0642] The immunoconjugates of the present invention can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art. The immunoconjugates and antibody constructs of the present invention can be characterized for their physical / chemical properties and / or biological activities by various assays known in the art. The immunoconjugates of the present invention can be characterized by a series of assays, including but not limited to polypeptide sequence determination, amino acid analysis, non - denaturing size - exclusion high - performance liquid chromatography (HPLC), mass spectrometry, ion - exchange chromatography, and papain digestion.

[0643] Antigen binding

[0644] The antigen - binding activity of the immunoconjugates of the present invention can be tested by methods known in the art such as ELISA, Western blotting, etc. The binding affinity of the antibody can be determined, for example, by Scatchard analysis as described in Munson et al., Anal Biochem. 107:220 (1980). In addition, the antigen - binding ability of the immunoconjugates of the present invention can be quantified using methods known in the art, such as quantitative ELISA, quantitative Western blotting, surface plasmon resonance assays, and / or Scatchard analysis.

[0645] In one embodiment, the K of the immunoconjugate is measured using radiolabeled antigen ELISA with the immunoconjugate. D . According to another embodiment, K D is measured by using surface plasmon resonance assays, using or instruments (BIAcore, Inc., Piscataway, N.J.), for example, by using an antigen - immobilized CM5 chip at 25 °C and 10 response units.

[0646] In another aspect, competitive binding assays can be used to identify immunoconjugates that competitively bind to the same antigen or its epitope. In some embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) of the immunoconjugates of the invention (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)).

[0647] Methods known to those of ordinary skill in the art can be used to identify or map epitopes and / or contacting residues within the antigen to which the immunoconjugates of the invention bind. Detailed exemplary methods for mapping antibody-binding epitopes are provided in Morris (1996) “Epitope Mapping Protocols,” Methods in Molecular Biology (3rd ed., Humana Press, Totowa, NJ).

[0648] Pharmaceutical compositions and formulations of the present invention

[0649] As will be recognized by those of ordinary skill in the art, certain teachings hereinbelow apply to the immunoconjugates and radioimmunoconjugates of the invention, but the specific text refers to one type of invention, and such applications are generally encompassed within the present invention.

[0650] In another aspect, the present invention provides a composition comprising an immunoconjugate or radioimmunoconjugate of the invention. The present invention also provides pharmaceutical compositions and formulations comprising at least one immunoconjugate of the invention and at least one pharmaceutically acceptable excipient or carrier. In some embodiments, the pharmaceutical formulation comprises (1) an immunoconjugate or radioimmunoconjugate of the invention and (2) a pharmaceutically acceptable carrier.

[0651] The immunoconjugate or radioimmunoconjugate is formulated in any suitable form for delivery to target cells / tissues. The pharmaceutical formulation of the immunoconjugate of the present invention is prepared in the form of a lyophilized preparation or an aqueous solution by mixing such an immunoconjugate having the desired purity with one or more optional pharmaceutically acceptable carriers, diluents, and / or excipients (Remington's Pharmaceutical Sciences, 16th Edition, edited by Osol, A. (1980)). Pharmaceutically acceptable carriers, diluents, and excipients are generally non-toxic to the recipient at the dosages and concentrations employed and include, but are not limited to: sterile water; buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as cetylpyridinium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).

[0652] The pharmaceutical formulation to be used for in vivo administration is generally sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0653] Examples of lyophilized antibody preparations are described in US 6,267,958. Aqueous antibody preparations include those described in US 6,171,586 and WO 2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0654] The pharmaceutically acceptable carriers herein also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20( Baxter International, Inc.). In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

[0655] The formulations of the present invention may also contain, if desired, more than one active ingredient directed against the particular indication being treated, preferably those having complementary activities which do not adversely affect each other. Such active ingredients are present in combination in a manner appropriate and in amounts effective for the intended purpose.

[0656] The active ingredient can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacrylate) microcapsules in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in coarse emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th Edition, A. Osol, Ed. (1980).

[0657] In some embodiments, the immunoconjugate can be formulated as an immunoliposome. An "immunoliposome" is a small vesicle composed of various types of lipids, phospholipids, and / or surfactants which can be used to deliver a drug to a mammal. The components of the liposome are generally arranged in a bilayer, similar to the lipid arrangement of biological membranes. Immunoliposomes containing the immunoconjugate are prepared by methods known in the art, such as those described in Epstein et al., Proc Natl Acad Sci USA 82:3688 (1985); Hwang et al., Proc Natl Acad Sci USA 77:4030 (1980); U.S. Patent Nos. 4,485,045 and 4,544,545; and WO1997 / 38731 published October 23, 1997. Particularly suitable immunoliposomes can be generated by the reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through filters with defined pore sizes to yield liposomes of the desired diameter. The chemotherapeutic agent is optionally included within the liposome (see Gabizon et al., J. National Cancer Inst. 81:1484 (1989)). Liposomes with increased circulation times are disclosed in U.S. Patent No. 5,013,556.

[0658] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, for example, films or microcapsules.

[0659] Methods of using immunoconjugates and radioimmunoconjugates and their compositions

[0660] In one aspect, the present invention provides a method of treating a disease, disorder or condition in a patient in need thereof, the method comprising administering to a subject in need a pharmaceutically effective amount of an immunoconjugate or radioimmunoconjugate or composition of the present invention. For some additional embodiments, the method is for inhibiting cancer cell or tumor growth and / or killing cancer cells or tumors. In another aspect, the present invention provides the use of an immunoconjugate as described herein in the preparation and / or manufacture of a medicament for treating a disease, disorder or condition (such as, for example, cancer) in a subject.

[0661] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the condition of the patient and the type and severity of the patient's disease, but appropriate doses can be determined by clinical trials.

[0662] In one embodiment, the immunoconjugate, or radioimmunoconjugate, or composition of the present invention can be used in a method for binding to a target antigen of an individual suffering from a condition associated with increased expression and / or activity of the target antigen, the method comprising administering to the individual the immunoconjugate, or radioimmunoconjugate, or composition such that the target antigen in the individual is bound. In one embodiment, the target antigen is a human target antigen and the individual is a human individual. The immunoconjugate or radioimmunoconjugate or composition of the present invention can be administered to a human for therapeutic purposes. In addition, the immunoconjugate, or radioimmunoconjugate, or composition of the present invention can be administered to a non-human mammal (e.g., a primate, pig, rat or mouse) that expresses a target antigen with which the immunoconjugate or radioimmunoconjugate cross-reacts, for veterinary purposes or as an animal model of a human disease. With respect to the latter, such animal models can be used to evaluate the therapeutic efficacy of the immunoconjugate or radioimmunoconjugate or composition of the present invention (e.g., testing the administered dose and duration).

[0663] The immunoconjugate or radioimmunoconjugate or composition of the present invention (and any additional therapeutic agent or adjuvant) can be administered by any suitable means, including parenterally, subcutaneously, intraperitoneally, intraluminally and intranasally, and, in cases where local treatment is required, intralesionally. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, antibodies are suitable for administration by pulse infusion, particularly in cases where the antibody dose is tapered. Administration can be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, which depends in part on whether the administration is short-term or long-term.

[0664] The immunoconjugates or radioimmunoconjugates or compositions of the present invention will be formulated, administered, and dispensed in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site to which the agent is to be delivered, the method of administration, the scheduling of administration, and other factors known to the practicing physician. The immunoconjugates of the present invention are administered to human patients according to known methods, such as intravenous administration, for example as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intrathecal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. For some embodiments, intravenous or subcutaneous administration of the immunoconjugates or radioimmunoconjugates or compositions of the present invention is preferred.

[0665] For the prevention or treatment of a disease, the dosage and mode of administration will be selected by the physician in accordance with known criteria. The appropriate dosage of the immunoconjugates or radioimmunoconjugates or compositions of the present invention will depend on the type of disease to be treated (as defined above), the severity and course of the disease, whether the immunoconjugates or radioimmunoconjugates or compositions of the present invention are being administered for prophylactic or therapeutic purposes, previous treatment, the clinical history of the patient, and response to the immunoconjugates or radioimmunoconjugates or compositions, as well as the judgment of the attending physician. The immunoconjugates or radioimmunoconjugates or compositions of the present invention are suitable for administration to a patient either as a single dose or in a series of treatments. Preferably, the immunoconjugates or radioimmunoconjugates or compositions are administered by intravenous infusion or by subcutaneous injection. Depending on the type and severity of the disease, an initial candidate dose of from about 1 μg / kg body weight to about 50 mg / kg body weight (e.g., about 0.1 - 15 mg / kg / dose) of the immunoconjugates or radioimmunoconjugates or compositions may be administered to the patient, either, for example, by one or more separate administrations or by continuous infusion. The dosing regimen may include an initial loading dose of about 4 mg / kg of the immunoconjugates or radioimmunoconjugates or compositions of the present invention, followed by a maintenance dose of about 2 mg / kg of the immunoconjugates or radioimmunoconjugates or compositions of the present invention administered weekly. However, other dosing regimens may be useful. The typical daily dose may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the condition, the treatment is continued until suppression of the desired disease symptoms occurs. The progress of such therapy can be readily monitored by conventional methods and assays and based on criteria known to the physician or other skilled persons in the art.

[0666] The dosage and administration schedule can be selected and adjusted based on the disease level or tolerance of the subject, which can be monitored during the course of treatment. The conjugates of the present invention can be administered once daily, once weekly, multiple times per week but less than once daily, multiple times per month but less than once daily, multiple times per month but less than once weekly, once monthly, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, or intermittently to alleviate or mitigate the symptoms of the disease. Administration can be continued at any of the disclosed intervals until remission of the tumor or the symptoms of the cancer being treated. In cases where remission or mitigation is prolonged by continued administration, administration can be continued after remission or mitigation of the symptoms has been achieved.

[0667] For some embodiments, an effective amount of the immunoconjugate or radioimmunoconjugate or composition can be provided as a single dose.

[0668] The immunoconjugates and radioimmunoconjugates of the present invention can be used in combination with conventional and / or novel treatments or therapies or administered alone as a single therapy. In some embodiments, the immunoconjugates and radioimmunoconjugates of the present invention can be used in combination with one or more radiosensitizers. Such agents include any agent that can increase the sensitivity of cancer cells to radiotherapy. In other embodiments, the immunoconjugates and radioimmunoconjugates of the present invention can be used in combination with novel and / or conventional agents that can enhance the biological effects of radiotherapy. Irradiation of tumors can cause a variety of biological consequences, which can be exploited by combining the immunoconjugates and radioimmunoconjugates of the present invention with agents that target relevant pathways. In some embodiments, such agents can reduce tumor angiogenesis, or inhibit local invasion and metastasis, or prevent repopulation, or enhance the immune response, or deregulate cellular energy, or reduce the population, or alter tumor metabolism, or increase tumor damage, or reduce DNA repair. In certain embodiments, the agents used in combination with the immunoconjugates and radioimmunoconjugates of the present invention can include: DDR inhibitors, e.g., PARP, ATR, Chk1, or DNA-PK; or survival signaling inhibitors, e.g., mTOR, PI3k, NF-kB; or anti-hypoxia agents, e.g., HIF-1-α, CAP, or UPR; or metabolic inhibitors, e.g., MCT1, MCT4 inhibitors; or immunotherapeutic agents, e.g., anti-CTLA4, anti-PD-1; or growth factor signaling inhibitors, e.g., EGFR or MAPK inhibitors; or anti-invasion agents, e.g., kinase inhibitors, chemokine inhibitors, or integrin inhibitors; or anti-angiogenic agents, e.g., VEGF inhibitors.

[0669] The immunoconjugates and radioimmunoconjugates of the present invention can (i) inhibit the growth or proliferation of the cells to which they bind; (ii) induce the death of the cells to which they bind; (iii) inhibit the stratification of the cells to which they bind; (iv) inhibit the metastasis of the cells to which they bind; or (v) inhibit the angiogenesis of tumors containing the cells to which they bind. In this context, "inhibit cell growth or proliferation" means reducing the growth or proliferation of cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%, and includes inducing cell death.

[0670] By way of example, an immunoconjugate that inhibits tumor cell growth is an immunoconjugate that results in a measurable growth inhibition of tumor cells (e.g., cancer cells). In one embodiment, the immunoconjugates or radioimmunoconjugates of the present invention are capable of inhibiting the growth of cancer cells that display the antigen to which the immunoconjugate or radioimmunoconjugate binds. Compared to an appropriate control, a preferred growth-inhibitory immunoconjugate or radioimmunoconjugate inhibits the growth of tumor cells expressing the antigen by more than 20%, preferably about 20% to about 50%, and even more preferably more than 50% (e.g., about 50% to about 100%), where the control is typically tumor cells not treated with the tested immunoconjugate or radioimmunoconjugate.

[0671] For some embodiments, most of the immunoconjugates or radioimmunoconjugates or compositions administered to a subject generally consist of unlabeled immunoconjugates, and a minority consist of labeled radioimmunoconjugates. The ratio of the labeled radioimmunoconjugate to the unlabeled immunoconjugate can be adjusted using known methods. Thus, in certain aspects of the present invention, immunoconjugate / radioimmunoconjugates can be provided having a total protein mass of up to 100 mg, such as less than 60 mg, or 5 mg to 45 mg, or having a total protein mass between 0.1 μg / kg and 1 mg / kg of patient body weight, such as 1 μg / kg to 1 mg / kg of patient body weight, or 10 μg / kg to 1 mg / kg of patient body weig...

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: Wherein: R 1 is a chelating moiety or a radionuclide complex thereof; X 1 is -O-, -S-, -S(=O)-, -S(=O) 2- , -NR a -, -C(=O)-, -NR a C(=O)-, -C(=O)NR a (=O)-, -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-, -(unsubstituted or substituted C 1 -C 6 -alkylene)-X 2 -, -(unsubstituted or substituted C 2 -C 10 -alkenylene)-X 2 -, -(unsubstituted or substituted C 2 -C 10 -alkynylene)-X 2 - or -(C 4 -C 20 -polyethylene glycol)-X 2 -; X 2 is absent, -C(=O)-, -NR a C(=O)-, -C(=O)NR a -, or -C(=O)X 4 -; Each R a is independently selected from hydrogen and C 1 -C 4 alkyl; X 4 is -NR a -, -NR a S(=O) 2 -, -NR a S(=O) 2 NR a -, or one or more independently selected natural or non-natural amino acids, wherein any free amine of the amino acid is optionally and independently substituted by R 5 or -C(=O)R 5 and wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 ; Each R 5 is independently selected from C 1 -C 10 alkyl, C 4 -C 30 polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; L is an optional linker; R 2 is a moiety capable of reacting with the amine (-NH 3 ) or thiol (-SH) of the tumor targeting moiety R 2 ; and v is 1, 2, 3 or 4.

2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, Wherein: R 2 is a moiety capable of reacting with the amine (-NH 3 ) of the tumor targeting moiety R 2 and includes tetrafluorophenyl ester, pentafluorophenyl ester, dinitrophenyl ester, succinimidyl ester, sulfo-succinimidyl ester or isothiocyanate.

3. The compound or a pharmaceutically acceptable salt thereof according to claim 1, Wherein: R 2 is a moiety capable of reacting with the amine (-NH 3 ) of said tumor targeting moiety R 2 and comprises: X is absent, -O-, -S-, -S(=O)-, -S(=O) 2- , -NR a -, -C(=O)-, -NR a C(=O)-, -C(=O)NR a -, -C(=O)O-, -OC(=O)-, -OC(=O)NR a -, -NR a C(=O)NR a -, -NR a C(=S)NR a -, -NR a C(=O)O-; and Each R a is independently selected from hydrogen and C 1 -C 4 alkyl groups.

4. A compound of formula (II), formula (III) or formula (IV) or a pharmaceutically acceptable salt thereof: Wherein: R 1 is a chelating moiety or a radionuclide complex thereof; X 1 is -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-, -(unsubstituted or substituted C 1 -C 6 -alkylene)-X 2 -, -(unsubstituted or substituted C 2 -C 10 -alkenylene)-X 2 -, -(unsubstituted or substituted C 2 -C 10 -alkynylene)-X 2 - or -(C 4 -C 20 -polyethylene glycol)-X 2 -; X 2 is absent, -C(=O)-, -NR a C(=O)-, -C(=O)NR a -, or -C(=O)X 4 -; Each R a is independently selected from hydrogen and C 1 -C 4 alkyl; X 4 is -NR a -, -NR a S(=O) 2 -, -NR a S(=O) 2 NR a -, or one or more independently selected natural or unnatural amino acids, wherein any free amine of the amino acid is optionally independently substituted by R 5 or -C(=O)R 5 and wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 ; Each R 5 is independently selected from C 1 -C 10 alkyl, C 4 -C 30 polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; L is an optional linker; -NH-R 3 is the tumor targeting moiety; and v is 1, 2, 3 or 4.

5. The compound or a pharmaceutically acceptable salt thereof according to claim 1, Wherein: R2 is a moiety capable of reacting with the thiol (-SH) of the tumor targeting moiety R3 and includes a maleimide group, a haloacetamide group, a haloacetyl group, a haloacetic acid group, a pyridylthio group, a vinylcarbonyl group, an aziridinyl group, a disulfide group, an acetylene group, a hydroxysuccinimide group or a thiol group.

6. The compound or a pharmaceutically acceptable salt thereof according to claim 1, Wherein: R 2 is a moiety capable of reacting with the thiol (-SH) of the tumor targeting moiety R 3 and comprises m is 0, 1, 2, 3, 4 or 5.

7. A compound of formula (V), formula (VI), formula (VII) or formula (VIII) or a pharmaceutically acceptable salt thereof: Wherein: R 1 is a chelating moiety or a radionuclide complex thereof; X 1 is -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-, -(unsubstituted or substituted C 1 -C 6 -alkylene)-X 2 -, -(unsubstituted or substituted C 2 -C 10 -alkenylene)-X 2 -, -(unsubstituted or substituted C 2 -C 10 -alkynylene)-X 2 - or -(C 4 -C 20 -polyethylene glycol)-X 2 -; X 2 is absent, -C(=O)-, -NR a C(=O)-, -C(=O)NR a -, or -C(=O)X 4 -; Each R a is independently selected from hydrogen and C 1 -C 4 alkyl; X 4 is -NR a -, -NR a S(=O) 2 -, -NR a S(=O) 2 NR a - or one or more independently selected natural or non-natural amino acids, wherein any free amine of the amino acid is optionally and independently substituted by R 5 or -C(=O)R 5 and wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 alternatively; Each R 5 is independently selected from C 1 -C 10 alkyl, C 4 -C 30 polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; L is an optional linker; -S-R 3 is the tumor targeting moiety; and v is 1, 2, 3 or 4.

8. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, wherein the tumor targeting moiety R 3 is a polypeptide comprising an antigen-binding region and an immunoglobulin heavy chain constant region, wherein the molecular weight of the polypeptide is between 60 and 110 kDa.

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

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

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

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

13. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 8-12, Wherein: The immunoglobulin heavy chain constant region comprises an alteration of one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region or alters the binding of the immunoconjugate to the neonatal Fc receptor (FcRn); or the immunoglobulin heavy chain constant region comprises an alteration of one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region and alters the binding of the immunoconjugate to the neonatal Fc receptor (FcRn).

14. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 8-13, Wherein: The constant region of the immunoglobulin heavy chain comprises alterations to one or more amino acid residues that reduce the effector function of the constant region of the immunoglobulin heavy chain; or the constant region of the immunoglobulin heavy chain comprises alterations to one or more amino acid residues that alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn); or both.

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

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

17. The compound or a pharmaceutically acceptable salt thereof according to claim 4 or any one of claims 8 - 15, wherein the compound of formula (II) has the structure of formula (IIa) or a pharmaceutically acceptable salt thereof: wherein: -NHCH 2 CH 2 CH 2 CH 2 - is the side chain of the lysine residue of the tumor targeting moiety R 3 of 18. The compound or a pharmaceutically acceptable salt thereof according to claim 4 or any one of claims 8 - 15, wherein the compound of formula (III) has the structure of formula (IIIa) or a pharmaceutically acceptable salt thereof: wherein: -NHCH 2 CH 2 CH 2 CH 2 - is the side chain of the lysine residue of the tumor targeting moiety R 3 thereof.

19. The compound or a pharmaceutically acceptable salt thereof according to claim 4 or any one of claims 8 - 15, wherein the compound of formula (II) has the structure of formula (IIa) or a pharmaceutically acceptable salt thereof: wherein: -NHCH 2 CH 2 CH 2 CH 2 - is the side chain of the lysine residue of the tumor targeting moiety R 3 of.

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

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

22. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 7 - 15, wherein the compound of formula (VI) has the structure of formula (VIa) or a pharmaceutically acceptable salt thereof: wherein: -SCH 2 - is the thiol (-SH) of the side chain of the cysteine residue of the tumor targeting moiety R 3 as described above.

23. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 7 - 15, wherein the compound of formula (VII) has the structure of formula (VIIa) or a pharmaceutically acceptable salt thereof: wherein: -SCH 2 - is the thiol (-SH) of the side chain of the cysteine residue of the tumor targeting moiety R 3 as described above.

24. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 7 - 15, wherein the compound of formula (VIII) has the structure of formula (VIIIa) or a pharmaceutically acceptable salt thereof: wherein: -SCH 2 - is the thiol (-SH) of the side chain of the cysteine residue of the tumor targeting moiety R 3 .

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

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

27. The compound according to any one of claims 1-24 or a pharmaceutically acceptable salt thereof, wherein R 1 is a chelating moiety 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'-(((Pyridine-2,6-diylbis(methylene))bis((carboxymethyl)azanediyl))-bis(methylene))dipicolinic acid (H 4 pypa); 6,6',6”,6”'-(((Pyridine-2,6-diylbis(methylene))bis(azanetriyl))tetrakis(methylene))-tetrapicolinic acid (H 4 py4pa); 10-((6-carboxypyridin-2-yl)methyl)-1,4,7,10-tetra-azacyclododecane-1,4,7-triacetic acid (DO3Apic); 3,6,9,12-tetra(carboxymethyl)-3,6,9,12-tetraazatetradecanedioic acid (TTHA); or a radionuclide complex thereof.

28. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-24, wherein R 1 is a chelating moiety selected from the following: or a radionuclide complex thereof.

29. The compound according to any one of claims 1-24, or a pharmaceutically acceptable salt thereof, wherein: R 1 is a chelating moiety selected from the following: or a radionuclide complex thereof.

30. The compound according to any one of claims 1-24, or a pharmaceutically acceptable salt thereof, wherein R 1 is a chelating moiety selected from the following: or a radionuclide complex thereof.

31. The compound according to any one of claims 1-30, or a pharmaceutically acceptable salt thereof, wherein: X 1 is -O-, -S-, -S(=O)-, -S(=O) 2- , -NR a -, -C(=O)-, -NR a C(=O)-, -C(=O)NR a -, -(C 1 -C 6 alkylene)-X 2 - or -(C 4 -C 20 polyethylene glycol)-X 2 -; X 2 is absent, -C(=O)-, -C(=O)NR a -, or -C(=O)X 4 -; X 4 is -NR a -, -NR a S(=O) 2 -, -NR a S(=O) 2 NR a -, or one or more independently selected natural or non-natural amino acids, wherein any free amine of the amino acid is optionally and independently substituted by R 5 or -C(=O)R 5 and wherein any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 alternatively.

32. The compound according to any one of claims 1-30, or a pharmaceutically acceptable salt thereof, wherein: X 1 is absent, -O-, -S-, -S(=O)-, -S(=O) 2- , -NR a -, -C(=O)-, -NR a C(=O)- or -C(=O)NR a -.

33. The compound according to any one of claims 1-30, or a pharmaceutically acceptable salt thereof, wherein: X 1 is absent, -O-, -S-, -NR a -, -C(=O)-, -NR a C(=O)- or -C(=O)NR a -.

34. The compound according to any one of claims 1-30, or a pharmaceutically acceptable salt thereof, wherein: X 1 is -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 -X 2 -, -CH 2 CH 2 -X 2 -, -CH 2 CH 2 CH 2 -X 2 -, -CH 2 CH 2 CH 2 CH 2 -X 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 -X 2 - or -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -X 2 -.

35. The compound according to any one of claims 1-30, or a pharmaceutically acceptable salt thereof, wherein: X 1 is -CH 2 CH 2 - or -CH 2 CH 2 -X 2 -; X 2 is -C(=O)X 4 -; X 4 is -NH-, -N(CH 3 )-, -N(CH 2 CH 3 )-, -NHS(=O) 2 -, -N(CH 3 )S(=O) 2 -, -N(CH 2 CH 3 )S(=O) 2 -, lysine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, serine, tyrosine, valine, citrulline, methionine - valine - lysine, glycine - phenylalanine - glycine - glycine, tyrosine - arginine - valine, arginine - valine or a combination thereof; wherein any free amine (-NH 2 ) of the amino acid is optionally independently substituted by R 5 or -C(=O)R 5 and any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 .

36. The compound according to any one of claims 1-35, or a pharmaceutically acceptable salt thereof, wherein: L is -L 1 -, -L 2 -, -L 3 -, -L 4 -, -L 5 -, -L 1 -L 2 -L 3 -L 4 -L 5 - or a combination thereof; L 1 is an unsubstituted or substituted C 1 -C 10 alkylene, unsubstituted or substituted C 1 -C 10 heteroalkylene, unsubstituted or substituted C 2 -C 20 alkenylene, unsubstituted or substituted C 2 -C 20 alkynylene, C 4 -C 20 polyethylene glycol, -(X 3 CH 2 CH 2 ) t -, unsubstituted or substituted cycloalkylene, unsubstituted or substituted heterocycloalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; Each X 3 is 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 is an unsubstituted or substituted C 1 -C 10 -alkylene, unsubstituted or substituted C 1 -C 10 -heteroalkylene, -C(=O)-(unsubstituted or substituted C 1 -C 10 -alkylene), -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 10 -alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 10 -alkylene)-, -C(=O)-(CH 2 CH 2 X 3 ) m -(CH 2 ) P -, -C(=O)NR 4 -(CH 2 CH 2 X 3 ) m -(CH 2 ) p -, -NR 4 C(=O)-(CH 2 CH 2 X 3 ) m -(CH 2 ) p - or -(CH 2 CH 2 X 3 ) m -(CH 2 ) p ; Each R 4 is independently selected from hydrogen and C 1 -C 6 alkyl; each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; each p is independently 0, 1 or 2; L 3 is absent or is one or more independently selected groups from: natural or unnatural amino acids and optionally amino(unsubstituted or substituted benzyl)carbamate; wherein any free amine (-NH 2 ) of the amino acid is optionally independently substituted by R 5 or -C(=O)R 5 , and any free carboxylic acid of any amino acid is optionally replaced by -C(=O)NH-R 5 , and the substituted benzyl is substituted by 1 or 2 groups selected from halogen, -OH, -OR 5 , -CO 2 H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C 1 -C 6 alkyl, wherein the substituted C 1 -C 6 alkyl is substituted by -OH, -CO 2 H, -NHR 5 , -C(=O)NHR 5 and -NHC(=O)R 5 ; L 4 is an unsubstituted or substituted C 1 -C 10 -alkylene, -C(=O)-(unsubstituted or substituted C 1 -C 10 -alkylene), -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 10 -alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 10 -alkylene)-, -C(=O)-(CH 2 CH 2 X 3 ) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 X 3 ) n -(CH 2 ) q -, -NR 4 C(=O)-(CH 2 CH 2 X 3 ) n -(CH 2 ) q -, -(CH 2 CH 2 X 3 ) n -(CH 2 ) q - or -(X 3 CH 2 CH 2 ) n -; 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 is absent, -C(=O)-(CH 2 ) n -, -C(=O)NR 4 -(CH 2 ) n -, -NR 4 C(=O)-(CH 2 ) n -, -C(=O)-(CH 2 CH 2 X 3 ) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 X 3 ) n -(CH 2 ) q -, -NR 4 C(=O)-(CH 2 CH 2 X 3 ) n -(CH 2 ) q -, -(CH 2 CH 2 X 3 ) n -(CH 2 ) q -, -C(=O)-(X 3 CH 2 CH 2 ) n - or -(X 3 CH 2 CH 2 ) n -; 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 is independently selected from hydrogen and C 1 -C 4 alkyl; Each R b is independently selected from hydrogen and C 1 -C 4 alkyl; wherein the heteroalkylene is an alkylene in which one carbon atom is replaced by -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(=N-R 5 )NR a - or -NR a C(=O)O- substituted alkylene; Wherein when -L 1 -, -L 2 -, -L 3 -, -L 4 - and -L 5 - is substituted by any one of 1 -, -L 2 -, -L 3 -, -L 4 - and -L 5 - is substituted by 1, 2, 3 or 4 groups selected from halogen, -OH, -OR 5 , -CO 2 H, -NHR 5 , -C(=O)NHR 5 , -NHC(=O)R 5 and substituted C 1 -C 6 alkyl groups, wherein the substituted C 1 -C 6 alkyl is substituted by -OH, -CO 2 H, -NHR 5 , -C(=O)NHR 5 and -NHC(=O)R 5 ; Each R 5 is independently selected from C 1 -C 10 alkyl, C 4 -C 30 polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene.

37. The compound according to claim 36, or a pharmaceutically acceptable salt thereof, wherein: A heteroalkylene group is an alkylene group in which one carbon atom is replaced by -O-, -S-, -S(=O)-, -S(=O) 2 -,-S(=O)(=NH)-,-S(=O)(=NR 5 )-,-NR 5 -,-P(=O)OH-,-NHC(=O)-,-C(=O)NH-,-OC(=O)NH-,-NHC(=N-CN)NH- or -NHC(=N-R 5 )NH-.

38. The compound according to claim 36, or a pharmaceutically acceptable salt thereof, wherein: A heteroalkylene group is an alkylene group in which one carbon atom is replaced by -S(=O)(=NH)-, -S(=O)(=NR 5 )-, -P(=O)OH-, -NHC(=N-CN)NH- or -NHC(=N-R 5 )NH-.

39. The compound according to any one of claims 36-38, or a pharmaceutically acceptable salt thereof, wherein: L 1 is unsubstituted or substituted C 1 -C 20 alkylene, unsubstituted or substituted C 1 -C 20 heteroalkylene, C 4 -C 20 polyethylene glycol, unsubstituted or substituted C 3 -C 8 cycloalkylene, unsubstituted or substituted monocyclic C 3 -C 8 heterocycloalkylene, unsubstituted or substituted phenylene, unsubstituted or substituted monocyclic heteroarylene.

40. The compound according to any one of claims 36-38, or a pharmaceutically acceptable salt thereof, wherein: L 1 is unsubstituted or substituted C 1 -C 6 -alkylene, unsubstituted or substituted C 1 -C 10 -heteroalkylene, C 4 -C 20 -polyethylene glycol, unsubstituted or substituted cyclohexylene or unsubstituted or substituted phenylene.

41. The compound according to any one of claims 36-40, or a pharmaceutically acceptable salt thereof, wherein: L 2 is absent, -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 10 -alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 10 -alkylene)-, -C(=O)-(CH 2 CH 2 O) m -(CH 2 ) P -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) P -, -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) P - or -(CH 2 CH 2 O) n -(CH 2 ) P -; each m is independently 1, 2, 3, 4, 5 or 6; each p is independently 1 or 2.

42. The compound according to any one of claims 1-41, or a pharmaceutically acceptable salt thereof, wherein: L 3 is absent or is one or more independently selected groups chosen from: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, tyrosine, valine, and (unsubstituted or substituted benzyl) carbamate; wherein any free amine of the amino acid is optionally substituted by R 5 or -C(=O)(R 5 ) and any free carboxylic acid of the amino acid is optionally replaced by -C(=O)NH(R 5 ).

43. The compound according to any one of claims 1-41 or a pharmaceutically acceptable salt thereof, wherein: L 3 is lysine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, serine, tyrosine, valine, citrulline, methionine-valine-lysine, glycine-phenylalanine-glycine-glycine, tyrosine-arginine-valine, arginine-valine, valine-citrulline-(p-aminobenzyl carbamate) or a combination thereof; wherein any free amine (-NH 2 ) of the amino acid is optionally substituted by -C(=O) (unsubstituted or substituted C 1 -C 20 -alkylene) or -C(=O)-C 4 -C 20 - polyethylene glycol; Any free carboxylic acid (-CO 2 H) of any of the amino acids is optionally replaced by -C(=O)NH-(2,4,6-trimethyl-3-bromophenyl), -C(=O)NH-(unsubstituted or substituted C 1 -C 20 -alkylene) or -C(=O)NH-(C 4 -C 20 -polyethylene glycol).

44. The compound according to any one of claims 1-43 or a pharmaceutically acceptable salt thereof, wherein: L 4 is absent, -C(=O)-(unsubstituted or substituted C 1 -C 6 -alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 6 -alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 6 -alkylene)-, -C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q -, -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q - or -(CH 2 CH 2 O) n -(CH 2 ) q -; each n is independently 1, 2, 3, 4, 5 or 6; each q is independently 1 or 2.

45. The compound according to any one of claims 1-44 or a pharmaceutically acceptable salt thereof, wherein: L 5 is absent, -C(=O)-(CH 2 ) n -, -C(=O)NR 4 -(CH 2 ) n -, -NR 4 C(=O)-(CH 2 ) n -, -C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q -, -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -, -(CH 2 CH 2 O) n -(CH 2 ) q -, -C(=O)-(OCH 2 CH 2 ) n - or -(OCH 2 CH 2 ) n -; each p is independently 0, 1 or 2.

46. The compound according to any one of claims 1-44 or a pharmaceutically acceptable salt thereof, wherein: L 5 is absent, -NR 4 C(=O)-(CH 2 ) n -, -C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q - or -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -; each q is independently 1 or 2.

47. The compound according to any one of claims 36-38 or a pharmaceutically acceptable salt thereof, wherein: L 1 is an unsubstituted or substituted C 1 -C 6 alkylene, unsubstituted or substituted C 1 -C 10 heteroalkylene, C 4 -C 20 polyethylene glycol, unsubstituted or substituted cyclohexylene or unsubstituted or substituted phenylene; L 2 is absent, -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 10 -alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 10 -alkylene)-, -C(=O)-(CH 2 CH 2 O) m -(CH 2 ) P -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) P -, -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) P - or -(CH 2 CH 2 O) n -(CH 2 ) P -; each m is independently 1, 2, 3, 4, 5 or 6; each p is independently 1 or 2; L 4 is absent, -C(=O)-(unsubstituted or substituted C 1 -C 6 -alkylene)-, -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 6 -alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 6 -alkylene)-, -C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q -, -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q - or -(CH 2 CH 2 O) n -(CH 2 ) q -; L 5 is -NR 4 C(=O)-(CH 2 ) n -, -C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q - or -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -; each n is independently 1, 2, 3, 4, 5 or 6; each q is independently 1 or 2.

48. The compound according to any one of claims 36-38 or a pharmaceutically acceptable salt thereof, wherein: L 1 is unsubstituted or substituted C 1 -C 6 -alkylene, unsubstituted or substituted C 1 -C 10 -heteroalkylene, C 4 -C 20 -polyethylene glycol, unsubstituted or substituted cyclohexylene or unsubstituted or substituted phenylene; L 2 is absent, -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 10 -alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 10 -alkylene)-, -C(=O)-(CH 2 CH 2 O) m -(CH 2 ) P -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) P -, -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) P - or -(CH 2 CH 2 O) n -(CH 2 ) P -; each m is independently 1, 2, 3, 4, 5 or 6; each p is independently 1 or 2; L 4 does not exist; L 5 is absent, -NR 4 C(=O)-(CH 2 ) n -, -C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q - or -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -; each n is independently 1, 2, 3, 4, 5 or 6; each q is independently 1 or 2.

49. The compound according to any one of claims 36-38 or a pharmaceutically acceptable salt thereof, wherein: L 1 is unsubstituted or substituted C 1 -C 6 -alkylene, unsubstituted or substituted C 1 -C 10 -heteroalkylene, C 4 -C 20 -polyethylene glycol, unsubstituted or substituted cyclohexylene or unsubstituted or substituted phenylene; L 2 is absent, -C(=O)NR 4 -(unsubstituted or substituted C 1 -C 10 -alkylene)-, -NR 4 C(=O)-(unsubstituted or substituted C 1 -C 10 -alkylene)-, -C(=O)-(CH 2 CH 2 O) m -(CH 2 ) P -、-C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) P -、-NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) P - or -(CH 2 CH 2 O) n -(CH 2 ) P -; each m is independently 1, 2, 3, 4, 5 or 6; each p is independently 1 or 2; L 3 is one or more independently selected groups chosen from: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, tyrosine, valine, and (unsubstituted or substituted benzyl) carbamate; wherein any free amine of the amino acid is optionally substituted by R 5 or -C(=O)(R 5 ) and any free carboxylic acid of the amino acid is optionally replaced by -C(=O)NH(R 5 ); Each R 5 is independently unsubstituted or substituted C 1 -C 10 -alkylene, C 4 -C 20 -polyethylene glycol or unsubstituted or substituted phenyl, wherein the substituted phenyl is substituted by 1, 2, 3, 4 or 5 groups independently selected from F, Cl, Br, I, -CH 3 and CF 3 ; L 4 does not exist; L 5 is -NR 4 C(=O)-(CH 2 ) n -, -C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -, -C(=O)NR 4 -(CH 2 CH 2 O) n -(CH 2 ) q -, or -NR 4 C(=O)-(CH 2 CH 2 O) n -(CH 2 ) q -; each n is independently 1, 2, 3, 4, 5 or 6; each q is independently 1 or 2.

50. The compound according to any one of claims 1-30 or a pharmaceutically acceptable salt thereof, wherein -X 1 -L- is:

51. The compound according to any one of claims 1-30 or a pharmaceutically acceptable salt thereof, wherein is: The compound according to any one of claims 1-30 or a pharmaceutically acceptable salt thereof, wherein is: or a radionuclide complex thereof.

53. The compound according to any one of claims 1-52 or a pharmaceutically acceptable salt thereof, wherein: the radionuclide is a lanthanide or an actinide.

54. The compound according to any one of claims 1-52 or a pharmaceutically acceptable salt thereof, wherein: the radionuclide is actinium, bismuth, cesium, cobalt, copper, dysprosium, erbium, gold, indium, iridium, gallium, lead, lutetium, manganese, palladium, platinum, radium, rhenium, samarium, strontium, technetium, ytterbium, yttrium or zirconium.

55. The compound according to any one of claims 1-52 or a pharmaceutically acceptable salt thereof, wherein: the radionuclide is a diagnostic or therapeutic radionuclide.

56. The compound according to any one of claims 1-52 or a pharmaceutically acceptable salt thereof, wherein: the radionuclide is an Auger electron-emitting radionuclide, an α-emitting radionuclide, a β-emitting radionuclide or a γ-emitting radionuclide.

57. The compound according to any one of claims 1-52 or a pharmaceutically acceptable salt thereof, wherein: the radionuclide is an α-emitting radionuclide.

58. The compound according to any one of claims 1-52 or a pharmaceutically acceptable salt thereof, wherein: The radionuclide is an Auger electron-emitting radionuclide, which is 111-indium ( 111 In), 67-gallium ( 67 Ga), 68-gallium ( 68 Ga), 99m-technetium ( 99m Tc) or 195m-platinum ( 195m Pt).

59. The compound according to any one of claims 1-52 or a pharmaceutically acceptable salt thereof, wherein: The radionuclide is an α-emitting radionuclide, which is actinium-225 ( 225 Ac), bismuth-213 ( 213 Bi), radium-223 ( 223 Ra), or lead-212 ( 212 Pb).

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

61. The compound according to any one of claims 1-52 or a pharmaceutically acceptable salt thereof, wherein: The radionuclide is a γ-emitting radionuclide, which is cobalt-60 ( 60 Co), palladium-103 ( 103 Pd), cesium-137 ( 137 Cs), ytterbium-169 ( 169 Yb), iridium-192 ( 192 Ir), or radium-226 ( 226 Ra). The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-61, wherein: the radionuclide is suitable for positron emission tomography (PET) analysis, single photon emission computed tomography (SPECT), or magnetic resonance imaging (MRI). The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-52, wherein: The radionuclide is 225-actinium ( 225 Ac).

64. A pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-63, and at least one pharmaceutically acceptable excipient.

65. The pharmaceutical composition according to claim 64, wherein the pharmaceutical composition is formulated for administration to a mammal by intravenous administration.

66. A method for treating a tumor in a mammal, comprising administering to the mammal a compound according to any one of claims 1 to 63 so as to treat the tumor.

67. The method according to claim 66, wherein the mammal is a human.

68. The method according to claim 66 or 67, wherein the tumor is a solid tumor.

69. The method according to any one of claims 66 to 68, wherein the tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer.

70. The method according to any one of claims 66 to 69, which comprises administering to the mammal 0.5 μCi to 30.0 μCi / kg.

71. The method according to any one of claims 66 to 69, which comprises administering to the mammal 10 mCi to 75 mCi / square meter of body surface area.

72. The method according to any one of claims 66 to 71, wherein the tumor expresses an antigen specifically bound by the antigen-binding region of the tumor-targeting polypeptide R 3 described above.

73. A method of targeting a radionuclide to a tumor in a mammal, which comprises administering to a mammal having a tumor a compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-63, wherein the tumor expresses an antigen specifically bound by the antigen-binding region of the tumor-targeting polypeptide R 3 to which the antigen-binding region binds specifically.

74. The method according to claim 73, wherein the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-63 comprises a diagnostic or therapeutic radionuclide.

75. A method for identifying a tissue or organ affected by a tumor in a mammal, which comprises: (i) Administering to the mammal a compound as claimed in any one of claims 1 - 63 or a pharmaceutically acceptable salt thereof, and (ii) performing positron emission tomography (PET) analysis, single photon emission computed tomography (SPECT) or magnetic resonance imaging (MRI); wherein the tumour cells express an antigen specifically bound by the antigen - binding region of the tumour - targeting polypeptide R 3 to which the antigen - binding region binds specifically.

76. A method for in vivo imaging of a tissue or organ affected by a tumor in a mammal, which comprises: i) administering to the mammal a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-63; and ii) performing positron emission tomography (PET) analysis, single photon emission computed tomography (SPECT), or magnetic resonance imaging (MRI); wherein the tumor cells express an antigen specifically bound by the antigen-binding region of the tumor-targeting polypeptide R 3 ​ 77. The method according to claim 75 or 76, wherein the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-63 comprises a diagnostic radionuclide.

78. The method according to any one of claims 75-77, wherein: step (ii) is started after a time period sufficient for the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-63 to interact with the antigen expressed on the tumor cells after step (i).

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