DLL3 binding immunoconjugates and uses thereof

Abstract

Described herein are heavy chain antibodies that bind to DLL3 and immunoconjugates of DLL3 heavy chain antibodies useful for cancer therapy.

Description

WSGR Attorney Docket No.: 60924-730.601 DLL3 BINDING IMMUNOCONJUGATES AND USES THEREOF CROSS-REFERENCE

[0001] The present application claims the benefit of U.S. Provisional Application No.63 / 556,334 filed on February 21, 2024, which is incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submittedelectronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 13, 2025, is named 60924-730.601_SL.xml and is 39,430 bytes in size. BACKGROUND

[0003] The exquisite specificity of antibodies, such as IgGs, to their antigens makesantibodies a premier targeting platform for therapeutics; however, the typical serum half-life of at least three weeks for an IgG is disadvantageous for the delivery of radioisotopes including alpha-emitting isotopes such as Ac-225 and beta-emitting isotopes such as Lu-177 and Y-90, in particular due to prolonged exposure and chronic off-target toxicities.225-Ac is among the most cytotoxic of the α-emitting radioisotopes, and a single decay event can effectively destroy a cancer cell 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 the acquired resistance observed in response to other therapies.

[0004] Moreover, there are additional issues for targeted radioscope delivering platforms,including for alpha-emitting and beta-emitting radioisotopes, requiring simultaneous optimization when designing such platforms, such as, e.g., immunogenicity, specificity, tissue penetration, stability, ease of manufacturing, and acceptable therapeutic window. SUMMARY

[0005] Provided and exemplified herein are immunoconjugates comprising an antibody thatbinds DLL3 attached (e.g., conjugated) a compound capable of killing a target cell (e.g., tumor cell expressing DLL3) and having advantageous PD / PK profiles. In certain instances, such advantageous PD and PK properties include faster body clearance, decreased liver accumulation and / or faster liver clearance, reduced accumulation in the kidney, bone or bone marrow, and / or increased tumor accumulation. In such instances, the PD and PK properties of the immunoconjugates provided herein are advantageous for improving clinical safety and / orWSGR Attorney Docket No.: 60924-730.601 efficacy profiles. In other embodiments, the immunoconjugates provided herein are also useful for the detection and labeling of target cells (e.g., a tumor cell expressing DLL3).

[0006] In some embodiments, provided herein are immunoconjugates comprising anantibody that binds DLL3 attached (e.g., conjugated) to a compound, wherein: the antibody comprises a VHH variable domain, wherein the VHH variable domain comprises: a heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence of SEQ ID NO: 14, the heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 15, and a heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence of SEQ ID NO: 16; a heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence of SEQ ID NO: 17, the heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 18, and the heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 19; a heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence of SEQ ID NO: 20, a heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence of SEQ ID NO: 21, and a heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence of SEQ ID NO: 22; a heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence of SEQ ID NO: 23, a heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence of SEQ ID NO: 24, and a heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence of SEQ ID NO: 25; or a heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence of SEQ ID NO: 26, a heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence of SEQ ID NO: 27, and a heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence of SEQ ID NO: 28; and the compound comprises a structure represented by Formula (I) or a pharmaceutically acceptable salt thereof:wherein: R1is a chelating moiety or a radionuclide complex thereof;WSGR Attorney Docket No.: 60924-730.601 X1is -O-, -S-, -S(=O)-, -S(=O)2-, -NRa-, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, -(C1-C6alkylene)- X2-, or -(C4-C20polyethylene glycol)-X2-; X2is absent, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, or -C(=O)X4-; each Rais independently selected from hydrogen, and C1-C4alkyl; X4is -NRa-, or -NRaS(=O)2-;L is an optional linker; R2is a moiety that is capable of reacting with an amine (-NH2) or thiol (-SH) of the antibody; and v is 1, 2, 3, or 4. ,, - OC(=O)-, -OC(=O)NRa-, -NRaC(=O)NRa-, -NRaC(=S)NRa-, -NRaC(=O)O-; and each Rais independently selected from hydrogen, and C1-C4alkyl.

[0007] In some embodiments, provided herein are immunoconjugates comprising anantibody that binds DLL3 attached (e.g., conjugated) to a compound, wherein: the antibody comprises a VHH variable domain, wherein the VHH variable domain comprises: a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 14, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 15, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 16; a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 17, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 18, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 19; a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 20, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 21, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 22; a heavy chainWSGR Attorney Docket No.: 60924-730.601 complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 23, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 24, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 25; or a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 26, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 27, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 28; and the compound comprises a structure represented by Formula (II), Formula (III), or Formula (IV), or a pharmaceutically acceptable salt thereof:wherein: R1is a chelating moiety or a radionuclide complex thereof; X1is -O-, -S-, -S(=O)-, -S(=O)2-, -NRa-, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, -(C1-C6alkylene)- X2-, or -(C4-C20polyethylene glycol)-X2-; X2is absent, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, or -C(=O)X4-; each Rais independently selected from hydrogen, and C1-C4alkyl; X4is -NRa-, or -NRaS(=O)2-; L is an optional linker;WSGR Attorney Docket No.: 60924-730.601 -NH-R3is the antibody; and v is 1, 2, 3, or 4.

[0008] In some embodiments, provided herein are immunoconjugates comprising anantibody that binds DLL3 attached (e.g., conjugated) to a compound, wherein: the antibody comprises a VHH variable domain, wherein the VHH variable domain comprises: a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 14, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 15, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 16; a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 17, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 18, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 19; a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 20, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 21, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 22; a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 23, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 24, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 25; or a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 26, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 27, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 28; and the compound comprises a structure represented by Formula (V), Formula (VI), Formula (VII), or Formula (VIII), or a pharmaceutically acceptable salt thereof:WSGR Attorney Docket No.: 60924-730.601wherein: R1is a chelating moiety or a radionuclide complex thereof; X1is -O-, -S-, -S(=O)-, -S(=O)2-, -NRa-, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, -(C1-C6alkylene)- X2-, or -(C4-C20polyethylene glycol)-X2-; X2is absent, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, or -C(=O)X4-; each Rais independently selected from hydrogen, and C1-C4alkyl; X4is -NRa-, or -NRaS(=O)2-; L is an optional linker; -S-R3is the antibody; and

[0009] In certain embodiments, R1 is a chelating moiety or a radionuclide complex thereof,wherein the chelating moiety is: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); 1,4,7,10-tetraazacyclododecane-1,4,7 -triacetic acid (DO3A); 1,4,7,10- tetraazacyclododecane-1,7-diacetic acid (DO2A); α,α',α'',α'''-tetramethyl-1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTMA); 1,4,7,10-tetrakis(carbamoylmethyl)- 1,4,7,10-tetraazacyclododecane (DOTAM); 1,4,7,10-tetraazacyclododecane-1,4,7,10- tetrapropionic acid (DOTPA); 2,2',2''-(10-(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-WSGR Attorney Docket No.: 60924-730.601 1,4,7-triyl)triacetic acid; 6,6'-(((pyridine-2,6- diylbis(methylene))bis((carboxymethyl)azanediyl))-bis(methylene))dipicolinic acid (H4pypa); 6,6',6'',6'''-(((pyridine-2,6-diylbis(methylene))bis(azanetriyl))tetrakis(methylene))-tetrapicolinic acid (H4py4pa); 10-((6-carboxypyridin-2-yl)methyl)-1,4,7,10-tetra-azacyclododecane-1,4,7- triacetic acid (DO3Apic); or 3,6,9,12-tetrakis(carboxymethyl)-3,6,9,12-tetraazatetradecanedioic acid (TTHA).

[0010] In certain embodiments, X1-L-is:.,or a

[0013] In certain embodiments, the radionuclide is a diagnostic or therapeutic radionuclide.In certain embodiments, the radionuclide is an Auger electron-emitting radionuclide, α-emitting radionuclide, β-emitting radionuclide, or γ-emitting radionuclide. In certain embodiments, the radionuclide is α-emitting radionuclide. In certain embodiments, the radionuclide is an AugerWSGR Attorney Docket No.: 60924-730.601electron-emitting radionuclide that is 111-indium (111In), 67-gallium (67Ga), 68-gallium (68Ga),99m-technetium (99mTc), or 195m-platinum (195mPt).; or the radionuclide is an α-emitting radionuclide that is 225-actinium (225Ac), 213-bismuth (213Bi), 223-Radium (223Ra), or 212-lead (212Pb). or the radionuclide is a β-emitting radionuclide that is 90-yttrium (90Y), 177-lutetium (177Lu), 186-rhenium (186Re), 188-rhenium (188Re), 64-copper (64Cu), 67-copper (67Cu), 153- samarium (153Sm), 89-strontium (89Sr), 198-gold (198Au), 169-Erbium (169Er), 165-dysprosium (165Dy), 99m-technetium (99mTc), 89-zirconium (89Zr), or 52-manganese (52Mn); or the radionuclide is a γ-emitting radionuclide that is 60-cobalt (60Co), 103-palldium (103Pd), 137- cesium (137Cs), 169-ytterbium (169Yb), 192-iridium (192Ir), or 226-radium (226Ra). In certain embodiments, the radionuclide is suitable for positron emission tomography (PET) analysis, single-photon emission computerized tomography (SPECT), or magnetic resonance imaging (MRI). In certain embodiments, the radionuclide is 225-actinium (225Ac).

[0014] In certain embodiments, the antibody further comprises an Fc domain. In certainembodiments, the Fc domain comprises an immunoglobulin CH2 domain, immunoglobulin CH3 domain, or both an immunoglobulin CH2 and immunoglobulin CH3 domain. In certain embodiments, the Fc domain comprises both the immunoglobulin CH2 and the immunoglobulin CH3 domain. In certain embodiments, the Fc domain is an IgA, IgG1, IgG2, IgG3, or IgG4 isotype. In certain embodiments, the Fc domain is an IgG1 isotype. In certain embodiments, Fc domain is an IgG4 isotype. In certain embodiments, the Fc domain comprises an alteration to one or more amino acid residues that reduces an effector function of the Fc domain or alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn) (e.g., relative to wild-type IgG1).

[0015] In certain embodiments, the Fc domain comprises an alteration to one or more aminoacid residues that reduces an effector function of the Fc domain and an alteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) (e.g., relative to wild-type IgG1). In certain embodiments, the Fc domain comprises an alteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) (e.g., relative to wild-type IgG1). In certain embodiments, Fc domain comprises an alteration to one or more amino acid residues that reduces an effector function of the Fc domain.

[0016] In certain embodiments, the Fc domain comprises an alteration to one or more aminoacid residues that reduces the effector function, wherein the alteration reduces complement dependent cytotoxicity (CDC), antibody-dependent cell-cytotoxicity (ADCC), antibody- dependent cell-phagocytosis ADCP, or a combination thereof (e.g., relative to wild-type IgG1).WSGR Attorney Docket No.: 60924-730.601 In certain embodiments, the Fc domain comprises an alteration to one or more amino acid residues that reduces the effector function of the Fc domain (e.g., relative to wild-type IgG1), wherein the alteration comprises L234A, L235E, G237A, A330S, and P331S per EU numbering. In certain embodiments, the Fc domain comprises an alteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) reduces the serum half-life of the immunoconjugate. In certain embodiments, the Fc domain comprises an alteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn), wherein the alteration comprises H310A, H435Q, or both H310A and H435Q per EU numbering. In certain embodiments, the Fc domain comprises an alteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn), wherein the alteration comprises H435Q. In certain embodiments, the Fc domain comprises an alteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn), wherein the alteration comprises H310A. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1A shows SHP-77 cell binding of humanized VHHFcs.

[0018] FIG. 1B shows SHP-77 cell internalization of humanized VHHFcs.

[0019] FIG. 2 shows a representative schematic of a VHHFc immunoconjugate.

[0020] FIG. 3A shows whole body SPECT / CT imaging at 1 hour post injection showingbiodistribution of mice injected with a 111In-labeled immunoconjugate comprising 126_zu2 and compound 3-16. Accumulation / presence in liver observable at 1 hour post injection.

[0021] FIG. 3B shows whole body SPECT / CT imaging at 24 hours post injection showingbiodistribution of mice injected with a 111In-labeled immunoconjugate comprising 126_zu2 and compound 3-16. Reduced liver distribution is observed at 24 hours post injection.

[0022] FIG. 3C shows whole body SPECT / CT imaging at 72 hours post injection showingbiodistribution of mice injected with a 111In-labeled immunoconjugate comprising 126_zu2 and compound 3-16. Minimal and / or substantially no liver accumulation is observed at 72 hours post-injection.

[0023] FIG. 3D shows whole body SPECT / CT imaging at 144 hours post injection showingbiodistribution of mice injected with a 111In-labeled immunoconjugate comprising 126_zu2 and compound 3-16. Substantially no liver accumulation is observed at 144 hours post-injection.WSGR Attorney Docket No.: 60924-730.601

[0024] FIG. 3E shows whole body SPECT / CT imaging at 240 hours post injection showingbiodistribution of mice injected with a 111In-labeled immunoconjugate comprising 126_zu2 and compound 3-16. Substantially no liver accumulation is observed at 240 hours post-injection.

[0025] FIG. 4 shows blood concentration vs. time following single bolus IV administrationof111In-126_zu2(compound 3-16) in naïve human FcRn transgenic (Tg32) mice.

[0026] FIG. 5 shows SPECT / CT derived whole-body clearance following single bolus IVadministration of111In-126_zu2(compound 3-16) in naïve human FcRn transgenic (Tg32) mice.

[0027] FIG. 6 shows SPECT / CT tissue biodistribution after a single bolus IV administrationof111In-126_zu2(compound 3-16) in naïve human FcRn transgenic (Tg32) mice.

[0028] FIG. 7 shows cumulative urinary and fecal excretion following a single bolus IVadministration of111In-126_zu2(compound 3-16) in naïve human FcRn transgenic (Tg32) mice.

[0029] FIG. 8 shows %Cmax vs. time profiles following a single bolus IV administration of111In-126_zu2(3-16) in naïve human FcRn transgenic (Tg32) mice.

[0030] FIG. 9 shows blood concentration vs time profiles following a single bolus IVadministration of111In-126_Zu2(compound 3-16) and225Ac-126_Zu2(compound 3-16) in naïve female CD-1 mice.

[0031] FIG. 10 shows tissue biodistribution of 111In-126_zu2(compound 3-16) following asingle bolus IV administration in naïve female CD-1 mice.

[0032] FIG. 11 shows tissue biodistribution of 225Ac-126_zu2(compound 3-16) following asingle bolus IV administration in naïve female CD-1 mice.

[0033] FIG. 12 shows blood concentration vs. time profiles following a single bolus IVadministration of111In-126_zu2(compound 3-16) in naïve male and female cynomolgus monkey.

[0034] FIG. 13 shows SPECT / CT derived whole-body 111In-126_zu2(compound 3-16)clearance following a single IV bolus administration in naïve male and female cynomolgus monkey.

[0035] FIG. 14 shows SPECT / CT biodistribution of 111In-126_zu2(compound 3-16)following a single IV bolus administration in naïve male and female cynomolgus monkey. DETAILED DESCRIPTIONWSGR Attorney Docket No.: 60924-730.601

[0036] Described and exemplified herein are immunoconjugates comprising an antibody thatbinds DLL3 attached (e.g., conjugated) to a compound capable of killing a target cell (e.g., tumor cell expressing DLL3) and having advantageous PD / PK profiles. In certain instances, such advantageous PD and PK properties include faster body clearance, decreased liver accumulation and / or faster liver clearance, reduced accumulation in the bone or bone marrow and kidney, and / or increased tumor accumulation. In other embodiments, the immunoconjugates provided herein are also useful for the detection and labeling of target cells (e.g., a tumor cell expressing DLL3). Antibodies that bind DLL3

[0037] The immunoconjugates provided and exemplified herein generally comprise anantibody that binds DLL3 attached to a compound, wherein the antibody comprises a variable domain (e.g., VHH). In certain embodiments, the immunoconjugate further comprises an Fc domain. In certain instances, the immunoconjugates described here can be used with or without the Fc domain.

[0038] Antibody generally refers to and / or includes single variable domain antibodies (e.g.,a VHH) that bind a DLL3 protein and comprises an Fc domain (collectively referred to as a single domain antibody). Antigen-binding fragments of antibodies (antibody fragments) generally refer to and / or include antibody-derived proteins that comprise a functional set of CDRs (e.g., the CDRH1-3 sequences described herein) that bind a DLL3 protein. The single domain antibodies provided and described herein generally have a molecule weight less than a full length human IgG antibody (e.g., a molecular weight less than ~150,00 Daltons). Antibodies and antigen-binding fragments of antibodies generally encompass genetically engineered, and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multi-specific antibodies, multi-valent antibodies, diabodies, triabodies, and tetrabodies.

[0039] Complementarity determining regions (CDRs) are synonymous with hypervariableregions (HVRs) generally refer to and / or include non-contiguous sequences of amino acids within antibody variable regions that confer antigen specificity and / or binding affinity (e.g., to DLL3). In general, for the single domain antibodies described herein (e.g., VHH-Fcs), there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3). Framework regions (FRs) generally refer to and / or include non-CDR regions of the heavy and light chain variable regions. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4).WSGR Attorney Docket No.: 60924-730.601

[0040] Variable regions (also referred to as variable domains) generally refer to and / orinclude a single variable domain that is involved in binding the antibody to antigen (e.g., a single variable domain comprises a CDR 1, CDR 2, and CDR 3). The VHH variable domains generally have similar structures comprising four conserved framework regions (FRs) and three CDRs As described herein, a single variable domain is sufficient to confer antigen-binding specificity (e.g., binding to DLL3).

[0041] In some embodiments, the VHH variable domain comprises: a heavy chaincomplementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 14, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 15, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, the VHH variable domain comprises: a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 17, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 18, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 19. In some embodiments, the VHH variable domain comprises: a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 20, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 21, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 22. In some embodiments, the VHH variable domain comprises: a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 23, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 24, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 25. In some embodiments, the VHH variable domain comprises: a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 26, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 27, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 28.

[0042] Variable domain sequences having the CDRH1-3 sequences described herein are alsoprovided. In certain embodiments, the VHH variable domain comprises an amino acid sequence having at least 85%, 90%, 95%, 98, 99% or greater sequence identity to SEQ ID NO: 29. In certain embodiments, the VHH variable domain comprises an amino acid sequence having atWSGR Attorney Docket No.: 60924-730.601 least 85%, 90%, 95%, 98, 99% or greater sequence identity to SEQ ID NO: 30. In certain embodiments, the VHH variable domain comprises an amino acid sequence having at least 85%, 90%, 95%, 98, 99% or greater sequence identity to SEQ ID NO: 31. In certain embodiments, the VHH variable domain comprises an amino acid sequence having at least 85%, 90%, 95%, 98, 99% or greater sequence identity to SEQ ID NO: 32. In certain embodiments, the VHH variable domain comprises an amino acid sequence having at least 85%, 90%, 95%, 98, 99% or greater sequence identity to SEQ ID NO: 33. In certain embodiments, the VHH variable domain comprises an amino acid sequence having at least 85%, 90%, 95%, 98, 99% or greater sequence identity to SEQ ID NO: 34. Fc Domains

[0043] The antibodies of the immunoconjugates described herein comprise an Fc domain.An Fc domain generally encompasses and / or refers to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region, such as an immunoglobulin CH2 and CH3 domain. The term includes native sequence Fc regions and variant Fc regions. For example, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In certain embodiments, the Fc region include IgG and sub-classes thereof (e.g., IgG1 and IgG4), IgM, IgE, IgA, and / or IgD heavy chain constant regions and / or heavy chain constant regions derived derived from IgG and sub-classes thereof (e.g., IgG1 and IgG4), IgM, IgE, IgA, and IgD.

[0044] In some embodiments, the Fc domain comprises an immunoglobulin (e.g., IgG1)CH2 domain, and immunoglobulin CH3 domain, or both of an immunoglobulin CH2 and CH3 domain. In certain embodiments, the Fc domain comprises an immunoglobulin CH2 and CH3 domain. The Fc domain is attached(e.g., linked or fused) to the VHH variable domain (e,g., VHH). In certain embodiments, the Fc domain is attached to the VHH variable domain via a hinge sequence (e.g., SEQ IS NO: 10). In certain embodiments, the Fc domain is attached to the VHH variable domain by an amino acid sequence having 70%, 80%, 90%, or greater sequence identity to SEQ ID NO: 11. In certain embodiments, the Fc domain is attached to the VHH variable domain by the amino acid sequence of SEQ ID NO: 11. Generally, the antibodies described herein have the format variable domain – hinge – Fc domain, wherein the hinge sequence is included as part of the Fc domain sequence.WSGR Attorney Docket No.: 60924-730.601

[0045] The antibodies that bind DLL3 are further useful in that they comprises one or morealterations (e.g., modification and / or mutation) within the Fc domain that reduce binding to FcRn. The alterations to Fc domains of the immunoconjugate can reduce the serum half-life of the immunoconjugate. In certain embodiments, the amino acid alteration that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) reduces the serum half-life of the immunoconjugate. In certain embodiments, the alteration that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 251, 252, 253, 254, 255, 288, 309, 310, 312, 385, 386, 388, 400, 415, 433, 435, 436, 439, 447, and combinations thereof per EU numbering. In certain embodiments, the alteration that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 253, 254, 310, 435, 436 and combinations thereof per EU numbering. In certain embodiments, the alteration that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: I253A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof per EU numbering. In certain embodiments, the alteration that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: I253A, S254A, H310A, H435Q, Y436A and combinations thereof per EU numbering. In certain embodiments, the alteration that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: I253A, H310A, H435Q, and combinations thereof per EU numbering. In certain embodiments, the alteration that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: H310A, H435Q, and combinations thereof per EU numbering.

[0046] In some embodiments, this disclosure contemplates a variant of an immunoconjugateof this disclosure that comprises a Fc region wherein the variant possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the immunoconjugate in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the immunoconjugate lacks FcγγR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcγγRIII only, whereas monocytes express FcγγRI, FcγγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol.9:457-492 (1991). Non-WSGR Attorney Docket No.: 60924-730.601 limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in US 5,500,362 (see e.g. Hellstrom, I. et al. Proc Natl Acad Sci USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc Natl Acad Sci USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non- radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc Natl Acad Sci USA 95:652-656 (1998). Clq binding assays may also be carried out to confirm that the immunoconjugate is unable to bind Clq and hence lacks CDC activity (see e.g., Clq and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402). To assess complement activation, a CDC assay may be performed (see e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, 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 determinations can also be performed using methods known in the art (see e.g., Petkova, S.B. et al., Int'l. Immunol.18(12): 1759-1769 (2006)).

[0047] The immunoglobulin heavy chain constant region can be a variant constant regionthat comprises one or more alterations to an amino acid residue present in a wild type constant region that confers additional utility and advantageous properties to the immunoconjugates described herein. In certain embodiments, the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region or alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn).In certain embodiments, the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region and reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region. In certain embodiments, the immunoglobulin heavy chain constant regionWSGR Attorney Docket No.: 60924-730.601 comprises an alteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn).

[0048] The alterations to heavy chain constant regions of the immunoconjugate can reduceeffector function associated with a heavy chain constant region, such as, the ability to fix complement, promote phagocytosis, or recruit other immune effector cells (e.g., NK cells) to the heavy chain constant region. In certain embodiments, the alteration to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region is an alteration that reduces complement dependent cytotoxicity (CDC), antibody-dependent cell- cytotoxicity (ADCC), antibody-dependent cell-phagocytosis ADCP, or a combination thereof. In certain embodiments, the alteration to 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: (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), per EU numbering. 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 per EU numbering.

[0049] In some embodiments, the Fc domain comprises an amino acid sequence having atleast 85%, 90%, 95%, 98, 99% or greater sequence identity to SEQ ID NO: 13. In someWSGR Attorney Docket No.: 60924-730.601 embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the Fc domain comprises an amino acid sequence having at least 85%, 90%, 95%, 98, 99% or greater sequence identity to SEQ ID NO: 12. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 12.

[0050] In some embodiments, the Fc domain comprises a 435Q, C220S, L234A, L235E,G237A, A330S, and P331S per EU numbering, and has an amino acid sequence having at least 85%, 90%, 95%, 98, 99% or greater sequence identity to SEQ ID NO: 13. In some embodiments, the Fc domain comprises a 435Q, L234A, L235E, G237A, A330S, and P331S per EU numbering, and has an amino acid sequence having at least 85%, 90%, 95%, 98, 99% or greater sequence identity to SEQ ID NO: 13. In some embodiments, the Fc domain comprises a 435Q per EU numbering, and has an amino acid sequence having at least 85%, 90%, 95%, 98, 99% or greater sequence identity to SEQ ID NO: 13. In some embodiments, the Fc domain comprises reduced binding to FcRn, reduces effector function, and has an amino acid sequence having at least 85%, 90%, 95%, 98, 99% or greater sequence identity to SEQ ID NO: 13.

[0051] In some embodiments, the antibody comprises an amino acid sequence having atleast 85%, 90%, 95%, 98, 99% or greater sequence identity to SEQ ID NO: 35. In some embodiments, the Fc domain comprises an amino acid sequence having at least 85%, 90%, 95%, 98, 99% or greater sequence identity to SEQ ID NO: 36. In some embodiments, the Fc domain comprises an amino acid sequence having at least 85%, 90%, 95%, 98, 99% or greater sequence identity to SEQ ID NO: 37. In some embodiments, the Fc domain comprises an amino acid sequence having at least 85%, 90%, 95%, 98, 99% or greater sequence identity to SEQ ID NO: 38. In some embodiments, the Fc domain comprises an amino acid sequence having at least 85%, 90%, 95%, 98, 99% or greater sequence identity to SEQ ID NO: 39.

[0052] In some embodiments, the antibody comprises the amino acid sequence of SEQ IDNO: 35. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO: 39.

[0053] As provided and exemplified herein, immunoconjugates comprising an antibodyhaving a molecular weight less than that of a standard human IgG1 antibody (e.g., ~150,000 Daltons) are useful for promoting tumor accumulation within a target tissue / organ (e.g., in the kidney).Accordingly, in some embodiments, the antibody comprises used in the immunoconjugates described herein have a molecular weight of less than 150,000 Daltons. In certain embodiments, the antibody has a molecular weight less than 140,000 Daltons. In certainWSGR Attorney Docket No.: 60924-730.601 embodiments, the antibody has a molecular weight less than 130,000 Daltons. In certain embodiments, the antibody has a molecular weight less than 125,000 Daltons. In certain embodiments, the antibody has a molecular weight less than 120,000 Daltons. In certain embodiments, the antibody has a molecular weight less than 110,000 Daltons. In certain embodiments, the antibody has a molecular weight less than 100,000 Daltons.

[0054] In certain embodiments, the antibody has a molecular weight between 60,000Daltons and 125,000 Daltons. In certain embodiments, the antibody has a molecular weight between 60,000 Daltons and 120,000 Daltons. In certain embodiments, the antibody has a molecular weight between 60,000 Daltons and 110,000 Daltons. In certain embodiments, the antibody has a molecular weight between 60,000 Daltons and 100,000 Daltons. Immunoconjugates

[0055] The immunoconjugates provided and exemplified herein generally comprise anantibody that binds DLL3 attached to a compound, wherein the compound comprises a linker and chelator that, in certain instances, gives rise to advantageous PD / PK properties for killing a tumor cell when complexed with a radionuclide. Accordingly, in some embodiments, provided herein are immunoconjugates comprising an antibody that binds DLL3 attached to a compound. In some embodiments, the immunoconjugate binds DLL3 with a KD of ≤ 1 μM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g.10-8M or less, e.g. from 10-8M to 10-13M, e.g., from 10-9M to 10-13M). In certain embodiments, the immunoconjugate binds DLL3 with a KDof 10 nanomolar or less. In certain embodiments, the immunoconjugate binds DLL3 with a KD of 5 nanomolar or less. In certain embodiments, the immunoconjugate binds DLL3 with a KD of 2 nanomolar or less. In certain embodiments, the immunoconjugate binds DLL3 with a KD of 1 nanomolar or less. In certain embodiments, the immunoconjugate binds DLL3 with a KD of 0.1 nanomolar or greater. In certain embodiments, the immunoconjugate binds DLL3 with a KDof 0.5 nanomolar or greater. Compounds

[0056] In some embodiments, the compound comprises a structure represented by Formula(I) or a pharmaceutically acceptable salt thereof: :WSGR Attorney Docket No.: 60924-730.601R1is a chelating moiety or a radionuclide complex thereof; X1is -O-, -S-, -S(=O)-, -S(=O)2-, -NRa-, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, -(C1-C6alkylene)-X2-, or -(C4-C20polyethylene glycol)-X2-; X2is absent, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, or -C(=O)X4-; each Rais independently selected from hydrogen, and C1-C4alkyl; X4is -NRa-, or -NRaS(=O)2-;L is an optional linker; R2is a moiety that is capable of reacting with an amine (-NH2) or thiol (- SH) of the antibody; and v is 1, 2, 3, or 4.

[0057] In certain embodiments, R2 is a moiety that is capable of reacting with an amine (-NH2) of the antibody and comprises a tetrafluorophenyl ester, pentafluorophenyl ester, dinitrophenyl ester, succinimide ester, sulfosuccinimide ester, or isothiocyanate. In certain embodiments, R2is a moiety that is capable of reacting with an amine (-NH2) of the antibody and comprises: , or-, -C(=O)O-, -OC(=O)-, -OC(=O)NRa-, -NRaC(=O)NRa-, -NRaC(=S)NRa-, - NRaC(=O)O-; and each Rais independently selected from hydrogen, and C1-C4alkyl.WSGR Attorney Docket No.: 60924-730.601

[0058] In some embodiments, the compound comprises a structure represented by Formula(II), Formula (III), or Formula (IV), or a pharmaceutically acceptable salt thereof:Formula (III) wherein:R1is a chelating moiety or a radionuclide complex thereof; X1is -O-, -S-, -S(=O)-, -S(=O)2-, -NRa-, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, -(C1-C6alkylene)-X2-, or -(C4-C20polyethylene glycol)-X2-; X2is absent, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, or -C(=O)X4-; each Rais independently selected from hydrogen, and C1-C4alkyl; X4is -NRa-, or -NRaS(=O)2-; L is an optional linker; -NH-R3is the antibody; and v is 1, 2, 3, or 4.

[0059] In certain embodiments, R2 is a moiety that is capable of reacting with a thiol (-SH)of the antibody R3and comprises a maleimide group, a haloacetamide group, a haloacetyl group, a haloacetate group, a pyrdinylthio group, a vinylcarbonyl group, an aziridinyl group, a disulfide group, an acetylene group, a hydroxysuccinimide group or a thiol group. In certain embodiments, R2is a moiety that is capable of reacting with a thiol (-SH) of the antibody R3and comprises:WSGR Attorney Docket No.: 60924-730.601 , ,

[0060] In a(IIa), or a pharmaceutically acceptable salt thereof:wherein: NHCH2CH2CH2CH2-is the side chain of a lysine residue of the antibody R3

[0061] In some embodiments, the compound comprises a structure represented by Formula(V), Formula (VI), Formula (VII), or Formula (VIII), or a pharmaceutically acceptable salt thereof:WSGR Attorney Docket No.: 60924-730.601 Formula (VI)wherein: R1is a chelating moiety or a radionuclide complex thereof; X1is -O-, -S-, -S(=O)-, -S(=O)2-, -NRa-, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, -(C1-C6alkylene)-X2-, or -(C4-C20polyethylene glycol)-X2-; X2is absent, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, or -C(=O)X4-; each Rais independently selected from hydrogen, and C1-C4alkyl; X4is -NRa-, or -NRaS(=O)2-; L is an optional linker; -S-R3is the antibody; and v is 1, 2, 3, or 4.

[0062] In some embodiments, X1 is absent, -O-, -S-, -NRa-, -C(=O)-, -NRaC(=O)-, or -C(=O)NRa-; or X1is -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, - CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -CH2-X2-, -CH2CH2-X2-, -CH2CH2CH2-X2-, -CH2CH2CH2CH2-X2-, -CH2CH2CH2CH2CH2-X2-, or -CH2CH2CH2CH2CH2CH2-X2-. In some embodiments, X1is -CH2CH2-or -CH2CH2-X2-; X2is -C(=O)X4-; X4is -NH-, -N(CH3)-, or - N(CH2CH3)-.

[0063] In some embodiments, L is -L1-, or -L1-L2-L3-L4-L5-; L1 is unsubstituted orsubstituted C1-C20alkylene, unsubstituted or substituted C1-C20heteroalkylene, C4- C20polyethylene glycol, unsubstituted or substituted C3-C8cycloalkylene, unsubstituted or substituted monocyclic C3-C8heterocycloalkylene, unsubstituted or substituted phenylene, unsubstituted or substituted monocyclic heteroarylene; L2is absent, -C(=O)NR4-(unsubstituted or substituted C1-C10alkylene)-, -NR4C(=O)-(unsubstituted or substituted C1-C10alkylene)-, - C(=O)-(CH2CH2O)m-(CH2)P-, -C(=O)NR4-(CH2CH2O)n-(CH2)P-, -NR4C(=O)-(CH2CH2O)n- (CH2)P-, or -(CH2CH2O)n-(CH2)P-; each R4is independently selected from hydrogen, and C1- C6alkyl; each m is independently 1, 2, 3, 4, 5, or 6; each p is independently 1, or 2; L3is absent;WSGR Attorney Docket No.: 60924-730.601 L4is absent, -C(=O)-( unsubstituted or substituted C1-C6alkylene)-, -C(=O)NR4-(unsubstituted or substituted C1-C6alkylene)-, -NR4C(=O)-(unsubstituted or substituted C1-C6alkylene)-, - C(=O)-(CH2CH2O)n-(CH2)q-, -C(=O)NR4-(CH2CH2O)n-(CH2)q-, -NR4C(=O)-(CH2CH2O)n- (CH2)q-, or -(CH2CH2O)n-(CH2)q-; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2;L5is absent, -C(=O)-(CH2)n-, -C(=O)NR4-(CH2)n-, -NR4C(=O)-(CH2)n-, - C(=O)-(CH2CH2O)n-(CH2)q-, -C(=O)NR4-(CH2CH2O)n-(CH2)q-, -NR4C(=O)-(CH2CH2O)n- (CH2)q-, -(CH2CH2O)n-(CH2)q-, -C(=O)-(OCH2CH2)n-, or -(OCH2CH2)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; wherein heteroalkylene is an alkylene where one carbon atom is replaced with -S(=O)(=NH)-, - S(=O)(=NR5)-, -P(=O)OH-, -NHC(=N-CN)NH-, or -NHC(=N-R5)NH-; wherein when any one of -L1-, -L2-, -L3-, -L4-, and -L5-is substituted then -L1-, -L2-, -L3-, -L4-, and -L5-is substituted with 1, 2, 3, or 4 groups selected from halogen, -OH, -OR5, -CO2H, -NHR5, -C(=O)NHR5, - NHC(=O)R5and substituted C1-C6alkyl, wherein the substituted C1-C6alkyl is substituted with, -OH, -CO2H, -NHR5, -C(=O)NHR5, and -NHC(=O)R5;each R5is independently selected from C1-C10alkyl, C4-C30polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene. In some embodiments, L is -L1-L5- or -L2-L5-

[0064] In some embodiments, L is -L1-, or -L1-L2-L3-L4-L5-; L1 is absent, unsubstituted orsubstituted C1-C20alkylene, unsubstituted or substituted C1-C20heteroalkylene, or C4- C20polyethylene glycol; L2is -C(=O)NR4-(unsubstituted or substituted C1-C10alkylene)-, - NR4C(=O)-(unsubstituted or substituted C1-C10alkylene)-, -C(=O)-(CH2CH2O)m-(CH2)P-, - C(=O)NR4-(CH2CH2O)n-(CH2)P-, -NR4C(=O)-(CH2CH2O)n-(CH2)P-, or -(CH2CH2O)n-(CH2)P-; each R4is independently selected from hydrogen, and C1-C6alkyl; each m is independently 1, 2,3, 4, 5, or 6; each p is independently 1, or 2; L3 is absent; L4 is absent, -C(=O)-( unsubstituted orsubstituted C1-C6alkylene)-, -C(=O)NR4-(unsubstituted or substituted C1-C6alkylene)-, - NR4C(=O)-(unsubstituted or substituted C1-C6alkylene)-, -C(=O)-(CH2CH2O)n-(CH2)q-, - C(=O)NR4-(CH2CH2O)n-(CH2)q-, -NR4C(=O)-(CH2CH2O)n-(CH2)q-, or -(CH2CH2O)n-(CH2)q-; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2; L5is -C(=O)-(CH2)n-, - C(=O)NR4-(CH2)n-, -NR4C(=O)-(CH2)n-, -C(=O)-(CH2CH2O)n-(CH2)q-, -C(=O)NR4- -or 12; each q is independently 0, 1, or 2; wherein heteroalkylene is an alkylene where one carbon atom is replaced with -S(=O)(=NH)-, -S(=O)(=NR5)-, -P(=O)OH-, -NHC(=N-CN)NH-, or -NHC(=N- R5)NH-; wherein when any one of -L1-, -L2-, -L3-, -L4-, and -L5-is substituted then -L1-, -L2-, - L3-, -L4-, and -L5-is substituted with 1, 2, 3, or 4 groups selected from halogen, -OH, -OR5, - CO2H, -NHR5,-C(=O)NHR5, -NHC(=O)R5and substituted C1-C6alkyl, wherein the substitutedWSGR Attorney Docket No.: 60924-730.601 C1-C6alkyl is substituted with, -OH, -CO2H, -NHR5, -C(=O)NHR5, and -NHC(=O)R5;each R5is independently selected from C1-C10alkyl, C4-C30polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene.

[0065] In some embodiments, L1 is unsubstituted or substituted C1-C6alkylene, unsubstitutedor substituted C1-C10heteroalkylene, C4-C20polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene. In some embodiments, L5 is absent, - NR4C(=O)-(CH2)n-, -C(=O)-(CH2CH2O)n-(CH2)q-, -C(=O)NR4-(CH2CH2O)n-(CH2)q-, or - NR4C(=O)-(CH2CH2O)n-(CH2)q-; each q is independently 1, or 2.

[0066] In some embodiments, L1 is unsubstituted or substituted C1-C6alkylene, unsubstitutedor substituted C1-C10heteroalkylene, C4-C20polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L2is absent, -C(=O)NR4- (unsubstituted or substituted C1-C10alkylene)-, -NR4C(=O)-(unsubstituted or substituted C1- C10alkylene)-, -C(=O)-(CH2CH2O)m-(CH2)P-, -C(=O)NR4-(CH2CH2O)n-(CH2)P-, -NR4C(=O)- (CH2CH2O)n-(CH2)P-, or -(CH2CH2O)n-(CH2)P-; each m is independently 1, 2, 3, 4, 5, or 6; each p is independently 1 or 2; L4is absent; L5is -NR4C(=O)-(CH2)n-, -C(=O)-(CH2CH2O)n-(CH2)q- , -C(=O)NR4-(CH2CH2O)n-(CH2)q-, or -NR4C(=O)-(CH2CH2O)n-(CH2)q-; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2.

[0067] In some embodiments, L1 is unsubstituted or substituted C1-C6alkylene, unsubstitutedor substituted C1-C10heteroalkylene, C4-C20polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L2is absent, -C(=O)NR4- (unsubstituted or substituted C1-C10alkylene)-, -NR4C(=O)-(unsubstituted or substituted C1- C10alkylene)-, -C(=O)-(CH2CH2O)m-(CH2)P-, -C(=O)NR4-(CH2CH2O)n-(CH2)P-, -NR4C(=O)- (CH2CH2O)n-(CH2)P-, or -(CH2CH2O)n-(CH2)P-; each m is independently 1, 2, 3, 4, 5, or 6; each p is independently 1 or 2; L4is absent; L5is absent, -NR4C(=O)-(CH2)n-, -C(=O)-(CH2CH2O)n- (CH2)q-, -C(=O)NR4-(CH2CH2O)n-(CH2)q-, or -NR4C(=O)-(CH2CH2O)n-(CH2)q-; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2.

[0068] In some embodiments, L1 is unsubstituted or substituted C1-C6alkylene, unsubstitutedor substituted C1-C10heteroalkylene, C4-C20polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L2is absent; L4is absent; L5is absent, -NR4C(=O)-(CH2)n-, -C(=O)-(CH2CH2O)n-(CH2)q-, -C(=O)NR4-(CH2CH2O)n-(CH2)q-, or -NR4C(=O)-(CH2CH2O)n-(CH2)q-; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2.WSGR Attorney Docket No.: 60924-730.601

[0069] In some embodiments, L1 is unsubstituted or substituted C1-C20alkylene,unsubstituted or substituted C1-C20heteroalkylene, or C4-C20polyethylene glycol.

[0070] In some embodiments, L1 is absent.

[0071] In some embodiments, L2 is -(CH2CH2O)n-(CH2)P-; n is 3, 4, 5, or 6; p is 2.

[0072] In some embodiments, L2 is -(CH2CH2O)n-(CH2)P-; n is 3; p is 2. In someembodiments, L2is -(CH2CH2O)n-(CH2)P-; n is 4; p is 2. In some embodiments, L2is - (CH2CH2O)n-(CH2)P-; n is 5; p is 2. In some embodiments, L2is -(CH2CH2O)n-(CH2)P-; n is 6; p is 2.

[0073] In some embodiments, L4 is absent.

[0074] In some embodiments, L5 is -C(=O)NR4-(CH2)n-, -NR4C(=O)-(CH2)n-, -C(=O)NR4-(CH2CH2O)n-(CH2)q-, or -NR4C(=O)-(CH2CH2O)n-(CH2)q-; n is 3, 4, 5, or 6; q is 2. In some embodiments, L5is -NR4C(=O)-(CH2)n-, or -NR4C(=O)-(CH2CH2O)n-(CH2)q-; n is 3, 4, 5, or 6; q is 2. In some embodiments, L5is -NR4C(=O)-(CH2)n-; n is 3, 4, 5, or 6. In some embodiments, L5is -NR4C(=O)-(CH2)n-; n is 4.

[0075] In some embodiments, L is -L2-L5-; L2 is -(CH2CH2O)n-(CH2)P-, wherein n is 1, 2, 3,4, 5, or 6, and p is 2; L5is -C(=O)NR4-(CH2)n-, -NR4C(=O)-(CH2)n-, -C(=O)NR4-(CH2CH2O)n- (CH2)q-, or -NR4C(=O)-(CH2CH2O)n-(CH2)q-, wherein n is 1, 2, 3, 4, 5, or 6, q is 2, and R4is selected from hydrogen, and C1-C4alkyl.

[0076] In some embodiments, L is -L2-L5-; L2 is -(CH2CH2O)n-(CH2)2-, wherein n is 2, 3, 4,5, or 6; L5is -C(=O)NR4-(CH2)4-; and R4is C1-C4alkyl. In some embodiments, R4is -CH3, - CH2CH3, -CH2CH2CH3, or -CH2CH2CH2CH3. In some embodiments, R4is -CH3. In some embodiments, R4is -CH2CH3. In some embodiments, R4is -CH2CH2CH3. In some embodiments, R4is -CH2CH2CH2CH3.

[0077] In some embodiments, L is -(CH2CH2O)n-(CH2)2-C(=O)NR4-(CH2)4-; wherein R4 is -CH3, -CH2CH3, -CH2CH2CH3, or -CH2CH2CH2CH3, and n is 1, 2, 3, 4, 5, or 6. In some embodiments, L is -(CH2CH2O)n-(CH2)2-C(=O)NR4-(CH2)4-; wherein R4is -CH3, -CH2CH3, - CH2CH2CH3, or -CH2CH2CH2CH3, and n is 1. In some embodiments, L is -(CH2CH2O)n- (CH2)2-C(=O)NR4-(CH2)4-; wherein R4is -CH3, -CH2CH3, -CH2CH2CH3, or -CH2CH2CH2CH3, and n is 2. In some embodiments, L is -(CH2CH2O)n-(CH2)2-C(=O)NR4-(CH2)4-; wherein R4is - CH3, -CH2CH3, -CH2CH2CH3, or -CH2CH2CH2CH3, and n is 3. In some embodiments, L is - (CH2CH2O)n-(CH2)2-C(=O)NR4-(CH2)4-; wherein R4is -CH3, -CH2CH3, -CH2CH2CH3, or - CH2CH2CH2CH3, and n is 4. In some embodiments, L is -(CH2CH2O)n-(CH2)2-C(=O)NR4- (CH2)4-; wherein R4is -CH3, -CH2CH3, -CH2CH2CH3, or -CH2CH2CH2CH3, and n is 5. In someWSGR Attorney Docket No.: 60924-730.601 embodiments, L is -(CH2CH2O)n-(CH2)2-C(=O)NR4-(CH2)4-; wherein R4is -CH3, -CH2CH3, - CH2CH2CH3, or -CH2CH2CH2CH3, and n is 6. In some embodiments, R4is -CH3. In some embodiments, R4is -CH2CH3. In some embodiments, R4is -CH2CH2CH3. In some embodiments, R4is -CH2CH2CH2CH3.

[0078] In some embodiments, wherein R1 is a chelating moiety or a radionuclide complexthereof, wherein the chelating moiety is: 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-tetra- azacyclododecane-1,4,7,10-tetraacetic acid (DOTMA); 1,4,7,10-tetrakis(carbamoylmethyl)- 1,4,7,10-tetraazacyclododecane (DOTAM); 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra- propionic acid (DOTPA); 2,2',2''-(10-(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane- 1,4,7-triyl)triacetic acid; 6,6'-(((pyridine-2,6- diylbis(methylene))bis((carboxymethyl)azanediyl))-bis(methylene))dipicolinic acid (H4pypa); 6,6',6'',6'''-(((pyridine-2,6-diylbis(methylene)) bis(azanetriyl))tetrakis(methylene))-tetrapicolinic acid (H4py4pa); 10-((6-carboxypyridin-2-yl)methyl)-1,4,7,10-tetra-azacyclododecane-1,4,7- triacetic acid (DO3Apic); or 3,6,9,12-tetrakis(carboxymethyl)-3,6,9,12-tetraazatetradecanedioic acid (TTHA).

[0079] In some embodiments, R1 is a chelating moiety or a radionuclide complex thereof,wherein the chelating moiety is: .complex thereof,wherein the chelating moiety is: .WSGR Attorney Docket No.: 60924-730.601

[0081] In certain embodiments, R1 is a chelating moiety or a radionuclide complex thereof,wherein the chelating moiety is: complex thereof;; X2is -C(=O)X4-; X4is -NH-, -N(CH3)-, or - N(CH2CH3)-, L is -L1-, or -L1-L2-L3-L4-L5-; L1is unsubstituted or substituted C1-C6alkylene, unsubstituted or substituted C1-C10heteroalkylene, C4-C20polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L2is absent; L3is absent; L4is absent; L5is -NR4C(=O)-(CH2)n-, -C(=O)-(CH2CH2O)n-(CH2)q-, -C(=O)NR4- (CH2CH2O)n-(CH2)q-, or -NR4C(=O)-(CH2CH2O)n-(CH2)q-; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2.

[0083] In some embodiments, R1 is:a radionuclide complex thereof;- X2is -C(=O)X4-; X4is -NRa-; L is -L1-L5-; L1is unsubstituted or substituted C1-C10heteroalkylene, or C4-C20polyethylene glycol; L5is -NR4C(=O)-(CH2)n- or - NR4C(=O)-(CH2CH2O)n-(CH2)2-; R4is C1-C4alkyl; R4is C1-C4alkyl; n is 4, 5, or 6.WSGR Attorney Docket No.: 60924-730.601

[0084] In some embodiments, R1 is:; or a radionuclide complex thereof; X2-2 4 4 1; X is -C(=O)X -; X is -NH-, -N(CH3)-, or -N(CH2CH3)-; L is -L - L5-; L1is unsubstituted or substituted C1-C10heteroalkylene, or C4-C20polyethylene glycol; L5is - NR4C(=O)-(CH2)2-; n is 1, 2, 3, 4, 5, or 6.

[0085] In some embodiments, R1 is:a radionuclide complex thereof;X2is -C(=O)X4-; X4is -NH-, -N(CH3)-, or -N(CH2CH3)-; L is -L2- L5-; L2is -(CH2CH2O)n-(CH2)2-; L5is -C(=O)NR4-(CH2)n-; R4is C1-C4alkyl; and each n is independently 3, 4, 5, or 6. In some embodiments, R4is -CH3, -CH2CH3, -CH2CH2CH3, or - CH2CH2CH2CH3. In some embodiments, R4is -CH3. In some embodiments, R4is -CH2CH3. In some embodiments, R4is -CH2CH2CH3. In some embodiments, R4is -CH2CH2CH2CH3. In some embodiments, each n is independently 4, 5, or 6. In some embodiments, each n is independently 4 or 5.

[0086] In some embodiments, R1 is:a radionuclide complex thereof;WSGR Attorney Docket No.: 60924-730.601 wherein, X1is -CH2CH2-X2-; X2is -C(=O)X4-; X4is -NH-, -N(CH3)-, or -N(CH2CH3)-; L is - (CH2CH2O)n-(CH2)2-C(=O)NR4-(CH2)n-; R4is C1-C4alkyl; and each n is independently 3, 4, 5, or 6. In or -CH2CH2CH2CH3. In someIn some embodiments, R4is - CH2CH2CH3. In some embodiments, R4is -CH2CH2CH2CH3. In some embodiments, each n is independently 4, 5, or 6. In some embodiments, each n is independently 4 or 5. In some embodiments, L is -(CH2CH2O)5-(CH2)2-C(=O)NR4-(CH2)4-.

[0087] In some embodiments, R1 is:a radionuclide complex thereof;X4-; X4is -NRa-; L is -(CH2CH2O)n-(CH2)2-C(=O)NR4-(CH2)2-; Rais C1-C4alkyl; R4is C1-C4alkyl; and n is 3, 4, 5, or 6. In some embodiments, Rais -CH3, - CH2CH3, -CH2CH2CH3, or -CH2CH2CH2CH3; R4is -CH3, -CH2CH3, -CH2CH2CH3, or - CH2CH2CH2CH3. In some embodiments, Rais -CH3or -CH2CH3; R4is -CH3or -CH2CH3. In some embodiments, Rais -CH2CH3; R4is -CH2CH3.

[0088] In some embodiments, each Ra is independently selected from C1-C4alkyl; each R4 isindependently selected from C1-C4alkyl. In some embodiments, Rais -CH3, -CH2CH3, - CH2CH2CH3, or -CH2CH2CH2CH3. In some embodiments, Rais -CH3. In some embodiments, Rais -CH2CH3. In some embodiments, Rais -CH2CH2CH3. In some embodiments, Rais - CH2CH2CH2CH3. In some embodiments, R4is -CH3, -CH2CH3, -CH2CH2CH3, or - CH2CH2CH2CH3. In some embodiments, R4is -CH3. In some embodiments, R4is -CH2CH3. In some embodiments, R4is -CH2CH2CH3. In some embodiments, R4is -CH2CH2CH2CH3. In some embodiments, Rais -CH2CH3, and R4is -CH2CH3.

[0089] In some embodiments, X1-L-is:.someWSGR Attorney Docket No.: 60924-730.601 . ,

[0092] In some embodiments, the radionuclide is a diagnostic or therapeutic radionuclide. Insome embodiments, the radionuclide is an Auger electron-emitting radionuclide, α-emitting radionuclide, β-emitting radionuclide, or γ-emitting radionuclide. In some embodiments, the radionuclide is α-emitting radionuclide.

[0093] In some embodiments, the radionuclide is an Auger electron-emitting radionuclidethat is 111-indium (111In), 67-gallium (67Ga), 68-gallium (68Ga), 99m-technetium (99mTc), or 195m-platinum (195mPt).

[0094] In some embodiments, the radionuclide is an α-emitting radionuclide that is 225-actinium (225Ac), 213-bismuth (213Bi), 223-Radium (223Ra), or 212-lead (212Pb). In some embodiments, the radionuclide is 225-actinium (225Ac).

[0095] In some embodiments, the radionuclide is a β-emitting radionuclide that is 90-yttrium(90Y), 177-lutetium (177Lu), 186-rhenium (186Re), 188-rhenium (188Re), 64-copper (64Cu), 67- copper (67Cu), 153-samarium (153Sm), 89-strontium (89Sr), 198-gold (198Au), 169-Erbium (169Er), 165-dysprosium (165Dy), 99m-technetium (99mTc), 89-zirconium (89Zr), or 52-manganese (52Mn);.WSGR Attorney Docket No.: 60924-730.601

[0096] In some embodiments, the radionuclide is a γ-emitting radionuclide that is 60-cobalt(60Co), 103-palldium (103Pd), 137-cesium (137Cs), 169-ytterbium (169Yb), 192-iridium (192Ir), or 226-radium (226Ra).

[0097] In some embodiments, the radionuclide is suitable for positron emission tomography(PET) analysis, single-photon emission computerized tomography (SPECT), or magnetic resonance imaging (MRI). Chelating moieties

[0098] As described herein, in some embodiments a chelating agent (i.e., R1) is coupled to theantibody (e.g., the polypeptides comprising an antigen binding region and an immunoglobulin heavy chain constant region des cribbed herein). The chelating moiety allows for the tumor targeting moieties to be loaded with an appropriate radioisotope, such as a beta emitter or an alpha emitter. The chelator can be coupled to the antigen binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof. Such coupling can suitably be by a covalent attachment to one or more amino acids of the immunoconjugate, the antigen binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof.

[0099] In one embodiment, a chelating agent of the immunoconjugate is covalently linked toan antigen binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof. In one embodiment, a chelating agent is covalently linked to the antigen binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof directly (e.g., without the use of a spacer, stretcher or linker). In one embodiment the chelating agent is covalently linked to the antigen binding arm through a linker that is covalently linked to the chelating agent and covalently linked to the antigen binding arm. In one embodiment, the linker is hydrophilic (e.g., a PEG chain). In one embodiment, the linker is hydrophobic (e.g., an alkyl or alkene chain). Chelators may be linked or coupled to the immunoconjugates as described in Sadiki, A. et al. “Site-specific conjugation of native antibody.” Antibody Therapeutics 2020, 3, 271–284.

[0100] In some embodiments, the immunoconjugate is formed through the attachment of thechelator-linker in a site-specific manner, directed into a specific amino acid or glycan residue. In some embodiments, the site-specific conjugation involves directed functionalization of a specific lysine residue in the framework region with the chelator-linker. In other embodiments, this residue may be functionalized with a different reactive functional group which then reacts in aWSGR Attorney Docket No.: 60924-730.601 second step with chelator-linker to furnish the immunoconjugate. In some embodiments, this reactive functional group is thiopropionate.

[0101] In some embodiments, a non-native cysteine residue is engineered into theframework of the antibody as a site for thiol directed conjugation to furnish the immunoconjugate. In some embodiments, other non-native amino acids or an amino acid sequence is engineered into the framework to serve as the attachment site for the chelator-linker or for a secondary reactive group upon which the chelator-linker will be conjugated to furnish the immunoconjugate.

[0102] In some embodiments, a non-natural amino acid containing a cross-linking group isengineered into the framework for attachment of the chelator-linker. In some embodiments, this non-natural amino-acid contains an azide.

[0103] In some embodiments, the chelator-linker is attached to a glutamine residue throughthe action of a transglutaminase enzyme. In other embodiments, a secondary reactive group is attached by transglutaminase upon which the chelator-linker is added to furnish the immunoconjugate.

[0104] In some embodiments, the chelator-linker is attached by modifying one or more N-glycans with a reactive functional group through the action of a glycosidase, then conjugation of the chelator-linker to that site. In some embodiments, the glycan is modified through the action of β-galactosidase. In some embodiments, the glycan is modified with a glycoside that contains an azide for attachment of a properly functionalized chelator-linker.

[0105] In one embodiment, the immunoconjugate comprises more than one chelating agent,which are the same or different.

[0106] In one embodiment, an immunoconjugate having more than one chelating agent hasmore than one chelating agent attached to the same antigen binding arm.

[0107] In one embodiment, an immunoconjugate having more than one chelating agent andless than eleven chelating agents has more than two chelating agents, more than three chelating agents, more than four chelating agents, more than five chelating agents, more than six chelating agents, more than seven chelating agents, more than eight chelating agents, or more than nine chelating agents. In one embodiment, the chelating agents are the same. In one embodiment, each antigen binding arm is linked directly or indirectly to more than one chelating agent.

[0108] In one embodiment, the chelating agent comprises a radioisotope chelatingcomponent and a functional group that allows for covalent attachment to the antigen binding arm. In one embodiment, the functional group is directly attached to the radioisotope chelating component. In one embodiment the chelating agent further comprises a linker between the functional group and the radioisotope chelating component.WSGR Attorney Docket No.: 60924-730.601

[0109] In one embodiment, the radioisotope chelating component comprises DOTA or aDOTA derivative. In one embodiment, the radioisotope chelating component comprises DOTAGA. In one embodiment, the radioisotope chelating component comprises macropa or a macropa derivative. In one embodiment, the radioisotope chelating component comprises Py4Pa or a Py4Pa derivative.

[0110] In a preferred embodiment, the chelating agent of an immunoconjugate is notattached to the antigen binding region in the antigen binding arm of the immunoconjugate.

[0111] In one embodiment, the chelating agent of the immunoconjugate is non-covalentlyassociated with an antigen binding arm. In a preferred embodiment, the chelator is not associated with the antigen binding region in the antigen binding arm of the immunoconjugate.

[0112] In one embodiment, the chelating agent comprises DOTA or a DOTA derivative. Inone embodiment, the chelating agent comprises DOTAGA. In one embodiment, the chelating agent comprises macropa or a macropa derivative. In one embodiment, the chelating agent comprises Py4Pa or a Py4Pa derivative. In one embodiment, the chelating agent comprises siderocalin or a siderocalin derivative.

[0113] In certain embodiments, the chelating agent is a radioisotope chelating agent. Incertain embodiments, the radioisotope chelating agent is selected from the list consisting of: tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), α-(2-Carboxyethyl)-1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTAGA), or (Py4Pa). In certain embodiments, the radioisotope chelating agent is DOTA. In certain embodiments, the radioisotope chelating agent is DOTAGA. In certain embodiments, the radioisotope chelating agent is Py4Pa. In certain embodiments, the radioisotope wherein the radioisotope chelating agent is directly coupled to the antigen binding region and / or the immunoglobulin heavy chain constant region. In certain embodiments, the radioisotope chelating agent is coupled to the antigen binding region or the immunoglobulin heavy chain constant region by a linker. In certain embodiments, the linker is selected from: 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and those resulting from conjugation with linker reagents: N-Succinimidyl 4-(2- pyridylthio) pentanoate forming linker moiety 4-mercaptopentanoic acid (SPP), Succinimidyl 4- (N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-Succinimidyl 4-(2- pyridyldithio)butanoate (SPDB), N-Succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB), polyethylene glycol (PEG), a polyethylene glycol polymers (PEGn), and S-2-(4- Isothiocyanatobenzyl) (SCN). In certain embodiments, the linker is selected from: polyethylene glycol (PEG), a polyethylene glycol polymers (PEG), and S-2-(4-isothiocyanatobenzyl) (SCN). In certain embodiments, the linker is PEG5. In certain embodiments, the linker is SCN. InWSGR Attorney Docket No.: 60924-730.601 certain embodiments, the radioisotope chelating agent is a linker-chelator selected from the list consisting of: TFP-Ad-PEG5-DOTAGA, p-SCN-Bn-DOTA, p-SCN-Ph-Et-Py4Pa, and TFP-Ad- PEG5-Ac-Py4Pa.

[0114] In some embodiments, the chelator is conjugated at a predefined ratio of polypeptide(i.e., antigen binding region and / or the immunoglobulin heavy chain constant) to chelator. In certain embodiments, the radioisotope chelating agent is coupled to the antigen binding region and / or the immunoglobulin heavy chain constant region at a ratio of 1:1 to 8:1. In certain embodiments, the radioisotope chelating agent is coupled to the antigen binding region and / or the immunoglobulin heavy chain constant region at a ratio of 1:1 to 6:1. In certain embodiments, the radioisotope chelating agent is coupled to the antigen binding region and / or the immunoglobulin heavy chain constant region at a ratio of 2:1 to 6:1.

[0115] For example, a bifunctional chelator is used to conjugate a radioisotope to aradioisotope delivery platform of the invention to create an immunoconjugate of the invention. (See e.g., Scheinberg D, McDevitt M, Curr Radiopharm 4: 306-20 (2011)). Examples of bifunctional chelators known in the art include DOTA, DTPA, DO3A-NHS, DOTAGA-NHS, DOTAGA-anhydride DOTAGA-TFP, p-SCN-Bn-DOTA, p-SCN-Bn-DTPA, p-SCN-Bn- CHX’A”-DTPA, p-SCN-Bn-TCMC, macropa-NCS, crown, p-SCN-Ph-Et-Py4Pa, 3,2-HOPO, and TCMC.

[0116] Examples of bifunctional chelators are 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylene triamine pentaacetic acid (DTPA), and related analogs of the aforementioned. Such chelators are suitable for coordinating metal ions like α and β-emitting radionuclides.

[0117] In some embodiments the chelating agent of an immunoconjugate orradioimmunoconjugate of the invention is selected from the group comprising bifunctional chelator, 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. Onc. Biol. Phys.105:410 (2019)) (For a review of these and other bifunctional chelators See e.g., Price EW and Orvig C Chem. Soc. Rev., 2014,43:260 (2014) and Brechbiel MW Q. J. Nucl. Med. Mol. Imaging 52:166 (2008)).

[0118] In some embodiments the chelating agent of an immunoconjugate orradioimmunoconjugate of the invention is selected from the group consisting of bifunctional chelator, 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-NCSWSGR Attorney Docket No.: 60924-730.601 (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. Onc. Biol. Phys.105:410 (2019)) (For a review of these and other bifunctional chelators see e.g., Price EW and Orvig C Chem. Soc. Rev., 2014,43:260 (2014) and Brechbiel MW Q. J. Nucl. Med. Mol. Imaging 52:166 (2008)).

[0119] For 225Ac immunoconjugates, there are a variety of acyclic and cyclic ligands knownin the art as suitable chelators (see e.g., Davis I, et al., Nucl Med Biol 26: 581 (1999); Chappell L, et al., Bioconjug Chem 11: 510 (2000); Chappell, L, et al., Nucl Med Biol 30: 581 (2003); McDevitt M, et al., Appl Radiat Isot 57: 841 (2002); Gouin S, et al., Org Biomol Chem 3: 453 (2005); Thiele N, et al., Angew Chem Int Ed Engl 56: 14712 (2017)).

[0120] In certain embodiments, the chelator is a chelator suitable for alpha emitter chelation.Some chelators suitable for alpha emitters are described in Yang et al, “Harnessing α-Emitting Radionuclides for Therapy: Radiolabeling Method Review.” J Nucl Med.2022 Jan;63(1):5-13.

[0121] In certain embodiments the, chelator suitable for alpha emitter chelation is selectedfrom 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 is 6,6'-((((4-isothiocyanatopyridine-2,6- diyl)bis(methylene))bis((carboxymethyl)azanediyl))-bis(methylene))dipicolinic acid; Crown 2,2',2'',2'''-(1,10-dioxa-4,7,13,16-tetraazacyclo-octadecane-4,7,13,16-tetrayl)tetraacetic acid; Macropa 6,6'-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))- dipicolinic acid; Macropa-NCS 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16- diazacyclooctadecan-7-yl)methyl)-4-isothiocyanatopicolinic acid; HEHA 1,4,7,10,13,16- hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid; CHXoctapa 6,6′-[(1R,2R)-1,2- Cyclohexanediylbis[[(carboxymethyl)imino]methylene]]bis[2-pyridinecarboxylic acid]; Bispa 3,7-Diazabicyclo[3.3.1]nonane-1,5-dicarboxylic acid, 7-[(6-carboxy-2-pyridinyl)methyl]-9- hydroxy-3-methyl-2,4-di-2-pyridinyl-, 1,5-dimethyl ester; Noneunpa 6,6'-(((oxybis(ethane-2,1- diyl))bis((carboxymethyl)azanediyl))bis(methylene))-dipicolinic acid; and combinations thereof.

[0122] In certain embodiments, the chelator is a chelator suitable for an beta- or gamma-emitter chelation. In certain embodiments the, chelator suitable for an beta- or gamma-emitter chelation is 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- tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane); DOTPA 1,4,7,10-tetraazacyclo-WSGR Attorney Docket No.: 60924-730.601 dodecane-1,4,7,10-tetrapropionic acid; DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)- 1,4,7,10-tetraazacyclododecan-1-yl)acetic acid); DOTP 1,4,7,10- tetraazacyclododecane-1 ,4,7,10-tetra(methylene phosphonic acid); DOTMP 1,4,6,10-tetraazacyclodecane-1 ,4,7,10- tetramethylene phosphonic acid; DOTA-4AMP 1,4,7,10-tetraazacyclododecane- 1 ,4,7, 10- tetrakis(acetamido-methylenephosphonic acid); CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexa- decane-4,11-diacetic acid); NOTA 1,4,7-triazacyclononane-1 ,4,7-triacetic acid; NOTP 1,4,7- triazacyclononane-1 ,4,7-tri(methylene phosphonic 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-Ν,Ν',Ν'',Ν'",N""-pentaacetic acid; H4Octapa Ν,Ν'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid; H2Dedpa 1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane; H6phospa Ν,Ν'- (methylenephosphonate)- N,N'-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2- diaminoethane; TTHA triethylene- tetramine-Ν,Ν,Ν',Ν'',Ν'",N"'-hexaacetic acid; DO2P tetraazacyclododecane dimethane- phosphonic acid; HP-DO3A hydroxypropyltetraazacyclododecanetriacetic acid; EDTA ethylenediaminetetraacetic acid; DTPA diethylenetriaminepentaacetic acid; DTPA-BMA diethylenetriaminepentaacetic acid-bismethylamide; HOPO octadentate hydroxypyridinones; 3,2,3-LI(HOPO) N,N'-(butane-1,4-diyl)bis(1-hydroxy-N-(3-(1-hydroxy-6-oxo-1,6-dihydro- pyridine-2-carboxamido)propyl)-6-oxo-1,6-dihydropyridine-2-carboxamide); 3,2-HOPO N,N'- (((2-(4-aminobenzyl)-3-((2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4- carboxamido)ethyl)(2-(3-hydroxy-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)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[16.2.1.13,6.18,11.113,16]tetracosa- 1,3,5,7,9,11(23),12,14,16,18(21),19-undecaene; Deferoxamine 30-Amino-3,14,25-trihydroxy- 3,9,14,20,25-pentaazatriacontane-2,10,13,21,24-pentaone; DFO* N1-[5-(Acetylhydroxyamino)- pentyl]-N26-(5-aminopentyl)-N26,5,16-trihydroxy-4,12,15,23-tetraoxo-5,11,16,22- tetraazahexacosanediamide; and combinations thereof.

[0123] In some embodiments, R1 comprises a chelating moiety selected from the listconsisting of: DOTA, DO3A, DO3Apic, DOTAGA, DOTAGA anhydride, Py4Pa, Py4Pa-NCS,WSGR Attorney Docket No.: 60924-730.601 Crown, Macropa, Macropa-NCS, HEHA, CHXoctapa, Bispa, and Noneunpa; or a radionuclide complex thereof.

[00124] In some embodiments, R1 comprises a chelating moiety selected from the listconsisting of: DOTMA, DOTPA, DO3Apic, DO3AM-acetic acid, DOTP, DOTMP, DOTA- 4AMP, CB-TE2A, NOTA, NOTP, TETPA, TETA, PEPA, H4Octapa, H2Dedpa, DO2P, EDTA, DTPA-BMA, 3,2,3-LI(HOPO), 3,2-HOPO, Neunpa, Neunpa-NCS, Octapa, PyPa, Porphyrin, and Deferoxamine; or a radionuclide complex thereof.

[00125] In some embodiments, R1 is a chelating moiety selected from the list consisting of:1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A); 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A); α,α',α'',α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTMA); 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM); 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid (DOTPA); 2,2',2''-(10-(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid; 10-((6-carboxypyridin-2-yl)methyl)-1,4,7,10-tetra-azacyclododecane-1,4,7-triacetic acid (DO3Apic); 6,6'-(((pyridine-2,6-diylbis(methylene))bis((carboxymethyl)azanediyl))- bis(methylene))dipicolinic acid (H4pypa); 6,6',6'',6'''-(((pyridine-2,6-diylbis(methylene))bis(azanetriyl))tetrakis(methylene))-tetrapicolinic acid (H4py4pa); and 3,6,9,12-tetrakis(carboxymethyl)-3,6,9,12-tetraazatetradecanedioic acid (TTHA); or a radionuclide complex thereof.

[00126] In some embodiments, R1 comprises a chelating moiety with one of the followingstructures: ,WSGR Attorney Docket No.: 60924-730.601 ,WSGR Attorney Docket No.: 60924-730.601 ,WSGR Attorney Docket No.: 60924-730.601 ,WSGR Attorney Docket No.: 60924-730.601 ,WSGR Attorney Docket No.: 60924-730.601 , .orO OH O OH O OH O OH . orcompound at the position .WSGR Attorney Docket No.: 60924-730.601

[0129] In some embodiments, R1 comprises DOTA and is attached to the antibody orcompound at the position .

[0130] It is understood that herein comprise one or moreR1moieties. In some embodiments, the immunoconjugates described herein comprise one or more than one R1moieties, wherein each R1moiety is the same. Radionuclides

[0131] In some embodiments, the radionuclide in the immunoconjugates described herein isan 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 targeted therapeutic compound can be tailored to the specific type of cancer, the type of targeting moiety (e.g., non-peptide ligand), etc. Radionuclides that undergo α-decay emit α-particles (helium ions with a +2 charge) from their nuclei. As a result of α-decay the daughter nuclide has 2 protons less and 2 neutrons less than the parent nuclide. This means that in α-decay, the proton number is reduced by 2 while the nucleon number is reduced by 4. Radionuclides that undergo β-decay emit β-particles (electrons) from their nuclei. During β-decay, one of the neutrons changes into a proton and an electron. The proton remains in the nucleus while the electron is emitted as a β-particle. This means that in β-decay, the nucleus loses a neutron but gains a proton. In γ-decay, a nucleus in an excited state (higher energy state) emits a γ-ray photon to change to a lower energy state. There is no change in the proton number and nucleon number during the γ-decay. The emission of γ-rays often accompanies the emission of α-particles and β-particles.

[0132] Auger electrons (AEs) are very low energy electrons that are emitted byradionuclides that decay by electron capture (EC) (e.g.,111In , gallium-67 (67Ga), technetium-99m (99mTc), platinum-195m (195mPt), iodine-125 (125I), and iodine-123 (123I). This energy isdeposited over nanometre-micrometre distances, resulting in high linear energy transfer that is potent for causing lethal damage in cancer cells. Thus, AE-emitting radiotherapeutic agents have great potential for treatment of cancer.WSGR Attorney Docket No.: 60924-730.601

[0133] β-Particles are electrons emitted from the nucleus. They typically have a longer rangein tissue (of the order of 1–5 mm) and are the most frequently used.

[0134] α-Particles are helium nuclei (two protons and two neutrons) that are emitted fromthe nucleus of a radioactive atom. Depending on their emission energy, they can travel 50–100 µm in tissue. They are positively charged and are orders of magnitude larger than electrons. The amount of energy deposited per path length travelled (designated ‘linear energy transfer’) of α- particles is approximately 400 times greater than that of electrons. This leads to substantially more damage along their path than that caused by electrons. An α-particle track leads to a preponderance of complex and largely irreparable DNA double-strand breaks. The absorbed dose required to achieve cytotoxicity relates to the number of α-particles traversing the cell nucleus. With use of this as a measure, cytotoxicity may be achieved with a range of 1 to 20 α- particle traversals of the cell nucleus. The resulting high potency, combined with the short range of α-particles (which reduces normal organ toxicity), has led to substantial interest in developing α-particle-emitting agents. The α-particle emitters typically used include212Bi ,212Pb,213Bi ,225Ac,223Ra, and229Th.

[0135] In some embodiments, the radionuclide is a diagnostic or therapeutic radionuclide.For example, representative radionuclides include: Isotope t1 / 2 (h) Decay mode ) ) ) )

[0136] In some embodiments, the radionuclide is an Auger electron-emitting radionuclide.In some embodiments, the radionuclide is an Auger electron-emitting radionuclide that is 111In,67Ga,68Ga, 999mTc, or195mPt. In some embodiments, the radionuclide is an Auger electron-emitting radionuclide that is 111In, 67Ga, 68Ga, or-99mTc.WSGR Attorney Docket No.: 60924-730.601

[0137] In some embodiments, the radionuclide is an α-emitting radionuclide. In someembodiments, the radionuclide is an α-emitting radionuclide that is225Ac,213Bi,223Ra, or212Pb. In some embodiments, the radionuclide is an α-emitting radionuclide that is225Ac. In some embodiments, the radionuclide is an β-emitting radionuclide. In some embodiments, the radionuclide is a β-emitting radionuclide that is90Y,177Lu, rhenium-186 (186Re), rhenium-188 (188Re),64Cu,67Cu,153Sm,89Sr, gold-198 (198Au), erbium-169 (169Er), dysprosium-165 (165Dy),99mTc,89Zr, or manganese-52 (52Mn). In some embodiments, the radionuclide is a β-emitting radionuclide that is90Y,177Lu,99mTc, or89Zr. In some embodiments, the radionuclide is a γ-emitting radionuclide. In some embodiments, the radionuclide is a γ-emitting radionuclide that is60Co cobalt-60 (60Co), palldium-103 (103Pd), cesium-137 (137Cs), ytterbium-169 (169Yb), iridium-192 (192Ir),212B1,213Bi, or226Ra. Immunoconjugate Production; Host Cells and Expression Vectors of This Disclosure

[0138] The description below relates primarily to production of the antibody constructs ofthis disclosure by culturing cells transformed or transfected with a vector-containing immunoconjugate of this disclosure-encoding nucleic acid. It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare the antibody constructs of this disclosure. For instance, the appropriate amino acid sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques (e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J, Am. Chem. Soc., 85: 2149-54 (1963)). In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer’s instructions. Various portions of the immunoconjugate of this disclosure may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired immunoconjugate of this disclosure.

[0139] Antibody constructs may be produced using recombinant methods and compositions,e.g., as described in US 4,816,567. In one embodiment, isolated nucleic acid encoding an antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VH of the antibody and / or comprising the VL amino acid sequence (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In some embodiments, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VHH of the antibody. In some other embodiments, a host cellWSGR Attorney Docket No.: 60924-730.601 comprises: (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., YO, NSO, Sp20 cell). In one embodiment, a method of making an immunoconjugate of this disclosure is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0140] For recombinant production of an immunoconjugate of the present disclosure,nucleic acid encoding an antibody construct, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and / or light chains of the antibody). Nucleic acid molecules encoding amino acid sequence of the immunoconjugate of the present disclosure (including sequence variants) may be prepared by a variety of methods known to the skilled worker. These methods include, but are not limited to, isolation from a natural source (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 an earlier prepared variant or a non-variant version of the antibody construct. Manipulation of Host Cells for Immunoconjugate Production

[0141] Host cells are transfected or transformed with expression or cloning vectorsdescribed herein for immunoconjugate of this disclosure production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

[0142] Suitable host cells for cloning or expression of immunoconjugate-encoding nucleicacids and vectors include prokaryotic or eukaryotic cells described herein. For example,WSGR Attorney Docket No.: 60924-730.601 antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides 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 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp.245–254, describing expression of antibody fragments in E. coli). After expression, the immunoconjugate may be isolated from the bacterial cell paste in a soluble fraction and can be further purified. Selection and Use of a Replicable Vector

[0143] For recombinant production of a radioisotope delivery platform of this disclosure, thenucleic acid (e.g., cDNA or genomic DNA) encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the immunoconjugate is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of an antibody). Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, suitable host cells are of either prokaryotic or eukaryotic (generally mammalian) origin.

[0144] The vector may, for example, be in the form of a plasmid, cosmid, viral particle, orphage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.

[0145] The immunoconjugate of the invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the immunoconjugate encoded by a DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces α-factor leaders, the latter described in U.S. Patent No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published 4 April 1990), or the signal described in WO 90 / 13646 published 15WSGR Attorney Docket No.: 60924-730.601 November 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders. Purification of an Immunoglobulin-derived Structure of This Disclosure

[0146] Forms of immunoconjugate of this disclosure may be recovered from culture mediumor from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton-X 100) or by enzymatic cleavage. Cells employed in expression of immunoconjugate of this disclosure can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.

[0147] It may be desired to purify immunoconjugate of this disclosure from recombinantcell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the immunoconjugate of this disclosure. Various methods of protein purification may 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 step(s) selected will depend, for example, on the nature of the production process used and the particular immunoconjugate of this disclosure produced.

[0148] When using recombinant techniques, the immunoconjugate can be producedintracellularly, in the periplasmic space, or directly secreted into the medium. If the immunoconjugate is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-7 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the immunoconjugate is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of theWSGR Attorney Docket No.: 60924-730.601 foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

[0149] The immunoconjugate composition prepared from the cells can be purified using, forexample, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the immunoconjugate. Protein A can be used to purify antibodies that are based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth.62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J.5: 15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the immunoconjugate comprises a CH3 domain, the Bakerbond ABX™resin (J. T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification such as fractionation on an ion- exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the immunoconjugate to be recovered.

[0150] Following any preliminary purification step(s), the mixture comprising theimmunoconjugate of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, and generally at low salt concentrations (e.g., from about 0-0.25M salt). Immunoconjugation using Chelators and / or Linkers

[0151] Methods for affixing a radioisotope to an immunoconjugate or antibody construct(i.e., “labeling” an antibody with a radioisotope) are well known to the skilled worker. Certain of these methods are described, for example, in WO 2017 / 155937.

[0152] Bifunctional chelators, such as, e.g., DOTA, DTPA, and related analogs are suitablefor coordinating metal ions like α and β-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., a functional group of a lysine residue) and a carboxylate on the DOTA / DTPA. In the case of peptide synthesis, characterization and purification of theWSGR Attorney Docket No.: 60924-730.601 linker addition can be part of the overall synthesis of an antibody platform or immunoconjugate for radioisotope conjugation.

[0153] For some embodiment, the method of producing an immunoconjugate involves a clickchemistry step described by Poty, S et al., Chem Commun. (Camb) 54: 2599 (2018).

[0154] For some embodiments, a peptide may be biosynthesized or may be synthesized bychemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. In some embodiments, radiolabels may be incorporated into peptide. In some embodiments, radiolabels may be linked to peptide. The IODOGEN method (Fraker et al. (1978) Biochem Biophys Res Commun.80: 49-57 can be used to incorporate iodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989) describes other methods in detail. Characterization of Immunoconjugates of the Present Invention

[0155] Immunoconjugates of the present invention may be identified, screened for, orcharacterized for their physical / chemical properties and / or biological activities by various assays known in the art. The immunoconjugates and antibody constructs of this disclosure may be characterized for their physical / chemical properties and / or biological activities by various assays known in the art. Immunoconjugates of this disclosure can be characterized by a series of assays including, but not limited to, polypeptide sequence determination, amino acid analysis, non- denaturing size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion. Antigen Binding

[0156] An immunoconjugate of the present invention may be tested for its antigen bindingactivity by methods known in the art, e.g., ELISA, Western blot, etc. The binding affinity of an antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal Biochem.107: 220 (1980). Further, the antigen binding ability of an immunoconjugate of this disclosure may be quantitated using methods known in the art, e.g., a quantitative ELISA, quantitative Western blot, surface plasmon resonance assay, and / or a Scatchard analysis.

[0157] In one embodiment, the KD of an immunoconjugate is measured using a radiolabeledantigen ELISA performed with the immunoconjugate. According to another embodiment, the KDis measured by using surface-plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 instrument (BIAcore, Inc., Piscataway, N.J.), e.g., using immobilized antigen CM5 chips at 25° C and 10 response units.WSGR Attorney Docket No.: 60924-730.601

[0158] In another aspect, binding competition assays may be used to identifyimmunoconjugates that compete for binding to the same antigen, or epitope thereof. In some embodiments, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) of an immunoconjugate of this disclosure (see e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)).

[0159] The epitope and / or contact residues within an antigen bound by an immunoconjugateof this disclosure can be identified or mapped using methods known to the skilled worker. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology (3rded., Humana Press, Totowa, NJ). Pharmaceutical Compositions and Formulations of the Present Disclosure

[0160] As will be recognized by the person of ordinary skill in the art, certain teachingsherein below apply to immunoconjugates and radioimmunoconjugates of this disclosure, notwithstanding the specific textual reference to one type of invention, and such applications are embraced in entirety by this disclosure.

[0161] In another aspect, this disclosure provides a composition comprising animmunoconjugate or radioimmunoconjugate of the present invention. This disclosure further provides pharmaceutical compositions and formulations comprising at least one immunoconjugate of the present invention and at least one pharmaceutically acceptable excipient or carrier. In some embodiments, a pharmaceutical formulation comprises (1) an immunoconjugate or radioimmunoconjugate of this disclosure, and (2) a pharmaceutically acceptable carrier.

[0162] An immunoconjugate or radioimmunoconjugate is formulated in any suitable formfor delivery to a target cell / tissue. Pharmaceutical formulations of an immunoconjugate of the present invention are prepared by mixing such immunoconjugate having the desired degree of purity with one or more optional pharmaceutically acceptable carriers, diluents, and / or excipients (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers, diluents, and excipients are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: sterile water, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkoniumWSGR Attorney Docket No.: 60924-730.601 chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating moietys such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0163] Pharmaceutical formulations to be used for in vivo administration are generallysterile. This is readily accomplished by filtration through sterile filtration membranes.

[0164] Examples of lyophilized antibody formulations are described in US 6,267,958.Aqueous antibody formulations include those described in US 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0165] Pharmaceutically acceptable carriers herein further include insterstitial drugdispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX ®, Baxter International, Inc.). In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

[0166] The formulation herein may also contain more than one active ingredient asnecessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0167] The active ingredients may also be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington’s Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[0168] In some embodiments, immunoconjugates may be formulated as immunoliposomes.A “liposome” is a small vesicle composed of various types of lipids, phospholipids and / or surfactant which is useful for delivery of a drug to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biologicalWSGR Attorney Docket No.: 60924-730.601 membranes. Liposomes containing the immunoconjugate are prepared by methods known in the art, such as described in Epstein et al., Proc Natl Acad Sci USA 82: 3688 (1985); Hwang et al., Proc Natl Acad Sci USA 77: 4030 (1980); U.S. Pat. Nos.4,485,045 and 4,544,545; and WO1997 / 38731 published October 23, 1997. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. A chemotherapeutic agent is optionally contained within the liposome (see Gabizon et al., J. National Cancer Inst.81: 1484 (1989)). Liposomes with enhanced circulation time are disclosed in U.S. Patent No.5,013,556.

[0169] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Methods of Using Immunoconjugates and Radioimmunoconjugates and Compositions Thereof

[0170] In one aspect, this disclosure provides a method of treating a disease, disorder, orcondition in a patient in need thereof, the method comprising administering to a subject in need thereof a pharmaceutically effective amount of an immunoconjugate or radioimmunoconjugate or composition of the present invention. For some further embodiments, the method is for inhibiting the growth and / or the killing of a cancer cell or tumor. In another aspect, this disclosure provides for the use of an immunoconjugate described herein for the preparation and / or manufacture of a medicament for treating a disease, disorder, or condition in a subject, such as, e.g., cancer.

[0171] Pharmaceutical compositions of the present invention may be administered in amanner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by clinical trials.

[0172] In one embodiment, an immunoconjugate or radioimmunoconjugate or compositionof this disclosure can be used in a method for binding target antigen in an individual suffering from a disorder associated with increased target antigen expression and / or activity, the method comprising administering to the individual the immunoconjugate or radioimmunoconjugate or composition such that target antigen in the individual is bound. In one embodiment, the targetWSGR Attorney Docket No.: 60924-730.601 antigen is human target antigen, and the individual is a human individual. An immunoconjugate or radioimmunoconjugate or composition of this disclosure can be administered to a human for therapeutic purposes. Moreover, an immunoconjugate or radioimmunoconjugate or composition of this disclosure can be administered to a non-human mammal expressing target antigen with which the immunoconjugate or radioimmunoconjugate cross-reacts (e.g., a primate, pig, rat, or mouse) for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of an immunoconjugate or radioimmunoconjugate or composition of this disclosure (e.g., testing of dosages and time courses of administration).

[0173] An immunoconjugate or radioimmunoconjugate or composition of this disclosure(and any additional therapeutic agent or adjuvant) can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the antibody is suitably administered by pulse infusion, particularly with declining doses of the antibody. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

[0174] Immunoconjugate or radioimmunoconjugate or compositions of this disclosurewould be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The immunoconjugates of this disclosure are administered to a human patient, in accordance with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. For some embodiments, intravenous or subcutaneous administration of the immunoconjugate or radioimmunoconjugate or composition of this disclosure is preferred.

[0175] For the prevention or treatment of disease, the dosage and mode of administrationwill be chosen by the physician according to known criteria. The appropriate dosage of immunoconjugate or radioimmunoconjugate or composition of this disclosure will depend on the type of disease to be treated, as defined above, the severity and course of the disease,WSGR Attorney Docket No.: 60924-730.601 whether the immunoconjugate or radioimmunoconjugate or composition of this disclosure is administered for preventive or therapeutic purposes, previous therapy, the patient’s clinical history and response to the immunoconjugate or radioimmunoconjugate or composition, and the discretion of the attending physician. The immunoconjugate or radioimmunoconjugate or composition of this disclosure is suitably administered to the patient at one time or over a series of treatments. Preferably, the immunoconjugate or radioimmunoconjugate or composition is administered by intravenous infusion or by subcutaneous injections. Depending on the type and severity of the disease, about 1 μg / kg to about 50 mg / kg body weight (e.g., about 0.1-15 mg / kg / dose) of immunoconjugate or radioimmunoconjugate or composition can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A dosing regimen can comprise administering an initial loading dose of about 4 mg / kg, followed by a weekly maintenance dose of about 2 mg / kg of the immunoconjugate or radioimmunoconjugate or composition of this disclosure. However, other dosage regimens may be useful. A typical daily dosage might range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. The progress of this therapy can be readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art.

[0176] The dose and administration schedule may be selected and adjusted based on thelevel of disease, or tolerability in the subject, which may be monitored during the course of treatment. The conjugates of the present invention may administered once per day, once per week, multiple times per week, but less than once per day, multiple times per month but less than once per day, multiple times per month but less than once per week, once per month, once per five weeks, once per six weeks, once per seven weeks, once per eight weeks, once per nine weeks, once per ten weeks, or intermittently to relieve or alleviate symptoms of the disease. Administration may continue at any of the disclosed intervals until remission of the tumor or symptoms of the cancer being treated. Administration may continue after remission or relief of symptoms is achieved where such remission or relief is prolonged by such continued administration.

[0177] For some embodiments, the effective amount of the immunoconjugate orradioimmunoconjugate or composition may be provided as a single dose.WSGR Attorney Docket No.: 60924-730.601

[0178] The Immunoconjugates and radioimmunoconjugates of the present invention maybeused in combination with conventional and / or novel methods of treatment or therapy or separately as a monotherapy.

[0179] Immunoconjugates and radioimmunoconjugates of the present invention may (i)inhibit the growth or proliferation of a cell to which they bind; (ii) induce the death of a cell to which they bind; (iii) inhibit the delamination of a cell to which they bind; (iv) inhibit the metastasis of a cell to which they bind; or (v) inhibit the vascularization of a tumor comprising a cell to which they bind. In this context, “inhibiting cell growth or proliferation” means decreasing a cell’s growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death.

[0180] By way of example, an immunoconjugate that inhibits the growth of a tumor cell isone that results in measurable growth inhibition of a tumor cell (e.g., a cancer cell). In one embodiment, an immunoconjugate or radioimmunoconjugate of this disclosure is capable of inhibiting the growth of cancer cells displaying the antigen bound by the immunoconjugate or radioimmunoconjugate. Preferred growth inhibitory immunoconjugates or radioimmunoconjugates inhibit growth of antigen-expressing tumor cells by greater than 20%, preferably from about 20% to about 50%, and even more preferably, by greater than 50% (e.g., from about 50% to about 100%) as compared to the appropriate control, the control typically being tumor cells not treated with the immunoconjugate or radioimmunoconjugate being tested.

[0181] For some embodiments, a majority of the immunoconjugate orradioimmunoconjugate or composition administered to a subject typically consists of non- labeled immunoconjugate, with the minority being labeled radioimmunoconjugate. The ratio of labeled radioimmunoconjugate to non-labeled immunoconjugate can be adjusted using known methods. Thus, accordingly to certain aspects of the present invention, the immunoconjugate / radioimmunoconjugate may be provided in a total protein amount of up to 100 mg, such as less than 60 mg, or from 5 mg to 45 mg, or a total protein amount of between 0.l µg / kg to l mg / kg patient weight, such as l µg / kg to l mg / kg patient weight, or l0 µg / kg to l mg / kg patient weight, or l00 µg / kg to l mg / kg patient weight, or 0.l µg / kg to l00 µg / kg patient weight, or 0.l µg / kg to 50 µg / kg patient weight, or 0.l µg / kg to l0 µg / kg patient weight, or 0.l µg / kg to 40 µg / kg patient weight, or l µg / kg to 40 µg / kg patient weight, or 0.1 mg / kg to 1.0 mg / kg patient weight, such as from 0.2 mg / kg patient weight to 0.6 mg / kg patient weight.

[0182] In certain embodiments, the immunoconjugate / radioimmunoconjugate may beadministered from about 0.5 mg / kg to about 30 mg / kg. In certain embodiments, the immunoconjugate / radioimmunoconjugate may be administered from about 0.5 mg / kg to about 1WSGR Attorney Docket No.: 60924-730.601 mg / kg, about 0.5 mg / kg to about 2 mg / kg, about 0.5 mg / kg to about 5 mg / kg, about 0.5 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 3 mg / kg, about 0.5 mg / kg to about 4 mg / kg, about 0.5 mg / kg to about 5 mg / kg, about 0.5 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 20 mg / kg, about 0.5 mg / kg to about 30 mg / kg, about 1 mg / kg to about 2 mg / kg, about 1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 3 mg / kg, about 1 mg / kg to about 4 mg / kg, about 1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 20 mg / kg, about 1 mg / kg to about 30 mg / kg, about 2 mg / kg to about 5 mg / kg, about 2 mg / kg to about 10 mg / kg, about 2 mg / kg to about 3 mg / kg, about 2 mg / kg to about 4 mg / kg, about 2 mg / kg to about 5 mg / kg, about 2 mg / kg to about 10 mg / kg, about 2 mg / kg to about 20 mg / kg, about 2 mg / kg to about 30 mg / kg, about 5 mg / kg to about 10 mg / kg, about 5 mg / kg to about 3 mg / kg, about 5 mg / kg to about 4 mg / kg, about 5 mg / kg to about 5 mg / kg, about 5 mg / kg to about 10 mg / kg, about 5 mg / kg to about 20 mg / kg, about 5 mg / kg to about 30 mg / kg, about 10 mg / kg to about 3 mg / kg, about 10 mg / kg to about 4 mg / kg, about 10 mg / kg to about 5 mg / kg, about 10 mg / kg to about 10 mg / kg, about 10 mg / kg to about 20 mg / kg, about 10 mg / kg to about 30 mg / kg, about 3 mg / kg to about 4 mg / kg, about 3 mg / kg to about 5 mg / kg, about 3 mg / kg to about 10 mg / kg, about 3 mg / kg to about 20 mg / kg, about 3 mg / kg to about 30 mg / kg, about 4 mg / kg to about 5 mg / kg, about 4 mg / kg to about 10 mg / kg, about 4 mg / kg to about 20 mg / kg, about 4 mg / kg to about 30 mg / kg, about 5 mg / kg to about 10 mg / kg, about 5 mg / kg to about 20 mg / kg, about 5 mg / kg to about 30 mg / kg, about 10 mg / kg to about 20 mg / kg, about 10 mg / kg to about 30 mg / kg, or about 20 mg / kg to about 30 mg / kg. In certain embodiments, the immunoconjugate / radioimmunoconjugate may be administered at about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, or about 30 mg / kg. In certain embodiments, the immunoconjugate / radioimmunoconjugate may be administered at least about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, or about 20 mg / kg. In certain embodiments, the immunoconjugate / radioimmunoconjugate may be administered at most about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, or about 30 mg / kg.

[0183] In some embodiments, the method comprises administering the effective amount of aradioimmunoconjugate comprising 225-Ac that is from 0.01-0.1 mCi, or 0.1 mCi to 1.0 mCi, or from 1.0 mCi to 2.0 mCi, or from 2.0 mCi to 4.0 mCi.

[0184] In some embodiments, the method comprises administering the effective amount of aradioimmunoconjugate comprising 225-Ac that is from 0.1 µCi / kg to 2.0 µCi / kg subject weight,WSGR Attorney Docket No.: 60924-730.601 or from 0.1 µCi / kg to 1.0 µCi / kg subject weight, or from 1.0 µCi / kg to 3.0 µCi / kg subject weight, or from 3.0 µCi / kg to 10.0 µCi / kg subject weight, or from 10.0 µCi / kg to 20.0 µCi / kg subject weight, or from 10.0 µCi / kg to 30.0 µCi / kg subject weight.

[0185] In certain embodiments, the effective amount of 225-Ac is about 0.1 microcurie toabout 20 microcurie. In certain embodiments, the effective amount of 225-Ac is about 0.1 microcurie to about 0.2 microcurie, about 0.1 microcurie to about 0.5 microcurie, about 0.1 microcurie to about 1 microcurie, about 0.1 microcurie to about 2 microcurie, about 0.1 microcurie to about 3 microcurie, about 0.1 microcurie to about 4 microcurie, about 0.1 microcurie to about 5 microcurie, about 0.1 microcurie to about 10 microcurie, about 0.1 microcurie to about 20 microcurie, about 0.2 microcurie to about 0.5 microcurie, about 0.2 microcurie to about 1 microcurie, about 0.2 microcurie to about 2 microcurie, about 0.2 microcurie to about 3 microcurie, about 0.2 microcurie to about 4 microcurie, about 0.2 microcurie to about 5 microcurie, about 0.2 microcurie to about 10 microcurie, about 0.2 microcurie to about 20 microcurie, about 0.5 microcurie to about 1 microcurie, about 0.5 microcurie to about 2 microcurie, about 0.5 microcurie to about 3 microcurie, about 0.5 microcurie to about 4 microcurie, about 0.5 microcurie to about 5 microcurie, about 0.5 microcurie to about 10 microcurie, about 0.5 microcurie to about 20 microcurie, about 1 microcurie to about 2 microcurie, about 1 microcurie to about 3 microcurie, about 1 microcurie to about 4 microcurie, about 1 microcurie to about 5 microcurie, about 1 microcurie to about 10 microcurie, about 1 microcurie to about 20 microcurie, about 2 microcurie to about 3 microcurie, about 2 microcurie to about 4 microcurie, about 2 microcurie to about 5 microcurie, about 2 microcurie to about 10 microcurie, about 2 microcurie to about 20 microcurie, about 3 microcurie to about 4 microcurie, about 3 microcurie to about 5 microcurie, about 3 microcurie to about 10 microcurie, about 3 microcurie to about 20 microcurie, about 4 microcurie to about 5 microcurie, about 4 microcurie to about 10 microcurie, about 4 microcurie to about 20 microcurie, about 5 microcurie to about 10 microcurie, about 5 microcurie to about 20 microcurie, or about 10 microcurie to about 20 microcurie. In certain embodiments, the effective amount of 225-Ac is about 0.1 microcurie, about 0.2 microcurie, about 0.5 microcurie, about 1 microcurie, about 2 microcurie, about 3 microcurie, about 4 microcurie, about 5 microcurie, about 10 microcurie, or about 20 microcurie. In certain embodiments, the effective amount of 225-Ac is at least about 0.1 microcurie, about 0.2 microcurie, about 0.5 microcurie, about 1 microcurie, about 2 microcurie, about 3 microcurie, about 4 microcurie, about 5 microcurie, or about 10 microcurie. In certain embodiments, the effective amount of 225-Ac is at most about 0.2 microcurie, about 0.5 microcurie, about 1 microcurie, about 2 microcurie, about 3 microcurie, about 4 microcurie, about 5 microcurie, about 10 microcurie, or about 20WSGR Attorney Docket No.: 60924-730.601 microcurie. According to aspects where the radioisotope of the radioimmunoconjugate is 111-In, the effective amount is below, for example, 15.0 mCi (i.e., where the amount of 111-In administered to the subject delivers a total body radiation dose of below 15.0 mCi).

[0186] According to aspects where the radioisotope of the radioimmunoconjugate is 111-In,the effective amount is below 15.0 mCi, below 14.0 mCi, below 13.0 mCi, below 12.0 mCi, below 11.0 mCi, below 10.0 mCi., below 9.0 mCi, below 8.0 mCi, below 7.0 mCi, below 6.0 mCi, below 5.0 mCi, below 4.0 mCi, below 3.5 mCi, below 3.0 mCi, below 2.5 mCi, below 2.0 mCi, below 1.5 mCi, below 1.0 mCi, below 0.5 mCi, below 0.4 mCi, below 0.3 mCi, below 0.2 mCi, or below 0.1 mCi.

[0187] According to aspects where the radioisotope of the radioimmunoconjugate is 111-In,the effective amount is from 0.1 mCi to 1.0 mCi, from 0.1 mCi to 2.0 mCi, from 1.0 mCi to 2.0 mCi, from 1.0 mCi to 3.0 mCi, from 1.0 mCi to 4.0 mCi, from 1.0 mCi to 5.0 mCi, from 1.0 mCi to 10.0 mCi, from 1.0 mCi to 15.0 mCi, from 1.0 mCi to 20.0 mCi, from 2.0 mCi to 3.0 mCi, from 3.0 mCi to 4.0 mCi, from 4.0 mCi to 5.0 mCi, from 5.0 mCi to 10.0 mCi, from 5.0 mCi to 15.0 mCi, from 5.0 mCi to 20.0 mCi, from 6.0 mCi to 14.0 mCi, from 7.0 mCi to 13.0 mCi, from 8.0 mCi to 12.0 mCi, from 9.0 mCi to 11.0 mCi, or from 10.0 mCi to 15.0 mCi.

[0188] According to aspects where the radioisotope of the radioimmunoconjugate is 111-In,the effective amount is 15.0 mCi, 14.0 mCi, 13.0 mCi, 12.0 mCi, 11.0 mCi, 10.0 mCi, 9.0 mCi, 8.0 mCi, 7.0 mCi, 6.0 mCi, 5.0 mCi, 4.0 mCi, 3.5 mCi, 3.0 mCi, 2.5 mCi, 2.0 mCi, 1.5 mCi, 1.0 mCi, 0.5 mCi, 0.4 mCi, 0.3 mCi, 0.2 mCi, or 0.1 mCi.

[0189] According to aspects where the radioisotope of the radioimmunoconjugate is 225-Ac,the effective amount is below, for example, 30.0 µCi / kg (i.e., where the amount of 225-Ac administered to the subject delivers a radiation dose of below 30.0 µCi per kilogram of subject’s body weight).

[0190] According to aspects where the radioisotope of the radioimmunoconjugate is 225-Ac,the effective amount is below 30 µCi / kg, 25 µCi / kg, 20 µCi / kg, 17.5 µCi / kg, 15.0 µCi / kg, 12.5 µCi / kg, 10.0 µCi / kg, 9 µCi / kg, 8 µCi / kg, 7 µCi / kg, 6 µCi / kg, 5 µCi / kg, 4.5 µCi / kg, 4.0 µCi / kg, 3.5 µCi / kg, 3.0 µCi / kg, 2.5 µCi / kg, 2.0 µCi / kg, 1.5 µCi / kg, 1.0 µCi / kg, 0.9 µCi / kg, 0.8 µCi / kg, 0.7 µCi / kg, 0.6 µCi / kg, 0.5 µCi / kg, 0.4 µCi / kg, 0.3 µCi / kg, 0.2 µCi / kg, 0.1 µCi / kg, or 0.05 µCi / kg.

[0191] According to aspects where the radioisotope of the radioimmunoconjugate is 225-Ac,the effective amount is from 0.05 µCi / kg to 0.1 µCi / kg, from 0.1 µCi / kg to 0.2 µCi / kg, from 0.2 µCi / kg to 0.3 µCi / kg, from 0.3 µCi / kg to 0.4 µCi / kg, from 0.4 µCi / kg to 0.5 µCi / kg, from 0.5WSGR Attorney Docket No.: 60924-730.601 µCi / kg to 0.6 µCi / kg, from 0.6 µCi / kg to 0.7 µCi / kg, from 0.7 µCi / kg to 0.8 µCi / kg, from 0.8 µCi / kg to 0.9 µCi / kg, from 0.9 µCi / kg to 1.0 µCi / kg, from 1.0 µCi / kg to 1.5 µCi / kg, from 1.5 µCi / kg to 2.0 µCi / kg, from 2.0 µCi / kg to 2.5 µCi / kg, from 2.5 µCi / kg to 3.0 µCi / kg, from 3.0 µCi / kg to 3.5 µCi / kg, from 3.5 µCi / kg to 4.0 µCi / kg, from 4.0 µCi / kg to 4.5 µCi / kg, or from 4.5 µCi / kg to 5.0 µCi / kg.

[0192] According to aspects where the radioisotope of the radioimmunoconjugate is 225-Ac,the effective amount is 0.05 µCi / kg, 0.1 µCi / kg, 0.2 µCi / kg, 0.3 µCi / kg, 0.4 µCi / kg, 0.5 µCi / kg, 0.6 µCi / kg, 0.7 µCi / kg, 0.8 µCi / kg, 0.9 µCi / kg, 1.0 µCi / kg, 1.5 µCi / kg, 2.0 µCi / kg, 2.5 µCi / kg, 3.0 µCi / kg, 3.5 µCi / kg, 4.0 µCi / kg or 4.5 µCi / kg, 5.0 µCi / kg, 6.0 µCi / kg, 7.0 µCi / kg, 8.0 µCi / kg, 9.0 µCi / kg, 10.0 µCi / kg, 12.5 µCi / kg, 15.0 µCi / kg, 17.5 µCi / kg, 20.0 µCi / kg, 25 µCi / kg, or 30 µCi / kg.

[0193] In certain embodiments where the radioisotope of the radioimmunoconjugate is 177-Lu the effective amount is from 0.1 uCi to 100 mCi per meter squared of body surface area.

[0194] In certain embodiments where the radioisotope of the radioimmunoconjugate is 177-Lu the effective amount is from 1 mCi to 100 mCi per meter squared of body surface area. Incertain embodiments, the effective amount is about 1 per meter squared to about 100 per meter squared. In certain embodiments, the effective amount is about 1 per meter squared to about 5 per meter squared, about 1 per meter squared to about 10 per meter squared, about 1 per meter squared to about 15 per meter squared, about 1 per meter squared to about 20 per meter squared, about 1 per meter squared to about 25 per meter squared, about 1 per meter squared to about 75 per meter squared, about 1 per meter squared to about 100 per meter squared, about 5 per meter squared to about 10 per meter squared, about 5 per meter squared to about 15 per meter squared, about 5 per meter squared to about 20 per meter squared, about 5 per meter squared to about 25 per meter squared, about 5 per meter squared to about 75 per meter squared, about 5 per meter squared to about 100 per meter squared, about 10 per meter squared to about 15 per meter squared, about 10 per meter squared to about 20 per meter squared, about 10 per meter squared to about 25 per meter squared, about 10 per meter squared to about 75 per meter squared, about 10 per meter squared to about 100 per meter squared, about 15 per meter squared to about 20 per meter squared, about 15 per meter squared to about 25 per meter squared, about 15 per meter squared to about 75 per meter squared, about 15 per meter squared to about 100 per meter squared, about 20 per meter squared to about 25 per meter squared, about 20 per meter squared to about 75 per meter squared, about 20 per meter squared to about 100 per meter squared, about 25 per meter squared to about 75 per meter squared, about 25 per meter squared to about 100 per meter squared, or about 75 per meter squared to about 100 per meter squared. In certainWSGR Attorney Docket No.: 60924-730.601 embodiments, the effective amount is about 1 per meter squared, about 5 per meter squared, about 10 per meter squared, about 15 per meter squared, about 20 per meter squared, about 25 per meter squared, about 75 per meter squared, or about 100 per meter squared. In certain embodiments, the effective amount is at least about 1 per meter squared, about 5 per meter squared, about 10 per meter squared, about 15 per meter squared, about 20 per meter squared, about 25 per meter squared, or about 75 per meter squared. In certain embodiments, the effective amount is at most about 5 per meter squared, about 10 per meter squared, about 15 per meter squared, about 20 per meter squared, about 25 per meter squared, about 75 per meter squared, or about 100 per meter squared.

[0195] According to certain aspects of the present invention, a preparation ofradioimmunoconjugate of this disclosure, or a composition thereof (e.g., a pharmaceutical composition), may comprise a radiolabeled fraction (radioimmunoconjugate) and an unlabeled fraction (immunoconjugate), wherein the ratio of labeled:unlabeled may be from about 1:1000 to 1:1.

[0196] Moreover, the pharmaceutical compositions may be provided as a single dosecomposition tailored to a specific patient, i.e., as a patient specific therapeutic composition, wherein the amount of labeled and unlabeled immunoconjugate (labeled immunoconjugate, for clarity, being the same as radioimmunoconjugate herein) in the composition may depend on at least a patient weight, height, body surface area, age, gender, and / or disease state or health status. As such, a total volume of the patient specific therapeutic composition may be provided in a vial that is configured to be wholly administered to the patient in one treatment session, such that little to no composition remains in the vial after administration.

[0197] Currently, depending on the stage of the cancer, cancer treatment involves one or acombination of the following therapies: surgery to remove the cancerous tissue, radiation therapy, and chemotherapy. Therapy using radioimmunoconjugate of this disclosure (interchangeably, “radiolabeled immunoconjugate”) may be especially desirable in elderly patients who do not tolerate the toxicity and side effects of chemotherapy well and in metastatic disease where radiation therapy has limited usefulness. For some embodiments, therapy using radiolabeled immunoconjugate of this disclosure are useful to alleviate target antigen-expressing cancers upon initial diagnosis of the disease or during relapse.

[0198] In some embodiments, determining whether a cancer is amenable to treatment bymethods disclosed herein involves detecting the presence of the target antigen in a subject or in a sample from a subject. To determine target antigen expression in a cancer, various detection assays are available. In one embodiment, target antigen overexpression is analyzed byWSGR Attorney Docket No.: 60924-730.601 immunohistochemistry (IHC). Parrafin embedded tissue sections from a tumor biopsy are subjected to the IHC assay and accorded a target antigen staining intensity criteria. Alternatively, or additionally, FISH assays such as the INFORM® (sold by Ventana, AZ, U.S.A.) or PATHVISION® (Vysis, IL, U.S.A.) may be carried out on formalin-fixed, paraffin- embedded tumor tissue to determine the extent (if any) of target antigen overexpression in the tumor.

[0199] Target antigen overexpression or amplification may be evaluated using an in vivodetection assay, e.g., by administering a molecule (such as an antibody construct or immunoconjugate of this disclosure) which binds the molecule to be detected and is tagged with a detectable label (e.g., a radioactive isotope or a fluorescent label) and externally scanning the patient for localization of the label.

[0200] An immunoconjugate or radioimmunoconjugate of this disclosure may be used in,for example, in vitro, ex vivo, and in vivo methods. In one aspect, this disclosure provides methods for inhibiting cell growth or proliferation, either in vivo or in vitro, the method comprising exposing a cell to an immunoconjugate or radioimmunoconjugate of this disclosure under conditions permissive for binding of the immunoconjugate or radioimmunoconjugate to a target antigen. The immunoconjugate or radioimmunoconjugate of this disclosure may also (i) inhibit the growth or proliferation of a cell to which they bind; (ii) induce the death of a cell to which they bind; (iii) inhibit the delamination of a cell to which they bind; (iv) inhibit the metastasis of a cell to which they bind; or (v) inhibit the vascularization of a tumor comprising a cell to which they bind.

[0201] In one aspect, this disclosure provides a method of killing an antigen expressing cell,the method comprising contacting the cell with an immunoconjugate or radioimmunoconjugate of the present invention (or a composition thereof). This method can be used, e.g., to kill, deplete, or eliminate target antigen-expressing cells from a population of mixed cells. This method can be used, e.g., to kill, deplete, or eliminate target antigen-expressing cells from a population of mixed cells as a step in the purification of other cells. This method can be performed in vitro or in vivo, including ex vivo on primary patient cell or tissue compositions to prepare such compositions for transplantation.

[0202] In one aspect, an immunoconjugate or radioimmunoconjugate of this disclosure isused to treat or prevent a cell proliferative disorder. In certain embodiments, the cell proliferative disorder comprises a solid tumor cancer. A solid tumor cancer is a cancer comprising an abnormal mass of tissue, e.g., carcinomas and sarcomas. In certain other embodiments, the cell proliferative disorder comprises a liquid tumor cancer or hematologicalWSGR Attorney Docket No.: 60924-730.601 cancer, Used interchangeably, such cancers present in the body fluid, e.g., leukemias and lymphomas. In certain embodiments, the cell proliferative disorder is associated with increased expression and / or activity of a target antigen. For example, in certain embodiments, the cell proliferative disorder is associated with increased expression of target antigen on the surface of a cell. In certain embodiments, the cell proliferative disorder is a tumor or a cancer. In certain embodiments, the cell proliferative disorder comprises a solid tumor cancer. A solid tumor cancer is a cancer comprising an abnormal mass of tissue, e.g., carcinomas and sarcomas. In certain other embodiments, the cell proliferative disorder comprises a liquid tumor cancer or hematological cancer, Used interchangeably, such cancers present in the body fluid, e.g., leukemias and lymphomas.

[0203] In one aspect, this disclosure provides methods for treating a cell proliferativedisorder comprising administering to an individual an effective amount of an immunoconjugate or radioimmunoconjugate of this disclosure.

[0204] In addition to direct cell killing of target cells expressing cell-surface antigenspecifically bound by the immunoconjugate or radioimmunoconjugate of this disclosure, the immunoconjugate or radioimmunoconjugate of the present invention optionally may be used for delivery of additional cargos to the vicinity of or the interiors of target cells. The delivery of additional exogenous materials may be used, e.g., for cytotoxic, cytostatic, information gathering, and / or diagnostic functions. Non-cytotoxic variants of the immunoconjugate or radioimmunoconjugate of this disclosure, or optionally toxic variants, may be used to deliver cargos to and / or label the interiors of cells expressing the target antigen. Non-limiting examples of cargos include cytotoxic agents, detection-promoting agents, and small molecule chemotherapeutic agents.

[0205] As described herein, in some embodiments, the antibody constructs,immunoconjugates, radioimmunoconjugates and targeted imaging complexes of the present invention have various non-therapeutic applications. In some embodiments, the compositions of this disclosure may be used to identify patient populations predicted to benefit from a specific therapeutic approach or modality, such as, e.g., treatment with an immunoconjugates or radioimmunoconjugates of this disclosure. In some embodiments, the compositions of this disclosure can be useful for staging of target antigen expressing cancers (e.g., by radioimaging) or as prognostic indicators of disease progression. In some embodiments, the compositions are also useful for detection and quantitation of a target epitope in vitro, e.g., in an ELISA or a Western blot, as well as purification or immunoprecipitation of a target antigen from cells or a tissue sample.WSGR Attorney Docket No.: 60924-730.601

[0206] For some embodiments, the immunoconjugate or radioimmunoconjugate of thisdisclosure is used in a method to detect the presence of or level of an antigen, such as, e.g., in vitro in a biological sample or in vivo using an imagine technique. Immunoconjugate and radioimmunoconjugate detection can be achieved via different techniques known to the skilled worker and as described herein, e.g., IHC and PET imaging. When an immunoconjugate or radiolabeled immunoconjugate of this disclosure is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example 99m-Tc or 111-In.

[0207] Another embodiment of the present invention is directed to a method of diagnosingthe presence of a tumor in a subject, wherein the method comprises (a) contacting a test sample comprising tissue cells obtained from the mammal with an immunoconjugate that binds to a target antigen and (b) detecting the formation of a complex between the immunoconjugate and the target antigen in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in the mammal. Optionally, the immunoconjugate is detectably labeled, attached to a solid support, or the like, and / or the test sample of tissue cells is obtained from an individual suspected of having a cancerous tumor.

[0208] In some embodiments, the immunoconjugates of the present invention, includingcompositions comprising the aforementioned and / or provided herein are useful for detecting the presence of a target antigen, e.g., in vivo or in a biological sample. The immunoconjugates of this disclosure can be used in a variety of different assays, including but not limited to ELISA, bead-based immunoassays, and mass spectrometry. Methods of Producing the Immunoconjugates of the Present Disclosure

[0209] The present disclosure provides a composition comprising one or more of theimmunoconjugates according to any of the above embodiments or described herein. In another aspect, this disclosure provides an isolated nucleic acid encoding a radioisotope delivering platform as described herein. Also provided herein are nucleic acids encoding the protein components of the immunoconjugates of the present disclosure, expression vectors comprising the aforementioned nucleic acid, and host cells comprising the aforementioned expression vectors.

[0210] In another aspect, this disclosure provides a host cell comprising a nucleic acidand / or vector as provided herein. In some embodiments, the host cell of the present disclosure is isolated or purified. In some embodiments, the host cell of the present disclosure is in a cell culture medium. The nucleic acids, expression vectors, and host cells of this disclosure may be used to produce a composition comprising one or more of the immunoconjugates of thisWSGR Attorney Docket No.: 60924-730.601 disclosure. In some embodiments, the host cell is eukaryotic. In some embodiments, the host cell is mammalian. 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 E. coli.

[0211] A description follows as to illustrative techniques for the production of theimmunoconjugates and radioimmunoconjugates of the present disclosure for use in accordance with the methods of the present disclosure. In some embodiments, this disclosure provides a process for making an immunoconjugate of the present disclosure, the method comprising culturing a host cell as provided herein under conditions suitable for the expression vector encoding the radioisotope delivery platform and recovering or purifying the radioisotope delivery platform. In some embodiments, the method further comprises radiolabeling the radioisotope delivery platform with an appropriate isotope, such as, e.g., an alpha or beta particle emitter. Kits and Articles of Manufacture of the Present Invention

[0212] Another aspect of the present invention is an article of manufacture containingmaterials useful for the treatment, prevention and / or diagnosis of diseases and disorders characterized by target antigen-expressing cells (e.g., a cancer cell). The article of manufacture of this disclosure comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating, preventing and / or diagnosing the cancer condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an immunoconjugate of this disclosure. The label or package insert indicates that the composition is used for treating cancer. The label or package insert will further comprise instructions for administering the immunoconjugate composition to the cancer patient. Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer’s solution and dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0213] In another aspect, this disclosure provides a kit comprising any of theimmunoconjugates described herein and an additional reagent or pharmaceutical device. In some further embodiments, the kit comprises a composition as provided herein (e.g., a pharmaceutical or diagnostic composition). Another aspect of the present invention is a kit useful for variousWSGR Attorney Docket No.: 60924-730.601 purposes, e.g., target antigen-expressing cell killing; for target antigen-expressing cell detection; quantification, purification, or immunoprecipitation of target antigen from cells.

[0214] In some embodiments, the kit of this disclosure is an immunoassay kit forspecifically detecting an antigen in a biological sample, comprising: (a) an immunoconjugate as described herein and / or a composition thereof; and (b) instructions for detecting said immunoconjugate. A target antigen detection assays of the present invention can be provided in the form of a kit. In some embodiments, such a kit comprises an immunoconjugate of the present invention, or a composition comprising the aforementioned, such as one described herein. The kit may further comprise a solid support for the capture reagents, which may be provided as a separate element or to which the capture reagents are already immobilized. For isolation and purification of a target antigen, the kit may contain an immunoconjugate of this disclosure coupled to beads (e.g., sepharose beads). This disclosure provides kits that contain an antibody for the detection and / or quantitation of target antigen in vitro, e.g., in an ELISA or a Western blot. In some embodiments, the capture reagents (e.g., the immunoconjugate of this disclosure) are coated on or attached to a solid material (e.g., to beads, a microtiter plate, or a comb). The detectable antibodies may be labeled antibodies detected directly or unlabeled antibodies that are detected by labeled antibodies directed against the unlabeled antibodies, such as, e.g., antibodies raised in a different species. Where the label is an enzyme, the kit will ordinarily include substrates and cofactors required by the enzyme; where the label is a fluorophore, a dye precursor that provides the detectable chromophore; and where the label is biotin, an avidin such as avidin, streptavidin, or streptavidin conjugated to HRP or β- galactosidase with MUG.

[0215] As with the article of manufacture of this disclosure, the kit of this disclosurecomprises a container and a label or package insert on or associated with the container. The container holds a composition comprising at least one immunoconjugate of this disclosure. Additional containers may be included that contain, e.g., diluents and buffers, control immunconjugates or antibodies. The label or package insert may provide a description of the composition as well as instructions for the intended in vitro or detection use. The kit also typically contains additives such as stabilizers, washing and incubation buffers, and the like for performing the assay method(s). The components of the kit will be provided in predetermined ratios, with the relative amounts of the various reagents suitably varied to provide for concentrations in solution of the reagents that substantially maximize the sensitivity of the assay(s). Particularly, the reagents may be provided as dry powders, usually lyophilized,WSGR Attorney Docket No.: 60924-730.601 including excipients, which on dissolution will provide for a reagent solution having the appropriate concentration for combining with the sample to be tested.

[0216] The present invention is further illustrated by the following non-limiting examples ofimmunoconjugates comprising the aforementioned structures and functions, in particular platforms having VHH polypeptides, a molecular weight between 60 and 110 kDa, a serum half- life of less than 96 hours, which in some embodiments exhibit enhanced stability during the temperatures required for certain radiolabeling processes relative to other antibody fragment platforms, and which in some embodiments exhibit decreased loss of targeting capacity due to radiolysis as compared to other possible delivery platforms. Exemplary EmbodimentsExemplary embodiment 1. An immunoconjugate comprising an antibody that binds DLL3attached (e.g., conjugated) to a compound, wherein: the antibody comprises a VHH variable domain and optionally an Fc domain, wherein the VHH variable domain comprises: a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 14, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 15, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 16; a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 17, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 18, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 19; a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 20, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 21, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 22; a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 23, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ IDWSGR Attorney Docket No.: 60924-730.601 NO: 24, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 25; or a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 26, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 27, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 28; and the compound comprises a structure represented by Formula (I) or a pharmaceutically acceptable salt thereof: :wherein: R1is a chelating moiety or a radionuclide complex thereof; X1is -O-, -S-, -S(=O)-, -S(=O)2-, -NRa-, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, -(C1-C6alkylene)-X2-, or -(C4-C20polyethylene glycol)-X2-; X2is absent, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, or -C(=O)X4-; each Rais independently selected from hydrogen, and C1-C4alkyl; X4is -NRa-, or -NRaS(=O)2-;L is an optional linker; R2is a moiety that is capable of reacting with an amine (-NH2) or thiol (- SH) of the antibody; and v is 1, 2, 3, or 4.Exemplary embodiment 2. The immunoconjugate of embodiment 1, wherein: R2 is a moietythat is capable of reacting with an amine (-NH2) of the antibody and comprises a tetrafluorophenyl ester, pentafluorophenyl ester, dinitrophenyl ester, succinimide ester, sulfosuccinimide ester, or isothiocyanate.Exemplary embodiment 3. The immunoconjugate of embodiment 1, wherein: R2 is a moietythat is capable of reacting with an amine (-NH2) of the antibody and comprises:WSGR Attorney Docket No.: 60924-730.601 , or , -Exemplary embodiment 4. An immunoconjugate comprising an antibody that binds DLL3attached (e.g., conjugated) to a compound, wherein: the antibody comprises a VHH variable domain and optionally an Fc domain an Fc domain, wherein the VHH variable domain comprises: a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 14, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 15, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 16; a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 17, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 18, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 19; a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 20, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 21, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 22;WSGR Attorney Docket No.: 60924-730.601 a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 23, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 24, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 25; or a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 26, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 27, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 28; and the compound comprises a structure represented by Formula (II), Formula (III), or Formula (IV), or a pharmaceutically acceptable salt thereof: wherein:R1is a chelating moiety or a radionuclide complex thereof; X1is -O-, -S-, -S(=O)-, -S(=O)2-, -NRa-, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, -(C1-C6alkylene)-X2-, or -(C4-C20polyethylene glycol)-X2-; X2is absent, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, or -C(=O)X4-; each Rais independently selected from hydrogen, and C1-C4alkyl; X4is -NRa-, or -NRaS(=O)2-; L is an optional linker; -NH-R3is the antibody; and v is 1, 2, 3, or 4.WSGR Attorney Docket No.: 60924-730.601Exemplary embodiment 5. The immunoconjugate of embodiment 4, wherein: R2 is a moietythat is capable of reacting with a thiol (-SH) of a antibody R3and comprises a maleimide group, a haloacetamide group, a haloacetyl group, a haloacetate group, a pyrdinylthio group, a vinylcarbonyl group, an aziridinyl group, a disulfide group, an acetylene group, a hydroxysuccinimide group or a thiol group.Exemplary embodiment 6. The immunoconjugate of embodiment 4, wherein: R2 is a moietythat is capable of reacting with a thiol (-SH) of the antibody R3and comprises: , ,Exemplary embodiment 7. The immunoconjugate of embodiment 4, wherein the compoundcomprises a structure represented by Formula (IIa), or a pharmaceutically acceptable salt thereof:wherein: NHCH2CH2CH2CH2-is the side chain of a lysine residue of the antibody R3.Exemplary embodiment 8. An immunoconjugate comprising an antibody that binds DLL3attached (e.g., conjugated) to a compound, wherein: the antibody comprises a VHH variable domain and optionally an Fc domain, wherein the variable domain comprises:WSGR Attorney Docket No.: 60924-730.601 a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 14, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 15, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 16; a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 17, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 18, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 19; a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 20, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 21, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 22; a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 23, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 24, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 25; or a heavy chain complementarity determining region 1 (CDRH1) comprising an amino acid sequence of SEQ ID NO: 26, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 27, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 28; and the compound comprises a structure represented by Formula (V), Formula (VI), Formula (VII), or Formula (VIII), or a pharmaceutically acceptable salt thereof:WSGR Attorney Docket No.: 60924-730.601 Formula (VI)wherein: R1is a chelating moiety or a radionuclide complex thereof; X1is -O-, -S-, -S(=O)-, -S(=O)2-, -NRa-, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, -(C1-C6alkylene)-X2-, or -(C4-C20polyethylene glycol)-X2-; X2is absent, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, or -C(=O)X4-; each Rais independently selected from hydrogen, and C1-C4alkyl; X4is -NRa-, or -NRaS(=O)2-; L is an optional linker; -S-R3is the antibody; and v is 1, 2, 3, or 4.Exemplary embodiment 9. The immunoconjugate of any one of embodiments 1-8, wherein:wherein R1is a chelating moiety or a radionuclide complex thereof, wherein the chelating moiety is: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); 1,4,7,10-tetraazacyclododecane-1,4,7 -triacetic acid (DO3A); 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A); α,α',α'',α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTMA); 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM); 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid (DOTPA); 2,2',2''-(10-(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7- triyl)triacetic acid;WSGR Attorney Docket No.: 60924-730.601 6,6'-(((pyridine-2,6-diylbis(methylene))bis((carboxymethyl)azanediyl))- bis(methylene))dipicolinic acid (H4pypa); 6,6',6'',6'''-(((pyridine-2,6- diylbis(methylene))bis(azanetriyl))tetrakis(methylene))-tetrapicolinic acid (H4py4pa); 10-((6-carboxypyridin-2-yl)methyl)-1,4,7,10-tetra-azacyclododecane- 1,4,7-triacetic acid (DO3Apic); or 3,6,9,12-tetrakis(carboxymethyl)-3,6,9,12-tetraazatetradecanedioic acid (TTHA).Exemplary embodiment 10. The immunoconjugate of any one of embodiments 1-8, wherein:R1is a chelating moiety or a radionuclide complex thereof, wherein the chelating moiety is: .Exemplaryis a chelating moiety or a radionuclide complex thereof, wherein the chelating moiety is: ;Exemplaryof any one of embodiments 1-8, wherein:R1is a chelating moiety or a radionuclide complex thereof, wherein the chelating moiety is:WSGR Attorney Docket No.: 60924-730.601 .:Exemplary of any one of embodiments 1-12, wherein:- , - , - , -C(=O)-, -NRaC(=O)-, or -C(=O)NRa-; or X1is -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, - CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -CH2-X2-, -CH2CH2-X2-, - CH2CH2CH2-X2-, -CH2CH2CH2CH2-X2-, -CH2CH2CH2CH2CH2-X2-, or - CH2CH2CH2CH2CH2CH2-X2-.Exemplary embodiment 14. The immunoconjugate of any one of embodiments 1-12, wherein:X1is -CH2CH2-or -CH2CH2-X2-; X2is -C(=O)X4-; X4is -NH-, -N(CH3)-, or -N(CH2CH3)-.Exemplary embodiment 15. The immunoconjugate of any one of embodiments 1-14, wherein:L is -L1-, or -L1-L2-L3-L4-L5-; L1is unsubstituted or substituted C1-C20alkylene, unsubstituted or substituted C1- C20heteroalkylene, C4-C20polyethylene glycol, unsubstituted or substituted C3- C8cycloalkylene, unsubstituted or substituted monocyclic C3- C8heterocycloalkylene, unsubstituted or substituted phenylene, unsubstituted or substituted monocyclic heteroarylene; L2is absent, -C(=O)NR4-(unsubstituted or substituted C1-C10alkylene)-, - NR4C(=O)-(unsubstituted or substituted C1-C10alkylene)-, -C(=O)-(CH2CH2O)m- (CH2)P-, -C(=O)NR4-(CH2CH2O)n-(CH2)P-, -NR4C(=O)-(CH2CH2O)n-(CH2)P-, or -(CH2CH2O)n-(CH2)P-; each R4is independently selected from hydrogen, and C1-C6alkyl; each m is independently 1, 2, 3, 4, 5, or 6; each p is independently 1, or 2; L3is absent; L4is absent, -C(=O)-( unsubstituted or substituted C1-C6alkylene)-, -C(=O)NR4- (unsubstituted or substituted C1-C6alkylene)-, -NR4C(=O)-(unsubstituted or substituted C1-C6alkylene)-, -C(=O)-(CH2CH2O)n-(CH2)q-, -C(=O)NR4-WSGR Attorney Docket No.: 60924-730.601 (CH2CH2O)n-(CH2)q-, -NR4C(=O)-(CH2CH2O)n-(CH2)q-, or -(CH2CH2O)n- (CH2)q-; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2; L5is absent, -C(=O)-(CH2)n-, -C(=O)NR4-(CH2)n-, -NR4C(=O)-(CH2)n-, -C(=O)- (CH2CH2O)n-(CH2)q-, -C(=O)NR4-(CH2CH2O)n-(CH2)q-, -NR4C(=O)- (CH2CH2O)n-(CH2)q-, -(CH2CH2O)n-(CH2)q-, -C(=O)-(OCH2CH2)n-, or - (OCH2CH2)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; wherein heteroalkylene is an alkylene where one carbon atom is replaced with - S(=O)(=NH)-, -S(=O)(=NR5)-, -P(=O)OH-, -NHC(=N-CN)NH-, or -NHC(=N- R5)NH-; wherein when any one of -L1-, -L2-, -L3-, -L4-, and -L5-is substituted then -L1-, - L2-, -L3-, -L4-, and -L5-is substituted with 1, 2, 3, or 4 groups selected from halogen, -OH, -OR5, -CO2H, -NHR5, -C(=O)NHR5, -NHC(=O)R5and substituted C1- C6alkyl, wherein the substituted C1-C6alkyl is substituted with , -OH, -CO2H, - NHR5, -C(=O)NHR5, and -NHC(=O)R5; each R5is independently selected from C1-C10alkyl, C4-C30polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene.Exemplary embodiment 16. The immunoconjugate of any one of embodiments 1-15, wherein:L1is unsubstituted or substituted C1-C6alkylene, unsubstituted or substituted C1- C10heteroalkylene, C4-C20polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene.Exemplary embodiment 17. The immunoconjugate of any one of embodiments 1-15, wherein:L5is absent, -NR4C(=O)-(CH2)n-, -C(=O)-(CH2CH2O)n-(CH2)q-, -C(=O)NR4- (CH2CH2O)n-(CH2)q-, or -NR4C(=O)-(CH2CH2O)n-(CH2)q-; each q is independently 1, or 2.Exemplary embodiment 18. The immunoconjugate of any one of embodiments 1-14, wherein:L1is unsubstituted or substituted C1-C6alkylene, unsubstituted or substituted C1- C10heteroalkylene, C4-C20polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L2is absent, -C(=O)NR4-(unsubstituted or substituted C1-C10alkylene)-, - NR4C(=O)-(unsubstituted or substituted C1-C10alkylene)-, -C(=O)-(CH2CH2O)m-WSGR Attorney Docket No.: 60924-730.601 (CH2)P-, -C(=O)NR4-(CH2CH2O)n-(CH2)P-, -NR4C(=O)-(CH2CH2O)n-(CH2)P-, or -(CH2CH2O)n-(CH2)P-; each m is independently 1, 2, 3, 4, 5, or 6; each p is independently 1 or 2; L4is absent; L5is -NR4C(=O)-(CH2)n-, -C(=O)-(CH2CH2O)n-(CH2)q-, -C(=O)NR4- (CH2CH2O)n-(CH2)q-, or -NR4C(=O)-(CH2CH2O)n-(CH2)q-; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2.Exemplary embodiment 19. The immunoconjugate of any one of embodiments 1-14, wherein:L1is unsubstituted or substituted C1-C6alkylene, unsubstituted or substituted C1- C10heteroalkylene, C4-C20polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L2is absent, -C(=O)NR4-(unsubstituted or substituted C1-C10alkylene)-, - NR4C(=O)-(unsubstituted or substituted C1-C10alkylene)-, -C(=O)-(CH2CH2O)m- (CH2)P-, -C(=O)NR4-(CH2CH2O)n-(CH2)P-, -NR4C(=O)-(CH2CH2O)n-(CH2)P-, or -(CH2CH2O)n-(CH2)P-; each m is independently 1, 2, 3, 4, 5, or 6; each p is independently 1 or 2; L4is absent; L5is absent, -NR4C(=O)-(CH2)n-, -C(=O)-(CH2CH2O)n-(CH2)q-, -C(=O)NR4- (CH2CH2O)n-(CH2)q-, or -NR4C(=O)-(CH2CH2O)n-(CH2)q-; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2.Exemplary embodiment 20. The immunoconjugate of any one of embodiments 1-14, wherein:L1is unsubstituted or substituted C1-C6alkylene, unsubstituted or substituted C1- C10heteroalkylene, C4-C20polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L2is absent; L4is absent; L5is absent, -NR4C(=O)-(CH2)n-, -C(=O)-(CH2CH2O)n-(CH2)q-, -C(=O)NR4- (CH2CH2O) eacheach q is independently 1 or 2.Exemplary embodiment 21. The immunoconjugate of any one of embodiments 1-8, wherein:WSGR Attorney Docket No.: 60924-730.601 R1is a chelating moiety or a radionuclide complex thereof, wherein the chelating moiety is: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); 1,4,7,10-tetraazacyclododecane-1,4,7 -triacetic acid (DO3A); 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A); α,α',α'',α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTMA); 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM); 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid (DOTPA); 2,2',2''-(10-(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7- triyl)triacetic acid; 6,6'-(((pyridine-2,6-diylbis(methylene))bis((carboxymethyl)azanediyl))- bis(methylene))dipicolinic acid (H4pypa); 6,6',6'',6'''-(((pyridine-2,6-diylbis(methylene))bis(azanetriyl))tetrakis (methylene))-tetrapicolinic acid (H4py4pa); 10-((6-carboxypyridin-2-yl)methyl)-1,4,7,10-tetra-azacyclododecane- 1,4,7-triacetic acid (DO3Apic); or 3,6,9,12-tetrakis(carboxymethyl)-3,6,9,12-tetraazatetradecanedioic acid (TTHA).Exemplary embodiment 22. The immunoconjugate of any one of embodiments 1-8, wherein:R1a radionuclide complex thereof; X1isX1is -CH2CH2-X2-; X2is -C(=O)X4-; X4is -NH-, -N(CH3)-, or -N(CH2CH3)-. L is -L1-, or -L1-L2-L3-L4-L5-; L1is unsubstituted or substituted C1-C6alkylene, unsubstituted or substituted C1- C10heteroalkylene, C4-C20polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene;WSGR Attorney Docket No.: 60924-730.601 L2is absent; L3is absent; L4is absent; L5is -NR4C(=O)-(CH2)n-, -C(=O)-(CH2CH2O)n-(CH2)q-, -C(=O)NR4- (CH2CH2O)n-(CH2)q-, or -NR4C(=O)-(CH2CH2O)n-(CH2)q-; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2.Exemplary embodiment 23. The immunoconjugate of any one of embodiments 1-22, whereineach Rais independently selected from C1-C4alkyl; each R4is independently selected from C1-C4alkyl.Exemplary embodiment 24. The immunoconjugate of any one of embodiments 1-8, wherein:X1-L-is: .Exemplaryof embodiments 1-8, wherein:.Exemplary1-8, wherein:,WSGR Attorney Docket No.: 60924-730.601Exemplary embodiment 27. The immunoconjugate of any one of embodiments 1-26, whereinthe radionuclide is a diagnostic or therapeutic radionuclide.Exemplary embodiment 28. The immunoconjugate of any one of embodiments 1-26, whereinthe radionuclide is an Auger electron-emitting radionuclide, α-emitting radionuclide, β-emitting radionuclide, or γ-emitting radionuclide.Exemplary embodiment 29. The immunoconjugate of any one of embodiments 1-26, whereinthe radionuclide is α-emitting radionuclide.Exemplary embodiment 30. The immunoconjugate of any one of embodiments 1-26, wherein:the radionuclide is an Auger electron-emitting radionuclide that is 111-indium (111In), 67-gallium (67Ga), 68-gallium (68Ga), 99m-technetium (99mTc), or 195m-platinum (195mPt).; or the radionuclide is an α-emitting radionuclide that is 225-actinium (225Ac), 213-bismuth (213Bi), 223-Radium (223Ra), or 212-lead (212Pb). or the radionuclide is a β-emitting radionuclide that is 90-yttrium (90Y), 177-lutetium (177Lu), 186-rhenium (186Re), 188-rhenium (188Re), 64-copper (64Cu), 67-copper (67Cu), 153-samarium (153Sm), 89-strontium (89Sr), 198-gold (198Au), 169-Erbium (169Er), 165- dysprosium (165Dy), 99m-technetium (99mTc), 89-zirconium (89Zr), or 52-manganese (52Mn); or the radionuclide is a γ-emitting radionuclide that is 60-cobalt (60Co), 103-palldium (103Pd), 137-cesium (137Cs), 169-ytterbium (169Yb), 192-iridium (192Ir), or 226-radium (226Ra).Exemplary embodiment 31. The immunoconjugate of any one of embodiments 1-26, whereinthe radionuclide is suitable for positron emission tomography (PET) analysis, single-photon emission computerized tomography (SPECT), or magnetic resonance imaging (MRI).Exemplary embodiment 32. The immunoconjugate of any one of embodiments 1-26, whereinthe radionuclide is 225-actinium (225Ac).Exemplary embodiment 33. The immunoconjugate of any one of embodiments 1-32, whereinthe antibody comprises the Fc domain, and wherein the Fc domain comprises an immunoglobulin CH2 domain, immunoglobulin CH3 domain, or both an immunoglobulin CH2 and immunoglobulin CH3 domain.Exemplary embodiment 34. The immunoconjugate of any one of embodiments 1-33 wherein theantibody has molecular weight between 60 and 110 kDa.Exemplary embodiment 35. The immunoconjugate of embodiment 34, wherein the Fc domaincomprises both the immunoglobulin CH2 and the immunoglobulin CH3 domain.Exemplary embodiment 36. The immunoconjugate of any one of embodiments 1-35, whereinthe Fc domain is an IgA, IgG1, IgG2, IgG3, or IgG4 isotype.WSGR Attorney Docket No.: 60924-730.601Exemplary embodiment 37. The immunoconjugate of any one of embodiments 1-36, whereinthe Fc domain is an IgG1 isotype.Exemplary embodiment 38. The immunoconjugate of any one of embodiments 1-37, whereinFc domain is an IgG4 isotype.Exemplary embodiment 39. The immunoconjugate of any one of embodiments 1-38, whereinthe Fc domain comprises an alteration to one or more amino acid residues that reduces an effector function of the Fc domain or alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn) (e.g., relative to wild-type IgG1).Exemplary embodiment 40. The immunoconjugate of any one of embodiments 1-39, whereinthe Fc domain comprises an alteration to one or more amino acid residues that reduces an effector function of the Fc domain and an alteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) (e.g., relative to wild-type IgG1).Exemplary embodiment 41. The immunoconjugate of any one of embodiments 1-40, whereinthe Fc domain comprises an alteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) (e.g., relative to wild-type IgG1).Exemplary embodiment 42. The immunoconjugate of any one of embodiments 1-41, whereinthe Fc domain comprises an alteration to one or more amino acid residues that reduces an effector function of the Fc domain.Exemplary embodiment 43. The immunoconjugate of any one of embodiment 1-42, wherein theFc domain comprises an alteration to one or more amino acid residues that reduces the effector function, wherein the alteration reduces complement dependent cytotoxicity (CDC), antibody- dependent cell-cytotoxicity (ADCC), antibody-dependent cell-phagocytosis ADCP, or a combination thereof (e.g., relative to wild-type IgG1).Exemplary embodiment 44. The immunoconjugate of any one of embodiments 1-43, whereinthe Fc domain comprises an alteration to one or more amino acid residues that reduces the effector function of the Fc domain (e.g., relative to wild-type IgG1), wherein the alteration comprises L234A, L235E, G237A, A330S, and P331S per EU numbering.Exemplary embodiment 45. The immunoconjugate of any one of embodiments 1-44, whereinthe Fc domain comprises an alteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) reduces the serum half-life of the immunoconjugate.Exemplary embodiment 46. The immunoconjugate of any one of embodiments 1-45, whereinthe Fc domain comprises an alteration to one or more amino acid residues that reduces binding ofWSGR Attorney Docket No.: 60924-730.601 the immunoconjugate to the neonatal Fc receptor (FcRn), wherein the alteration comprises H310A, H435Q, or both H310A and H435Q per EU numbering.Exemplary embodiment 47. The immunoconjugate of any one of embodiments 1-46, whereinthe antibody comprises an hinge region connecting the VHH variable domain and the Fc domain.Exemplary embodiment 48. The immunoconjugate of embodiment 47, wherein the human IgGhinge region comprises the amino acid sequence set forth in SEQ ID NO: 10 or 11.Exemplary embodiment 49. The immunoconjugate of any one of embodiments 1-47, whereinthe VHH variable domain comprises an amino acid sequence having at least 90% or greater sequence identity to any one of SEQ ID NOs: 29-34.Exemplary embodiment 50. The immunoconjugate of any one of embodiments 1-49, whereinthe VHH variable domain comprises an amino acid sequence having at least 90% or greater sequence identity to SEQ ID NO: 31.Exemplary embodiment 51. The immunoconjugate of any one of embodiments 1-50, whereinthe VHH variable domain comprises the amino acid sequence of SEQ ID NO: 31.Exemplary embodiment 52. The immunoconjugate of any one of embodiments 1-51, whereinthe antibody comprises an amino acid sequence having at least 90% or greater sequence identity to any one of SEQ ID NOs: 35-39.Exemplary embodiment 53. The immunoconjugate of any one of embodiments 1-52, whereinthe antibody comprises an amino acid sequence having at least 90% or greater sequence identity to SEQ ID NO: 36.Exemplary embodiment 54. The immunoconjugate of any one of embodiments 1-53, whereinthe antibody comprises the amino acid sequence of SEQ ID NO: 36Exemplary embodiment 55. The immunoconjugate of any one of embodiments Error!Reference source not found. to 54, wherein the immunoconjugate binds DLL3 having an affinity of KD of 10 nanomolar or less.Exemplary embodiment 56. The immunoconjugate of any one of embodiments Error!Reference source not found. to 55, wherein the immunoconjugate binds DLL3 having an affinity of KD of 5 nanomolar or less.Exemplary embodiment 57. The immunoconjugate of any one of embodiments Error!Reference source not found. to 56, wherein the immunoconjugate binds DLL3 having an affinity of KDof 2 nanomolar or less.Exemplary embodiment 58. The immunoconjugate of any one of embodiments 1-57, whereinthe molecular weight of the antibody is between 60 and 90 kDa.Exemplary embodiment 59. The immunoconjugate of any one of embodiments 1-57, whereinthe molecular weight of the antibody is between 65 and 90 kDa.WSGR Attorney Docket No.: 60924-730.601Exemplary embodiment 60. The immunoconjugate of any one of embodiments 1-57, whereinthe molecular weight of the antibody is between 70 and 90 kDa.Exemplary embodiment 61. A pharmaceutical composition comprising the immunoconjugate ofany one of embodiments 1-60 and a pharmaceutically acceptable excipient or carrier.Exemplary embodiment 62. The pharmaceutical composition comprising the immunoconjugateof any one of embodiments 1-60 formulated for intravenous administration.Exemplary embodiment 63. A method of making the immunoconjugate of any one ofembodiments 1-60, comprising loading the immunoconjugate with a radioisotope.Exemplary embodiment 64. A method of treating a cancer or a tumor in an individualcomprising administering to the individual the immunoconjugate of any one of embodiments 1- 60 having the radionuclide complex as R1; thereby treating the cancer or the tumorExemplary embodiment 65. The method of embodiment 64, wherein the individual is a humanindividual.Exemplary embodiment 66. The method of embodiment 64 or 65, wherein the cancer or thetumor is a solid cancer or tumor.Exemplary embodiment 67. The method of embodiment 64 or 65, wherein the cancer or thetumor comprises lung cancer, breast cancer, ovarian cancer, or a neuroendocrine cancer.Exemplary embodiment 68. The method of any one of embodiments 64-67, comprisingadministering from 0.5 µCi to 30.0 µCi per kilogram to the individual.Exemplary embodiment 69. The method of any one of embodiments 64-68, comprisingadministering from 10 m Ci to 75 mCi per meter squared of body area to the individual.Exemplary embodiment 70. The method of any one of embodiments 64-69, wherein the canceror tumor expresses DLL3.Exemplary embodiment 71. A method of killing a cancer cell in an individual comprisingadministering to the individual the immunoconjugate of any one of embodiments 1-60 having the radionuclide complex as R1, thereby killing the cancer cell.Exemplary embodiment 72. The method of embodiment 71, wherein the individual is a humanindividual.Exemplary embodiment 73. The method of embodiment 71 or 72, wherein the cancer cellcomprises lung cancer cell, a breast cancer cell, an ovarian cancer cell, or a neuroendocrine cancer cell.Exemplary embodiment 74. The method of any one of embodiments 71-73, wherein the cancercell expresses DLL3.Exemplary embodiment 75. A method of delivering a radioisotope to a cancer cell or a tumorcell in an individual comprising administering to the individual the immunoconjugate of any oneWSGR Attorney Docket No.: 60924-730.601 of embodiments 1-60 having the radionuclide complex as R1, thereby delivering the radioisotope to the cancer cell or the tumor cell. Exemplary embodiment 76. A method of imaging a tumor in an individual comprising administering to the individual the immunoconjugate of any one of embodiments 1-60 having the radionuclide complex as R1. Certain definitions

[0217] In this description, certain specific details are set forth in order to provide a thoroughunderstanding of various embodiments. However, one skilled in the art will understand that the embodiments provided may be practiced without these details. Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0218] The practice of the present invention will employ, unless otherwise indicated,conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et al., 2001). The skilled worker will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, some terms are defined below.

[0219] As used in the specification and the appended claims, the terms “a,” “an” and “the”include both singular and the plural referents unless the context clearly dictates otherwise.WSGR Attorney Docket No.: 60924-730.601

[0220] Throughout this specification, the term “including” is used to mean “including but notlimited to.” “Including” and “including but not limited to” are used interchangeably.

[0221] The term “about” as used herein refers to the usual error range for the respective valuereadily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. The term “about” when used before a numerical designation, e.g., a numerical temperature, time, amount, or concentration, including a range, indicates approximations which may vary by ± 10%.

[0222] The term “amino acid residue” or “amino acid” includes reference to an amino acidthat is incorporated into a protein, polypeptide, and / or peptide. The term “polypeptide” includes any polymer of amino acids or amino acid residues. The term “polypeptide sequence” refers to a series of amino acids or amino acid residues which physically comprise a polypeptide and can be any length. A “protein” is a macromolecule comprising one or more polypeptides or polypeptide “chains.” A “peptide” is a small polypeptide of a size of 2 to 20 amino acid residues. The term “amino acid sequence” refers to a series of amino acids or amino acid residues which physically comprise a peptide or polypeptide depending on the length. Unless otherwise indicated, polypeptide and protein sequences disclosed herein are written from left to right representing their order from an amino terminus to a carboxy terminus.

[0223] The terms “amino acid,” “amino acid residue,” “amino acid sequence,” orpolypeptide sequence include naturally occurring amino acids (including L and D isosteriomers) and, unless otherwise limited, also include known analogs of natural amino acids that can function in a similar manner as the common natural amino acids, such as selenocysteine, pyrrolysine, N-formylmethionine, gamma-carboxyglutamate, hydroxyprolinehypusine,pyroglutamic acid, and selenomethionine (see, e.g., Ho J et al., ACS Synth Biol 5: 163-71(2016); Wang Y, Tsao M, Chembiochem 17: 2234-9 (2016)). The amino acids referred to herein are described by shorthand designations as follows in Table A:

[0224] As used herein, the term “radioisotope” includes, but is not limited to, an alphaemitting isotope (interchangeably, α-emitting isotope), beta-emitting isotope (interchangeably, β-emitting isotope), and / or gamma-emitting isotope (interchangeably, ɣ-emitting isotope), such as, e.g., any one of 86-Y, 90-Y, 177-Lu, 186-Re, 188-Re, 89-Sr, 153-Sm, 225-Ac, 213-Bi, 213- Po, 212-Bi, 223-Ra, 224-Ra, 227-Th, 149-Tb, 68-Ga, 64-Cu, 67-Cu, 89-Zr, 137-Cs, 212-Pb, 103-Pd, 111-In, 89-Zn, 123-I, and 99m-Tc.WSGR Attorney Docket No.: 60924-730.601

[0225] As used herein, the term “radioimmunoconjugate” refers to a molecular complexcomprising (1) an immunoconjugate according to the present disclosure and (2) a radioisotope. In a preferred embodiment, the radioisotope is an α-emitting radioisotope. In another embodiment, the radioisotope is a β-emitting radioisotope. In another embodiment, the radioisotope is a ɣ-emitting isotope. In another embodiment, this disclosure provides radioimmunoconjugates comprising α-emitting and β-emitting radioisotopes. The term “radioconjugate” is used interchangeably with the term “radioimmunoconjugate” herein. In one embodiment, the radioisotope is associated with a chelating moiety of the radioimmunoconjugate. In one embodiment, the radioisotope is directly linked to the immunoconjugate.

[0226] As used herein, the term “immunoconjugate” refers to a molecular complexcomprising an at least one antigen binding region derived from an antibody (e.g., variable regions or complementarity determining regions) further coupled to at least one non-antibody derived molecule, such as a chelator or cytotoxic agent. Non-antibody derived molecules may for example be conjugated to one or more lysine or cysteine resides of the antigen binding region or to a constant region coupled (by peptide linkage or otherwise) to the antigen binding region. In some embodiments, the immunoconjugate further comprises a chelating moiety (interchangeably, “chelator”). In one embodiment, an immunoconjugate comprises an antibody construct of this disclosure linked directly or indirectly to a cytotoxic agent or radioisotope.

[0227] The immunoconjugates and radioimmunoconjugates described herein compriseantigen binding regions. These antigen binding regions can be derived from an “antibody.” The term “antibody” herein is used in the broadest sense and includes monoclonal antibodies, and includes intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab')2fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain antibody fragments, including single chain variable fragments (sFv or scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. Antibodies may include VHH polypeptides comprising an immunoglobulin hinge region and CH2 and CH3 domains. In addition, VHH polypeptides comprising an immunoglobulin hinge region and a CH2 and / or CH3 domain may dimerize to form bivalent single domain Fc antibodies. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functionalWSGR Attorney Docket No.: 60924-730.601 antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD. The antibody can comprise a human IgG1 constant region. The antibody can comprise a human IgG4 constant region.

[0228] The terms “complementarity determining region,” and “CDR,” which aresynonymous with “hypervariable region” or “HVR,” are known in the art to refer to non- contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and / or binding affinity. In general, there are three CDRs in each variable domain (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). “Framework regions” and “FR” are known in the art to refer to the non- CDR portions of the VHH variable regions of the heavy and light chains. In general, there are four FRs in each full-length variable domain (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4). The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol.262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol.262, 732-745.” (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70, (“Aho” numbering scheme); and Whitelegg NR and Rees AR, “WAM: an improved algorithm for modelling antibodies on the WEB,” Protein Eng.2000 Dec;13(12):819-24 (“AbM” numbering scheme. In certain embodiments, the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or combinations thereof.

[0229] The boundaries of a given CDR or FR may vary depending on the scheme used foridentification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at differentWSGR Attorney Docket No.: 60924-730.601 positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.

[0230] The term “variable region” or “variable domain” refers to the domain of an antibodyheavy or light chain that is involved in binding the antibody to antigen. The VHH variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved frameworkregions (FRs) and three CDRs (See e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freemanand Co., page 91(2007)). A single VHor VLdomain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VLdomain from an antibody that binds the antigen to screen a library of complementary VLor VH domains, respectively (See e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0231] The antigen binding regions of the immunoconjugates described herein may behumanized. “Humanized” in reference to an immunoconjugate refers to an antigen binding region in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized immunoconjugate optionally may include at least a portion of an antibody constant region derived from a human antibody.

[0232] Among the provided immunoconjugates are human immunoconjugates. A “humanimmunoconjugates” is an immunoconjugates possessing an antigen binding region with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences, including human antibody libraries. The term excludes humanized forms of non- human antibodies comprising non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human.

[0233] The phrase “antigen binding arm”, as used herein, refers to a single polypeptidechain, comprising an “antigen binding region”, a hinge region, and a variant constant region. Other elements (e.g., a chelating moiety; an imaging metal) may be attached to the antigen binding arm directly or through one or more linkers in compositions of this disclosure. Immunoconjugates of this disclosure comprise two antigen binding arms that are covalently linked together. In one embodiment, the antigen binding arms are linked through the hinge region. In one embodiment, the antigen binding arms are linked through an immunoglobulin heavy chain constant region. In one embodiment, the antigen binding arms are linked throughWSGR Attorney Docket No.: 60924-730.601 the variant constant region. In one embodiment, the antigen binding arms are linked via a disulfide linkage (e.g., via a cysteine residue in a hinge region).

[0234] The phrase “antigen binding region”, as used herein, refers to the region of animmunoconjugate responsible for specific binding to an antigen, such region one or more antigen binding domains comprising complementarity determining regions, variable regions and framework regions, which may be derived from, modeled on, or may mimic, antibodies or fragments thereof, as are known by the person of ordinary skill in the art. In one embodiment, the “antigen binding region’ of an antigen binding arm contains one or two antigen binding domains. In a preferred embodiment, the “antigen binding region” of an antigen binding arm consists of a single antigen binding domain, which antigen binding domain is preferably a VHH polypeptide. In a preferred embodiment, the antigen binding regions of both antigen binding arms of an immunoconjugate independently consist of a single antigen binding domain, which antigen binding domain is preferably a VHH polypeptide, which VHH polypeptides are the same or different.

[0235] The term “VHH polypeptide” as used herein encompasses natural and syntheticcompositions and refers to a polypeptide constituting a VHH fragment as it is known in the art, i.e., a polypeptide that constitutes a single domain heavy chain only variable domain fragment, or a polypeptide that structurally and functionally resembles a VHH fragment, as such structure is further described below and has the ability to specifically bind antigen is described below, and as both are well known in the art. In preferred embodiments, the VHH polypeptides comprise a variable domain comprising three heavy chain CDR’s; in one embodiment the VHH polypeptide is derived from a camelid; in another embodiment the VHH polypeptide is derived from a library; VHH polypeptides bind to antigens with specificity and high affinity. In a preferred embodiment, the VHH polypeptide is a single variable domain comprising the arrangement: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. VHH polypeptides may be obtained, for example, as the antigen binding fragments of heavy chain only antibodies generated in vivo (e.g., in camelids). VHH polypeptides may also be obtained from synthetic libraries, e.g., phage display libraries. For example, see McMahon et al., Nature Structural & Molecular Biology | VOL 25 | MARCH 2018 | 289–296 Yeast surface display platform for rapid discovery of conformationally selective nanobodies; Moutel et al., eLife 2016;5:e16228 NaLi- H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. De Genst E, Saerens D, Muyldermans S, Conrath K. Antibody repertoire development in camelids. Dev Comp Immunol.2006;30(1-2):187-98. doi: 10.1016 / j.dci.2005.06.010. PMID: 16051357. Vincke C, Gutiérrez C, Wernery U, Devoogdt N,WSGR Attorney Docket No.: 60924-730.601 Hassanzadeh-Ghassabeh G, Muyldermans S. Generation of single domain antibody fragments derived from camelids and generation of manifold constructs. Methods Mol Biol.2012;907:145- 76. doi: 10.1007 / 978-1-61779-604-7_8. PMID: 22907350. Arbabi Ghahroudi M, Desmyter A, Wyns L, Hamers R, Muyldermans S. Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett.1997 Sep 15;414(3):521-6. doi: 10.1016 / s0014-5793(97)01062-4. PMID: 9323027.

[0236] For VHH humanization, see, for example, Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath K. General strategy to humanize a camelid single- domain antibody and identification of a universal humanized nanobody scaffold. J Biol Chem. 2009 Jan 30;284(5):3273-84. doi: 10.1074 / jbc.M806889200. Epub 2008 Nov 14. PMID: 19010777.

[0237] For VHH stability, see, for example, Kunz P, Flock T, Soler N, Zaiss M, Vincke C,Sterckx Y, Kastelic D, Muyldermans S, Hoheisel JD. Exploiting sequence and stability information for directing nanobody stability engineering. Biochim Biophys Acta Gen Subj.2017 Sep;1861(9):2196-2205. doi: 10.1016 / j.bbagen.2017.06.014. Epub 2017 Jun 20. PMID: 28642127; PMCID: PMC5548252; Kunz P, Zinner K, Mücke N, Bartoschik T, Muyldermans S, Hoheisel JD. The structural basis of nanobody unfolding reversibility and thermoresistance. Sci Rep.2018 May 21;8(1):7934. doi: 10.1038 / s41598-018-26338-z. PMID: 29784954; PMCID: PMC5962586.

[0238] A “linker” herein is also referred to as “linker sequence” “spacer” “tetheringsequence” or grammatical equivalents thereof. A “linker” as referred herein connects two distinct molecules that by themselves possess target binding, catalytic activity, or are naturally expressed and assembled as separate polypeptides or comprise separate domains of the same polypeptide. For example, two distinct binding moieties or a heavy-chain / light-chain pair or an antigen binding region and an immunoglobulin heavy chain constant region. A number of strategies may be used to covalently link molecules together. Linkers described herein may be utilized to join a light chain variable region and a variable domain in an scFv molecule; or may be used to tether an scFv or other antigen binding fragment on the N-or C-terminus of an antibody heavy chain. These include but are not limited to polypeptide linkages between N-and C-termini of proteins or protein domains, linkage via disulfide bonds, and linkage via chemical cross-linking reagents. In one aspect of this embodiment, the linker is a peptide bond, generated by recombinant techniques or peptide synthesis.

[0239] An antibody that “binds” an antigen or epitope of interest is one that binds theantigen or epitope with sufficient affinity that is measurably different from a non-specificWSGR Attorney Docket No.: 60924-730.601 interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity (e.g., an isotype control).

[0240] “Specific binding” refers to an antibody or immunoconjugate that is capable ofbinding antigen with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting that antigen. In one embodiment, the extent of binding of an antibody to an unrelated protein is less than about 10% of the binding of the antibody to its antigen as measured, e.g., by a radioimmunoassay. An “antigen specific” antibody or immunoconjugate, as used herein, is one that specifically binds to the antigen with sufficient specificity and affinity to be useful in targeting a therapeutic, targeting diagnostic, or method of detecting the antigen in a biological sample from a subject. In some embodiments, an immunoconjugate or antibody construct or target imaging complex or radioimmunoconjugate that binds to its target antigen has a dissociation constant (KD) of ≤ 1 μM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10-8M or less, e.g., from 10-8M to 10-13M, e.g., from 10-9M to 10-13M). In some embodiments, an immunoconjugate or antibody construct or target imaging complex or radioimmunoconjugate of the present invention binds to multiple antigens, such as, e.g., an epitope conserved among homologs from different species, such as wherein the amino acid identity of the epitope is non-identical in different species.

[0241] As used herein, the term “variant constant region” refers to a polypeptide comprisingof a portion of an immunoglobulin heavy chain constant region that has been modified from native immunoglobulin amino acid sequence, preferably at from one to several amino acid positions. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Modifications to Fc regions for various purposes are well known in the art. For example, see Kevin O. Saunders, Frontiers in Immunology, June 2019 | Volume 10 | Article 1296, titled “Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life”.

[0242] Percent (%) sequence identity with respect to a reference polypeptide sequence is thepercentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways usingWSGR Attorney Docket No.: 60924-730.601 available computer software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN- 2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0243] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the% amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0244] The term “cytotoxic agent” as used herein refers to a substance that inhibits orprevents a cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes; chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and the various cytotoxic agents described herein.

[0245] The term “affinity” refers to the strength of the sum total of noncovalent interactionsbetween a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope). Unless indicated otherwise, as used herein, “binding affinity” refers toWSGR Attorney Docket No.: 60924-730.601 intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen or epitope). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative embodiments for measuring binding affinity are described herein.

[0246] The term “antagonist” is used in the broadest sense, and includes any molecule thatpartially or fully blocks, inhibits, or neutralizes a biological activity of antigen. Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, or derivatives thereof.

[0247] A “blocking” antibody or an “antagonist” antibody is an antibody that inhibits orreduces biological activity of the antigen it binds or a protein complex comprising the antigen. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen or protein complex comprising the antigen.

[0248] The term “tumor” as used herein refers to all neoplastic cell growth and proliferation,whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0249] The terms “cancer” and “cancerous” as used herein refer to or describe thephysiological condition in mammals that is typically characterized by unregulated cell growth. A “tumor” comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), skin cancer, melanoma, lung cancer including small-cell lung cancer, non-small cell lung cancer (“NSCLC”), small cell neuroendocrine lung cancer, large cell neuroendocrine lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), glioblastoma, cervical cancer, ovarian cancer (e.g., high grade serous ovarian carcinoma), liver cancer (e.g., hepatocellular carcinoma (HCC)), bladder cancer (e.g., urothelial bladder cancer), testicular (germ cell tumor) cancer, hepatoma, breast cancer, brain cancer (e.g., astrocytoma), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer (e.g., renal cell carcinoma, nephroblastoma or Wilms’ tumor), prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer. Additional examples of cancer include, without limitation, retinoblastoma, thecomas, arrhenoblastomas, hepatoma, hematologic malignancies including non-Hodgkins lymphoma (NHL), multiple myeloma and acute hematologic malignancies, endometrial orWSGR Attorney Docket No.: 60924-730.601 uterine carcinoma, endometriosis, fibrosarcomas, choriocarcinoma, salivary gland carcinoma, vulval cancer, thyroid cancer, esophageal carcinomas, hepatic carcinoma, anal carcinoma, penile carcinoma, nasopharyngeal carcinoma, laryngeal carcinomas, Kaposi’s sarcoma, melanoma, skin carcinomas, Schwannoma, oligodendroglioma, neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, anaplastic astrocytoma, basal cell carcinoma (basal cell epithelioma), bile duct cancer, small cell bladder cancer, metastatic breast cancer, metastatic colorectal cancer, epithelial ovarian cancer, fallopian tube cancer, gastric adenocarcinoma, glioblastoma multiforme (GBM), recurrent glioblastoma multiforme (GBM), gliomas, gliosarcoma, head and neck squamous cell carcinoma (HNSCC), recurrent head and neck cancer squamous cell carcinoma, malignant pleural mesothelioma head and neck cancer, Hodgkin lymphoma, metastatic renal cell carcinoma, metastatic renal clear cell carcinoma, squamous non-small cell lung cancer, squamous carcinoma of the lung, relapsed or refractory small-cell lung cancer, treatment-resistant melanoma, metastatic melanoma, Merkel cell carcinoma, neuroendocrine cancer, large cell neuroendocrine cancer, neuroendocrine tumors (NETS), ovarian carcinoma, papillary carcinoma, peritoneal cancer, neuroendocrine prostate cancer, hormone-refractory prostate cancer, castration-resistant prostate cancer, soft tissue sarcoma, and squamous cell carcinoma.

[0250] The term “metastatic cancer” means the state of cancer where the cancer cells of atissue of origin are transmitted from the original site to one or more sites elsewhere in the body, by the blood vessels or lymphatics, to form one or more secondary tumors in one or more organs besides the tissue of origin. A prominent example is a metastatic breast cancer.

[0251] The terms “cell proliferative disorder” and “proliferative disorder” refer to disordersthat are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0252] The terms “associated,” “associating,” “linked,” or “linking” with regard to theclaimed invention refers to the state of two or more components of a molecule being joined, attached, connected, or otherwise coupled to form a single molecule (or single molecular complex) or the act of making two molecules associated with each other to form a single molecule (or single molecular complex) by creating an association, linkage, attachment, and / or any other connection between the two molecules. For example, the term “linked” may refer to two or more components associated by one or more atomic interactions such that a single molecule is formed and wherein the individual atomic interactions may be covalent or non- covalent. Non-limiting examples of covalent associations between two components includeWSGR Attorney Docket No.: 60924-730.601 peptide bonds and cysteine-cysteine disulfide bonds. Non-limiting examples of non-covalent associations between two molecular components include ionic bonds.

[0253] A “bispecific” antibody refers to an antibody that has binding specificities for at leasttwo different epitopes, regardless of whether the plurality of epitopes are in the same molecule and / or partially overlapping. In some embodiments, the bispecific immunoconjugate of the present invention binds to two different epitopes of a single antigen described herein.

[0254] As used herein, the terms “expressed,” “expressing,” or “expresses,” andgrammatical variants thereof, refer to translation of a polynucleotide or nucleic acid into a protein. The expressed protein may remain intracellular, become a component of the cell surface membrane or be secreted into an extracellular space.

[0255] For purposes of the present invention, the phrase “derived from” when referring to apolypeptide or polypeptide region means that the polypeptide or polypeptide region comprises highly similar amino acid sequences originally found in a “parental” protein and which may now comprise certain amino acid residue additions, deletions, truncations, rearrangements, or other alterations relative to the original polypeptide or polypeptide region as long as a certain function(s) (e.g., antigen binding affinity) and a structure(s) of the “parental” molecule are substantially conserved. The skilled worker will be able to identify a parental molecule (e.g., an antibody sequence) from which a polypeptide or polypeptide region (e.g., a VHH polypeptide, CDR, HVR, VH, and / or VL) was derived using techniques known in the art, e.g., protein sequence alignment software.

[0256] As used herein, cells which express an extracellular target biomolecule or antigen onat least one cellular surface are “target positive cells” or “target+ cells” and are cells physically coupled to the specified, extracellular target biomolecule. Additional target biomolecule description is provided below. “Target biomolecule”, “target antigen molecule”, “target antigen”, “antigen of interest”, and grammatical variants and equivalents are used interchangeably herein as will be recognized by the person of ordinary skill in the art viewing the context of usage, and include the molecular determinants of antibody binding. Such antigens can be bound by the immunoconjugates described herein though the antigen binding region or antigen binding arm of the immunoconjugate.

[0257] The term “pharmaceutical formulation” or “pharmaceutical composition” refers to apreparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.WSGR Attorney Docket No.: 60924-730.601

[0258] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceuticalformulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0259] An “isolated” antibody or immunoconjugate or radio immunoconjugate is one whichhas been separated from a component of its natural environment or artificial production. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). Routine methods for assessment of antibody purity in a composition are known to the skilled worker, see e.g., Flatman et al., J. Chromatogr. B 848:79–87 (2007). In particular, unwanted components (contaminants) to be purified away from are such components that would interfere with desired uses for the antibody, such as, e.g., a therapeutic use, and may include, inter alia, bacterial factors, enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0260] An “isolated” nucleic acid refers to a nucleic acid molecule that has been separatedfrom a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present at extrachromosomal location or at a chromosomal location that is different from its natural chromosomal location.

[0261] The terms “host cell,” “host cell line,” and “host cell culture” are usedinterchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0262] As used herein, the term “administer”, with respect to an immunoconjugate orcomposition thereof (e.g., a radioimmunoconjugate, a pharmaceutical composition, or a diagnostic composition), means to deliver the immunoconjugate, or composition thereof, to a subject’s body via any known method suitable for delivery of immunoconjugate or composition thereof. Specific modes of administration include, without limitation, intravenous, transdermal, subcutaneous, intraperitoneal and intrathecal administration.WSGR Attorney Docket No.: 60924-730.601

[0263] An “effective amount” of an agent, e.g., a pharmaceutical formulation, refers to anamount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0264] As used herein, “treatment” (and grammatical variations thereof such as “treat” or“treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, radioimmunoconjugates of this disclosure are used to delay development of a disease or to slow the progression of a disease.

[0265] A “therapeutically effective amount” is at least the minimum concentration requiredto effect a measurable improvement or prevention of a particular disorder. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of a composition of this disclosure to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition of this disclosure are outweighed by the therapeutically beneficial effects.

[0266] The terms “predictive” and “prognostic” as used herein are interchangeable. In onesense, the methods for prediction or prognostication are to allow the person practicing a predictive / prognostic method of this disclosure to select patients that are deemed (usually in advance of treatment, but not necessarily) more likely to respond to treatment with an immunoconjugate of the present invention or a composition of the aforementioned (e.g., a pharmaceutical composition).

[0267] The term “detecting” is used in the broadest sense to include both qualitative andquantitative measurements of a target antigen molecule. In one aspect, the detecting method as described herein is used to identify the mere presence of the antigen of interest in a biological sample. In another aspect, the method is used to test whether the antigen of interest in a sample is present at a detectable level. In yet another aspect, the method can be used to quantify the amount of the antigen of interest in a sample and further to compare the antigen levels from different samples. In another aspect, the method can be used in vivo to determine the location of a target cell, for example, using a targeted imaging complex of this disclosure.WSGR Attorney Docket No.: 60924-730.601

[0268] The term “biological sample” refers to any biological substance that might contain anantigen of interest. A sample can be biological fluid, such as whole blood or whole blood components including red blood cells, white blood cells, platelets, serum and plasma, ascites, itreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, saliva, sputum, tears, perspiration, mucus, cerebrospinal fluid, and other constituents of the body that might contain the antigen of interest. In various embodiments, the sample is a biological sample from any animal. In some embodiments, the sample is from a mammal. In some embodiments, the sample is from a human subject. In some embodiments, the biological sample is serum from a clinical patient. In some embodiments, the biological sample is biopsy material. In some embodiments, the biological sample is biopsy material from a clinical patient. In some embodiments, the biological sample is serum from a clinical patient. In some embodiments, the biological sample is primary cell culture material. In some embodiments, the biological sample is primary cell culture material from a clinical patient. In some embodiments, the biological sample is from clinical patients or patients treated with a composition of this disclosure e.g., a radioimmunoconjugate, or treated with a different therapeutic agent, such as an antibody-drug conjugate targeting the antigen of interest or β-irradiation or a small molecule therapeutic.

[0269] The term “package insert” is used to refer to instructions customarily included incommercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0270] The term “vector,” as used herein, refers to a nucleic acid molecule capable ofpropagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” EXAMPLES

[0271] The following illustrative examples are representative of embodiments of thecompositions and methods described herein and are not meant to be limiting in any way.WSGR Attorney Docket No.: 60924-730.601

[0272] The Examples below describe radioisotope-delivering platforms having sizesbetween 60 and 110 kDa and which have shorter half-lives (e.g., 4 days or less) compared to traditional IgGs but longer half-lives than smaller monomeric antibody fragment formats (e.g., greater than 10 hours). Furthermore, certain radioisotope-delivering platforms provided herein exhibit high stability in vitro or in vivo, low immunogenicity, and suitable therapeutic windows. These radioisotope-delivering platforms are preferred for targeting radioisotopes in vivo in order to treat disease. These radioisotope-delivering platforms are particularly useful for targeted delivery of alpha emitters safely and effectively in a subject by exhibiting reduced adverse effects as compared to antibodies having half-lives over 4 days and / or molecular weights under 60 kDa.

[0273] Below, in certain phrases, “Fc portion” is used in reference to variant constantdomain and “hinge” is used in reference to “hinge region” as will be understood by the person of ordinary skill in the art. Example 1. Generation and characterization of DLL3 binding regions

[0274] Camelids (llamas and alpacas) were immunized subcutaneously (SC) withrecombinant human DLL3 in complete Freud’s adjuvant (CFA) or incomplete Freud’s adjuvant (IFA) at 2-4 week intervals. Serum titer response was assessed using dilution series of serum. Sera samples were incubated with multiplexed beads differentially optically encoded to various DLL3 antigens (human, mouse, cynomolgus monkey). Binding of antigen-specific antibodies in the serum to the beads was then detected using a fluorescently labelled secondary antibody via high-throughput, plate-based flow cytometry. Samples were selected for screening and peripheral blood mononuclear cells were collected. Single-cell screening and recovery

[0275] Peripheral blood mononuclear cells were thawed, activated in culture to generatememory B cells, and enriched for heavy chain-only antibody-secreting B cells before screening. Single B cells secreting target-specific antibodies were identified and isolated using a multi-step assay assessing both internalization in cells and binding to DLL3 immobilized on beads. Cross- reactivity to species homologs was assessed using a multiplexed bead assay using differentially optically encoded beads, each conjugated to different species of DLL3 antigens (human, mouse, cynomolgus monkey) and binding was detected using a fluorescently labelled secondary antibody specific to alpaca IgG subclasses 2 and 3. Internalization was assessed by flowing in HEK293T cells expressing human DLL3 and internalizing antibodies were detected using a pH- sensitive fluorescent reagent.WSGR Attorney Docket No.: 60924-730.601 Single-cell sequencing and bioinformatic analysis

[0276] Single-cell polymerase chain reaction (PCR) and custom molecular biology protocolsgenerated NGS sequencing libraries (MiSeq, Illumina) using automated workstations (Bravo, Agilent). Sequencing data were analyzed using a custom bioinformatics pipeline to yield heavy chain sequences for recovered antibody-secreting cells. Each sequence was annotated with the closest germline (V(D)J) genes and degree of somatic hypermutation. Antibodies were considered members of the same clonal family if they shared the same inferred heavy V and J genes and had the same CDR3 length.

[0277] VHH-Fc plasmids were generated by cloning the VHH sequence, with a hinge andFc portion (human IgG1 CH2-CH3) into a mammalian expression vector. In some instances, mutations were introduced into the Fc portion. To produce recombinant VHH-Fc and variants thereof, plasmid was transfected into HEK293.SUS cells (ATUM, or similar). After 3-5 days of secretion, the antibody-containing supernatant was cleared of cells by centrifugation and sterile filtration. Antibodies were purified using Mab Select SuRe PCC column (GE, Cat#: 11003495) and buffer exchanged into PBS, pH 7.0. Proteins were quantified using A280 or BCA. The purity of the antibodies were tested by SDS-PAGE, capillary electrophoresis, HPLC-SEC and LC-MS using standard protocols.

[0278] A total of 209 VHH clones were tested for properties important for the developmentof immunoconjugates useful for the delivery of toxic payloads. These criteria included binding to murine DLL3, binding to cynoDLL3, binding to human DLL3, the ability to be internalized by target expressing cells, the absence of lysine residues in CDR regions, high sequence redundancy and absence of known sequence liabilities for developability.

[0279] Of these 209 clones, 46 were selected for expression and purification as VHH-Fc(with wildtype Fc and modified hinge region, SEQ ID: 12) for further analysis. A summary of the data generated on these 46 VHH.Fcs is shown in Tables 1-3. High-throughput antibody expression and purification

[0280] The VHH variable [V(D)J] region of each antibody heavy chain was synthesized andinserted into expression plasmids. Plasmids were verified by Sanger sequencing. Chimeric human Fc, camelid VHH (VHH-Fc) antibodies were recombinantly produced by transient transfection. Antibody-encoding plasmid DNA was transfected into Expi293F cells (Thermo Fisher Scientific). Antibody titers were measured by biolayer interferometry on an Octet HTX instrument (FortéBio). Antibodies were purified using protein A-based purification and quantified by UV / Vis Spectroscopy at 280 nm absorbance.WSGR Attorney Docket No.: 60924-730.601 Antibody bead binding and cell internalization validation

[0281] Recombinant VHH-Fc antibodies were confirmed to bind targets and induceinternalization via high-throughput flow cytometry using fluorescently labelled anti-human IgG. In a multiplexed bead-based assay, optically encoded beads were conjugated to one of the following antigens: human DLL3, mouse DLL3, or cynomolgus monkey DLL3. Purified VHH- Fc antibodies were incubated with target-conjugated beads at 25 nM antibody concentration for 30 minutes at 4ºC. Beads were washed and binding was detected using a fluorescently labelled secondary antibody. In a live cell-based internalization assay, purified VHH-Fc antibodies were incubated with HEK293T cells expressing human DLL3, parental HEK293T cells or SHP-77 cells at 5 nM antibody concentration and a pH-sensitive fluorescent reagent for two hours at 37ºC. Fluorescence was measured using high-throughput, plate-based flow cytometry. An irrelevant antibody, chimeric human Fc camelid VHH specific to HER2 (VHH-Fc anti-HER2) was used as a negative control. Median fluorescence intensity of each antibody was normalized over median fluorescence intensity of the negative control. SPR binding experiments

[0282] All SPR binding experiments were performed on a Carterra LSA instrumentequipped with an HC-30M chip type (Carterra-bio) using a 384-ligand array format as described herein. The HC-30M chip was prepared by immobilizing a goat anti-human IgG Fc antibody (Southern Biotech #2014-01) via direct coupling: The chip surface was first activated by flowing a freshly prepared 1:1:1 activation mix of 100 mM MES (pH 5.5), 100 mM sulfo–N- hydroxysuccinimide, and 400 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide for 7 minutes, and goat anti-human IgG Fc antibody diluted to 50 ug / ml in 10 mM NaOAc (pH 4.25) buffer + 0.01% Tween was injected onto the chip surface for 10 minutes. The chip surface was quenched by flowing 1 M ethanolamine for 7 minutes, followed by two wash steps of 15 seconds each in 25 mM MES (pH 5.5) buffer. The test antibodies diluted to 5 ug / ml in HEPES- buffered saline containing 0.05% Tween 20 and 3 mM EDTA (HBSTE) + 0.1% BSA running buffer were captured on the chip surface for 5 minutes. Relevant benchmarks and negative control antibodies (VHH-Fc anti-HER2, VHH-Fc anti-DLL3, Rovalpituzumab) were also captured on the chip surface.

[0283] For binding kinetics and affinity measurements, a threefold dilution series of theantigen of interest (human DLL3), starting at 300 nM in HEPES-buffered saline containing 0.05% Tween 20 and 3 mM EDTA (HBSTE) + 0.1% BSA running buffer, was sequentially injected onto the chip surface. For each concentration, the antigen was injected for 10 min (association phase), followed by running buffer injection for 15 min (dissociation phase). TheWSGR Attorney Docket No.: 60924-730.601 data were analyzed using the Carterra Kinetics analysis software using a 1:1 Langmuir binding model to determine apparent association (ka) and dissociation (kd) kinetic rate constants and binding affinity constants (KD). Capillary electrophoresis sodium dodecyl sulfate (CE-SDS)

[0284] The purity of the expressed and purified VHH-Fc antibodies was analyzed bydenaturing CE-SDS using the LabChip GXII Touch instrument (Perkin Elmer, Protein Express LabChip #760528) according to the manufacturer's protocol. Two (2) uL of VHH-Fc solution at a concentration of 0.35 mg / mL in PBS was mixed with a non-reducing denaturing buffer solution (Perkin Elmer Reagent Kit #CLS960008) and incubated at 70°C for 10 min. Separation was performed using the HT Antibody Analysis 200 assay setting on the LabChipGXII Touch instrument (Perkin Elmer). The data was analyzed using the LabChip GX Reviewer Software (Perkin Elmer). Dynamic light scattering (DLS)

[0285] Percent aggregation and polydispersity of VHH-Fc antibodies was assessed by DLSon a DynaPro® Plate Reader III instrument (Wyatt Technology). Seven (7) µL of each sample at 0.35 mg / mL in PBS were dispensed into glass-bottom 1536 well Sensoplates (Greiner Bio-One, # 783892) and covered with silicon oil. DLS of individual samples was then acquired at 20°C with 5 x 5 seconds acquisitions per sample. Data was analyzed in the Dynamics software (Wyatt Technology, v 7.10.1.21) using the regularization algorithm and replicate measurements with less than 60% of acquisitions unmarked were omitted from the analysis. Replicates were then averaged using an in-house developed Python script. Filter settings were a maximum sum of squares deviation of 50 between autocorrelation function and data fit, a minimum and maximum autocorrelation function amplitude of 0.05 and 1, respectively, and a baseline limit of 1 ± 0.005. Percent polydispersity and percent mass of soluble mAbs were calculated for the size range of 1–10 nm. Nanoscale differential scanning fluorimetry (nDSF)

[0286] All nano-DSF studies were performed using the Nanotemper Prometheus NT.Plexinstrument equipped with a Backreflection Optics and an NT.Robotic Autosampler for automated sample loading and measurement.

[0287] Samples at 350 ug / mL were loaded by capillarity into premium grade nDSFcapillaries (NanoTemper, Cat # PR-AC006). Capillaries were then placed on the Prometheus thermal element and subjected to a temperature ramping of 1°C / minute from 20°C to 95°C. The melting point (Tm, in °C) was obtained by monitoring the intrinsic tryptophan and tyrosineWSGR Attorney Docket No.: 60924-730.601 fluorescence at the emission wavelengths of 330 nm and 350 nm. To generate an unfolding curve, the ratio of the fluorescence intensities (F350 nm / F330 nm) was plotted versus the temperature. The Tm corresponds to the inflection point of the unfolding curve and was determined via the derivative of the curve using the NanoTemper PR.Stability Analysis software (version 1.1). The onset of aggregation (Tagg, in °C) was obtained by monitoring the light backreflection of protein aggregates and determined using the NanoTemper’s PR.Stability Analysis software (version 1.1). Analytical size-exclusion chromatography (aSEC)

[0288] The relative percentage of monomer, high molecular weight (HMW), and lowmolecular weight (LMW) species in purified VHH-Fc antibodies was assessed using aSEC. Using a Vanquish Duo UHPLC System for Dual LC (Thermo Fisher Scientific), 5 µL of each sample at 0.35 mg / mL was injected onto a size exclusion column (ACQUITY UPLC Protein BEH SEC Column, 200 Å, 1.7 µm, 4.6 mm X 150 mm, Waters # 186005226). The mobile phase (100 mM sodium phosphate pH 6.8, 250 mM NaCl; Fisher Scientific # S468-500, # S373-500, and # S271-500) was applied to the column for 10 minutes per injection at a flow rate of 0.3 mL / min to separate species based on their size. Chromatograms monitoring absorbance at 280 nm were acquired and analyzed using Chromeleon software (Thermo Fisher Scientific, v7.3). The relative percentage of each species was determined based on the integrated area of each peak. Analytical hydrophobic interaction chromatography (aHIC)

[0289] Relative surface hydrophobicity of the purified VHH-Fc antibodies was assessed byaHIC. Using a Vanquish Duo UHPLC System for Dual LC (Thermo Fisher Scientific), 5 µL of each sample at 0.35 mg / mL was injected onto a hydrophobic interaction column (TSKgel Butyl- NPR, 2.5 µm, 4.6 mm ID × 3.5 cm, TOSOH # 0014947). A linear gradient method from 42% to 0% buffer A over 6 minutes with a flow rate of 0.5 mL / min was used to separate samples based on their surface hydrophobicity properties (buffer A: 25 mM sodium phosphate pH 7.0, 2.5 M ammonium sulfate; buffer B: 25 mM sodium phosphate pH 7.0; Fisher Scientific # S468-500, # S373-500, and # A702-3). Chromatograms monitoring absorbance at 280 nm were acquired and analyzed using Chromeleon software (Thermo Fisher Scientific, v7.3). Relative hydrophobicity of each sample was determined based on retention time of the largest peak by integrated area.WSGR Attorney Docket No.: 60924-730.601 Table 1. Summary of initial screening of VHH clone 126 (SEQ ID NO: 29) having formatted ty ngTable 2. Summary of initial screening, biophysical characteristics of VHH clone 126 formatted on noTable 3. Summary of initial screening, binding properties of VHH clone 126 formatted on Wildtype Fc / modified hingeTable 4. binding characteristics of 126 clone DExpression and Purification of Humanized DLL3 -Fc variants

[00290] Humanized variants were expressed and purified as described above. All constructswere designed incorporating an Fc with mutations for FcRn binding and FcgR binding, and mutated hinge. (SEQ ID NO: 13 (435Q / AEASS-Fc / C220S IgG1-hinge). A number of variantsWSGR Attorney Docket No.: 60924-730.601 were highly aggregated or lost binding to target due to FW mutations made during humanization (Table 5). Table 5 ID % purity aSEC hDLL3 bindingmDLL3 bindingHumanized DLL3 in vitro Characterization

[0291] Humanized VHHFcs were tested for binding to DLL3 expressing SHP-77 cells (FIG.1A).126_zu1 showed lower binding to cells compared to all other clones. VHHFcs were tested for internalization on SHP-77 cells, and whilst all levels were low as seen previously, the 107 set of variants showed higher internalization signals (FIG.1B). Antibody Production

[0292] VHH-Fc plasmids were generated by cloning the VHH sequence, with a hinge andFc portion(human IgG1 CH2-CH3 ) into a mammalian expression vector. In some instances, mutations were introduced into the Fc portion. To produce recombinant VHH-Fc and variants thereof, plasmid was transfected into HEK293.SUS cells (ATUM, or similar). After 3-5 days of secretion, the antibody-containing supernatant was cleared of cells by centrifugation and sterile filtration. Antibodies were purified using Mab Select SuRe PCC column (GE, Cat#: 11003495) and buffer exchange into PBS, pH 7.0. Proteins were quantified using A280 or BCA. The purity of the antibodies were tested by SDS-PAGE, capillary electrophoresis, HPLC-SEC and LC-MS using standard protocols. Regarding VHH polypeptides, see, for example, McMahon et al., Nature Structural & Molecular Biology | VOL 25 | MARCH 2018 | 289–296 Yeast surfaceWSGR Attorney Docket No.: 60924-730.601 display platform for rapid discovery of conformationally selective nanobodies; Moutel et al., eLife 2016;5:e16228 NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. De Genst E, Saerens D, Muyldermans S, Conrath K. Antibody repertoire development in camelids. Dev Comp Immunol.2006;30(1-2):187-98. doi: 10.1016 / j.dci.2005.06.010. PMID: 16051357. Vincke C, Gutiérrez C, Wernery U, Devoogdt N, Hassanzadeh-Ghassabeh G, Muyldermans S. Generation of single domain antibody fragments derived from camelids and generation of manifold constructs. Methods Mol Biol. 2012;907:145-76. doi: 10.1007 / 978-1-61779-604-7_8. PMID: 22907350. Arbabi Ghahroudi M, Desmyter A, Wyns L, Hamers R, Muyldermans S. Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett.1997 Sep 15;414(3):521-6. doi: 10.1016 / s0014-5793(97)01062-4. PMID: 9323027.

[0293] For VHH humanization, see, for example, Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath K. General strategy to humanize a camelid single- domain antibody and identification of a universal humanized nanobody scaffold. J Biol Chem. 2009 Jan 30;284(5):3273-84. doi: 10.1074 / jbc.M806889200. Epub 2008 Nov 14. PMID: 19010777.

[0294] For VHH stability, see, for example, Kunz P, Flock T, Soler N, Zaiss M, Vincke C,Sterckx Y, Kastelic D, Muyldermans S, Hoheisel JD. Exploiting sequence and stability information for directing nanobody stability engineering. Biochim Biophys Acta Gen Subj.2017 Sep;1861(9):2196-2205. doi: 10.1016 / j.bbagen.2017.06.014. Epub 2017 Jun 20. PMID: 28642127; PMCID: PMC5548252; Kunz P, Zinner K, Mücke N, Bartoschik T, Muyldermans S, Hoheisel JD. The structural basis of nanobody unfolding reversibility and thermoresistance. Sci Rep.2018 May 21;8(1):7934. doi: 10.1038 / s41598-018-26338-z. PMID: 29784954; PMCID: PMC5962586. Example 2. Synthesis of Compound 3-16 Synthesis of Intermediate BWSGR Attorney Docket No.: 60924-730.601a- g,mmol) in methanol (500 mL) was added thionyl chloride (29.2 g, 246 mmol). The reaction mixture was stirred at 80°C for 2 h under nitrogen atmosphere and concentrated to obtain crude (S)-methyl 2-amino-3-(4-bromophenyl)propanoate hydrochloride (36.2 g, 100% yield) as a yellow solid. m / z found = 260.0 (M+H).1H NMR (400 MHz, MeOH - d4) δ = 7.53 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.0 Hz, 1H), 4.35 - 4.31 (m, 1H), 3.81 (s, 3H), 3.23 - 3.14 (m, 2H). Preparation of (S)-methyl 2-amino-3-(4-bromophenyl)propanoate hydrochloride

[0296] To a solution of (S)-methyl 2-amino-3-(4-bromophenyl)propanoate hydrochloride(005-2) (20.0 g, 77.5 mmol) and triethylamine (23.5 g, 232 mmol) in N,N-dimethylformamide (250 mL) was added methyl 2-bromoacetate (29.6 g, 194 mmol). The reaction mixture wasstirred at 25 ºC for 16 h under nitrogen atmosphere. After cooled, the reaction mixture wasdiluted with ethyl acetate (500 mL) and filtered. The filtrate was washed with water (2x 200 mL), brine (200 mL), dried over anhydrous sodium sulfate and concentrated to dryness. The residue was purified by column chromatography (10-20% ethyl acetate / petroleum ether) to the title compound (17 g, 66.5% yield) as a pale oil. m / z found = 331.7 (M+H).1H NMR (400 MHz, MeOH - d4) δ = 7.43 (d, J = 8.8 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 3.70 (s, 3H), 3.65 - 3.61 (m, 4H), 3.44 - 3.37 (m, 2H), 2.96 (d, J = 7.2 Hz, 2H). Preparation of (S)-3-(4-bromobenzyl)-1,4,7,10-tetraazacyclododecane-2,6-dione

[0297] To a solution of (S)-methyl 2-amino-3-(4-bromophenyl)propanoate hydrochloride(1.70 g, 5.15 mmol) and N'-(2-aminoethyl)ethane-1,2-diamine (637.0 mg, 6.18 mmol,) in tetrahydrofuran (30 mL) was added 3,4,6,7,8,9-hexahydro-2H-pyrimido[1,2-a]pyrimidine (215.0 mg, 1.54 mmol). The mixture was stirred at 75°C for 12 h under nitrogen atmosphere. After cooled, the mixture was diluted with ethyl acetate (30 mL) and filtrated. The collected solid was washed with methanol (10 mL) and dried to afford crude (S)-3-(4-bromobenzyl)-1,4,7,10-WSGR Attorney Docket No.: 60924-730.601tetraazacyclododecane-2,6-dione (1.02 g, 52% yield) as a white solid. m / z found = 370.9(M+H).1H NMR (400 MHz, MeOH - d4) δ = 7.45 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.4 Hz, 2H), 3.54 (d, J = 16.4 Hz, 1H), 3.50 - 3.37 (m, 2H), 3.27 - 3.17 (m, 2H), 3.02 - 2.94 (m, 3H), 2.83 - 2.74 (m, 3H), 2.66 - 2.57 (m, 2H). Preparation of (S)-2-(4-bromobenzyl)-1,4,7,10-tetraazacyclododecane

[0298] To a solution of compound (S)-3-(4-bromobenzyl)-1,4,7,10-tetraazacyclododecane-2,6-dione (1.0 g, 2.71 mmol) in tetrahydrofuran (200 mL) was added slowly BH3-Me2S (10 M, 8.1 mL 81.3 mmol). The mixture was stirred at 80°C for 16 h under nitrogen atmosphere. After cooled, the reaction was quenched by methanol (15 mL) and concentrated. The residue was diluted with water (30 mL) and concentrated hydrochloric acid (30 mL). The mixture was stirred at 100°C for 14 h and then adjusted to pH = 7 by addition of saturate aqueous sodium carbonate. All the volatiles was removed under reduce pressure. The residue was diluted with methanol and dichloromethane (methanol / dichloromethane = 1:10，100 mL) and filtered. The filtrate was concentrated to obtain crude compound (S)-2-(4-bromobenzyl)-1,4,7,10-tetraazacyclododecane (0.86 g, 80% yield) as a white solid. The crude product was used in next step without purification. m / z found = 342.8 (M+H). Preparation of (S)-tetra-tert-butyl 2,2',2'',2'''-(2-(4-bromobenzyl)-1,4,7,10-tetra- azacyclododecane-1,4,7,10-tetrayl)tetraacetate

[0299] To a solution of (S)-2-(4-bromobenzyl)-1,4,7,10-tetraazacyclododecane (2.0 g, 5.86mmol) and tert-butyl 2-bromoacetate (6.86 g, 35.2 mmol) in acetonitrile (50 mL) was added potassium carbonate (9.72 g, 70.3 mmol). The reaction mixture was stirred at 50°C for 16 h under nitrogen atmosphere. After cooled, the reaction mixture was partitioned between ethyl acetate (200 mL) and water (200 mL). The separated organic layer was washed with brine (50 mL), dried over anhydrous sodium sulfate and concentration to dryness. The residue was further purified by prep-HPLC (30%-60% MeCN / water + 0.2 % formic acid) to give the title compound (2.1 g, 44.9% yield) as a yellow solid. m / z found = 799.4 (M+H).1H NMR (400 MHz, MeOH - d4) δ = 7.46 (d, J = 7.6 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H), 4.19 - 3.44 (m, 11H), 3.13 - 2.53 (m, 13H), 1.53 - 1.40 (m, 36H). Synthesis of Intermediate CWSGR Attorney Docket No.: 60924-730.601g,dicyclohexylcarbodiimide (58.9 g, 0.29 mmol) in dichloromethane (200 mL) was added slowly over 1 h to a solution of prop-2-ynoic acid (20.0 g, 0.29 mmol) and benzyl alcohol (31.5 g, 0.29 mmol) in dichloromethane (200 mL) at 0°C. The suspension was stirred at 20°C for 12 h and concentrated to dryness. The residue was partitioned between ethyl acetate (500 mL) and water (200 mL). The separated organic layer was washed with brine (200 mL), dried over anhydrous sodium sulfate and concentration to dryness. The residue was purified by column chromatography (5%-30% ethyl acetate / petroleum ether) to give benzyl prop-2-ynoate (20.0 g, 43.7% yield) as a colorless oil. Preparation of (E)-benzyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)acrylate

[0301] To a mixture of cuprous chloride (278 mg, 2.81 mmol) and tert-butoxide (540 mg,5.62 mmol), (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine) (1.63 g, 2.81 mmol) and sodium tert-butoxide (540 mg, 5.62 mmol) in tetrahydrofuran (100 mL) was added bis(pinacolato)diboron (23.8 g, 93.70 mmol), followed by benzyl prop-2-ynoate (15.0 g, 93.70 mmol) and methanol (50 mL). The reaction mixture was stirred at 20°C for 12 h and concentrated to dryness. The residue was partitioned between ethyl acetate (100 mL) and water (100 mL). The separated organic layer was washed with brine (50 mL), dried over anhydrous sodium sulfate and concentrated to dryness. The residue was purified by column chromatography (5%-30% ethyl acetate / petroleum ether) to give the title compound (10.5 g, 38.9% yield) as a colorless oil.WSGR Attorney Docket No.: 60924-730.601 Preparation of (E)-tetra-tert-butyl 2,2',2'',2'''-(2-(4-(3-(benzyloxy)-3-oxoprop-1-en-1- yl)benzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetate

[0302] A mixture of Intermediate B (200.0 mg, 0.25 mmol), (E)-benzyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)acrylate (108.0 mg, 0.38 mmol), Pd(dppf)Cl2 (18.3 mg, 0.03 mmol) and sodium carbonate (79.7 mg, 0.75 mmol) in dioxane (3 mL) and water (0.5 mL) was stirred at 80°C for 4 h under nitrogen atmosphere and concentrated. The residue was purified by column chromatography (1%-8% methanol / dichloromethane) to give the title compound (150.0 mg, 68.1% yield) as a brown oil. m / z found = 879.4 (M+H). Preparation of 3-(4-((1,4,7,10-tetrakis(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10- tetraazacyclododecan-2-yl)methyl)phenyl)propanoic acid

[0303] A mixture of compound (E)-tetra-tert-butyl 2,2',2'',2'''-(2-(4-(3-(benzyloxy)-3-oxoprop-1-en-1-yl)benzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetate (150.0 mg, 0.17 mmol), 10% palladium on carbon (6.0 mg, 0.05 mmol) and 10% palladium hydroxide on carbon (6.2 mg, 0.05 mmol) in tetrahydrofuran (10 mL) was stirred at 20°C under hydrogen atmosphere (15 Psi) for 16 h and filtered. The filtrate was concentrated to obtain crude compound 006-5 (120.0 mg, 88.9% yield) as a brown solid. m / z found = 791.5 (M+H). Synthesis of compound 3-16 from Intermediate CWSGR Attorney Docket No.: 60924-730.601

[0304] a mg, (1.75 mL, 97.1mmol) was added potassium carbonate (847 mg, 6.13 mmol), tert-butylammonium bromide (18.9 mg, 123 µmol), and ethylamine (627 mg, 13.9 mmol) at 0 °C. After 30 min, the ice bath was removed and the reaction was stirred for 18 h. The solution was diluted with brine, then extracted six times with CH2Cl2. The organic layers were washed with brine, dried with anhydrous Na2SO4, filtered and concentrated to give the title compound as a colorless oil (412 mg, quant. yield). m / z found = 337.2 (M+H).1H (DMSO-d6): d 3.54-3.49 (m, 18H), 3.44 (t, J=5.8 Hz, 4H), 2.64 (t, J=5.8 Hz, 4H), 2.64 (q, J=7.1 Hz, 4H), 0.99 (t, J=7.2Hz, 6H). Preparation of tetra-tert-butyl 2,2',2'',2'''-(2-(4-(21-ethyl-22-oxo-6,9,12,15,18-pentaoxa-3,21- diazatetracosan-24-yl)benzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)(S)-tetraacetate 3-(4-((1,4,7,10-tetrakis(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-2- yl)methyl)phenyl)propanoic acid (100 mg, 0.126 mmol) was dissolved in DMF (3 mL) and placed under nitrogen atmosphere. DIPEA (96.9 µL, 556 µmol) and HATU (130 mg, 328 µmol) were then added to the solution, followed by a solution of the above intermediate (196 mg, 582 µmol) in DMF (2 mL). After 1 h at rt. SM is still evident, but major peak is desired product. The reaction mixture was stirred at 50 °C for 2 h, poured onto water, extracted with EtOAc, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude residue was then triturated with Et2O three times and concentrated under reduced pressure to give the title compound as an orange oil (108 mg, 77% yield). m / z found = 1109.6 (M+H). Preparation of tetra-tert-butyl 2,2',2'',2'''-(2-(4-(4,22-diethyl-3,23,28-trioxo-28-(2,3,5,6- tetrafluorophenoxy)-7,10,13,16,19-pentaoxa-4,22-diazaoctacosyl)benzyl)-1,4,7,10- tetraazacyclododecane-1,4,7,10-tetrayl)(S)-tetraacetateWSGR Attorney Docket No.: 60924-730.601

[0305] To a solution of tetra-tert-butyl 2,2',2'',2'''-(2-(4-(21-ethyl-22-oxo-6,9,12,15,18-pentaoxa-3,21-diazatetracosan-24-yl)benzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10- tetrayl)(S)-tetraacetate (0.1 g, 90.1 µmol) in dimethylformamide (9.01 mL, 116 mmol) was added triethylamine (105 µL, 750 µmol), followed by bis(2,3,5,6-tetrafluorophenyl) adipate (140 mg, 3.5 eq., 315 µmol). The reaction was stirred at rt for 1 h 20 min and concentrated under reduced pressure to give the crude TFP ester intermediate as a brown oil. m / z = 693.5 (M+2H). Preparation of (S)-2,2',2'',2'''-(2-(4-(4,22-diethyl-3,23,28-trioxo-28-(2,3,5,6-tetrafluoro- phenoxy)-7,10,13,16,19-pentaoxa-4,22-diazaoctacosyl)benzyl)-1,4,7,10-tetraazacyclo- dodecane-1,4,7,10-tetrayl)tetraacetic acid

[0306] TFP ester intermediate from above was dissolved in dichloromethane (1 mL) andtrifluoroacetic acid (2 mL) was added. The reaction was stirred for 18 h, concentrated, and purified by prep-HPLC (40-60% ACN / H2O + 0.1% TFA) to afford the title compound (Compound 3-16) as a white powder (12.5 mg, 12% yield). m / z found = 1161.4 (M+H).1H NMR (500 MHz, DMSO) δ 7.94 (tt, J = 10.8, 7.3 Hz, 1H), 7.18 (d, J = 5.5 Hz, 4H), 4.11 – 3.81 (m, 9H), 3.50 (m, 28H), 2.79 (m, 8H), 2.66 – 2.55 (m, 5H), 2.36 (dt, J = 12.6, 7.3 Hz, 4H), 1.69 (h, J = 7.6 Hz, 3H), 1.60 (p, J = 7.6 Hz, 3H), 1.13 – 0.96 (m, 8H). Example 3. Conjugation of Bifunctional Chelators

[0307] Conjugations can be carried out using many of the methods available for preparationof IgG radioconjugates and IgG antibody-drug conjugates. For information on the range of applicable methodologies, see PW Howard Antibody-Drug Conjugates (ADCs), Protein Therapeutics, First Edition, chapter 9, pp.278-279 (2017).

[0308] For a typical lysine-based conjugation, an antibody was buffer-exchanged into 0.1 MNaHCO¬3, pH 9.0 by either Microsep Advance Centrifugal Device (Pall, 10K MWCO, Omega modified PES membrane, Cat#: MCP010C41), Amicon Ultra Centrifugal Filter Unit (Millipore, 40 Kda MWCO, Regenerated Cellulose membrane, Cat#: UFC803096), or Vivaspin Turbo 4 Ultrafiltration Unit (Sartorius, 30kDa MWCO, High flux PES membrane, Cat#: VS04T21) followed by sterilization with a Costar Spin-X Centrifuge Tube, 0.22 μm (Corning, Cat#: 8160). The buffer-exchanged antibody was quantified by either Pierce BCA assay (Thermo Scientific, Cat#: 23225) or A280 (Unchained Labs, Lunatic). In some cases, the antibody may have been concentrated such that it could be adjusted to a concentration of 5 mg / mL after buffer exchange into the 0.1 M NaHCO¬3, pH 9.0; in most procedures the antibody was diluted to 2–5 mg / mL in 0.1 M NaHCO¬3, pH 9.0 by adding additional 0.1 M NaHCO¬3 after buffer exchange.WSGR Attorney Docket No.: 60924-730.601

[0309] Next, an appropriate molar excess (5-30 eq) of chelator-linker (20–50 mM in DMSO)was added to the mAb solution (2 or 5 mg / mL final concentration). In some cases, DMSO was added to make the conjugation solution a consistent 2–10% DMSO. The reaction was incubated at 25°C overnight in an Eppendorf thermomixer C (with constant shaking at 300rpm). After the reaction was complete, the sample was passed through a Zeba spin desalting column (40 KDa MWCO, ThermoFisher, Cat#: 87770) according to the manufacturer’s protocol to remove unused chelator-linker and buffer-exchange into PBS (pH 7.4) (LifeTechnologies, Cat#: 10010- 023). Each antibody-chelator conjugate (ACC) was stored at 4°C until analysis and purification.

[0310] Representative results of conjugation experiments are presented in Table 6.Table 6 – Conjugation results before purification PCT Chelator- Conjugatio C m nd VHH link r:Ab DMSO (%) n protein MS CAR(%) EC50 (nM) Cell lineExample 4. Purification by prep-SEC

[0311] To remove high molecular weight species (HMWS) and low molecular weightspecies (LMWS), VHH-Fcs were purified by SEC using an AKTA Pure FPLC system with a Cytiva HiLoad 16 / 600 Superdex 200pg column. TBS buffer (50mM Tris, 150mM NaCl, OmniTrace Ultra water [VWR, Cat#: CAWX0003-2]), pH 7.6 was used for the SEC buffer. The fractions containing intact VHH-Fcs were pooled together and concentrated using Microsep Advance Centrifugal Device (Pall 10k MWCO, Cat#: MCP010C41). The concentrated sample was transferred to an Ultrafree-MC GV Centrifugal Filter, 0.22µm 0.5mL (Millipore, Cat#: UFC30GV0S) and spun at 3,000 x g for 3 minutes. Results for the purification of 126_zu2- compound 3-16 are shown in Table 7. Example 5. Protein QuantificationWSGR Attorney Docket No.: 60924-730.601

[0312] For some samples, VHH-Fc protein or VHH-Fc conjugate content was quantifiedwith a Pierce BCA Protein Assay Kit (Thermo, Cat#: 23225) standardized by Cetuximab (LIST / E: 094822, DIN 02271249, 2 mg / mL).

[0313] For other samples, VHH-Fc protein or VHH-Fc conjugate content was quantified bymeasuring the absorbance of the solution at 280 nm and the protein’s calculated extinction coefficient at 280 nm using a 2 uL sample with a Lunatic UV / Vis spectrophotometer (Unchained Labs). Example 6. Chelator to VHH-Fc Ratio (CAR) Analysis

[0314] The chelator loading, herein described as CAR (chelator-antibody ratio), can beanalyzed through methods applicable to practitioners of the art of antibody conjugates. For a review of these methods in the context of ADCs, see A Wakankar et al., mAbs 3:161 (2011).

[0315] VHH-Fc antibodies were de-glycosylated prior to analysis with in-house Endo-Senzyme (final concentration of 10 μg / mL) of commercial Endo-S (New England BioLabs Inc., Cat#: P0741L) at 37 °C for 1 hour. For analysis of the intact mass, 8 μL samples were injected on to a Waters Acquity UPLC-Q-TOF with a BEH200 SEC 1.7 µM 4.6x150 mm column. These samples were eluted with a mobile phase of water / ACN (70 / 30, v / v) with 0.1% TFA and 0.1% FA (formic acid) for 11 min with a flow rate of 0.25 mL / min). Deconvoluted spectra showed a distribution of masses representing species with a certain number of chelator-linkers attached. The intensities of the peaks were used to calculate the average CAR of the conjugate preparation by taking the sum of each peak intensity multiplied by the number of modification represented by that mass, then dividing by the total intensity of all antibody masses. (could also put in the equation). In this case, a distribution of masses is obtained after spectrum deconvolution that allows calculation of the average CAR of the preparation. This analysis contributes to CAR results in Tables 6 and 7.

[0316] Alternatively, some conjugates were analyzed by UPLC-Q-TOF-MS using anAgilent AdvanceBio 6545XT LC / QTOF mass spectrometer with a mobile phase gradient of starting at 80% mobile phase A (Milli-Q water with 0.1 % Formic Acid) and 20% mobile phase B (Acetonitrile with 0.1 % Formic Acid) for 4 min. then linearly changing to 90% mobile phase B over the next 7 min. at a constant flow rate of 0.6 mL / min on an Agilent PLRP-S column (I.D. 2.1 mm / Length: 50 mm / Particle size: 5μm / Pore size: 1000 Å, Part#: PL1912-1502PK). De- convolution of raw data and CAR calculations were done with Agilent's BioConfirm software (Version 12.0) on Agilent's MassHunter workstation. This analysis contributes to CAR results in Tables 6 and 7.WSGR Attorney Docket No.: 60924-730.601 Example 7. Binding of VHH-Fc conjugates to cells expressing target protein

[0317] Conjugates were screened for binding to a range of target-positive cancer cell linesby flow cytometry. All cell lines were sourced from ATCC unless otherwise noted, and cultured according to manufacturer’s instructions and recommended media. SHP-77 (ATCC CRL-2195) cells were used as the DLL3-positive cell line. Primary antibodies diluted in same manner as for ELISA were added to cells and incubated for 1 hour on ice. Cells were washed twice with 1% FBS in PBS, centrifuged at 450G for 4 minutes and incubated with 2 μg / mL AlexaFluor 647 conjugated anti-human IgG (Jackson, Cat#109-605-098) or AlexaFluor 647 conjugated anti- mouse IgG (Jackson, Cat#115-605-164) with 1:1000 DAPI (Biolegend, Cat#422801) for 30 minutes on ice. Following two further washes, cells were resuspended, and analyzed by flow cytometry on the iQue screener platform (Intellicyt), and data was processed with Forecyt, according to standard protocols.

[0318] Table 6 above also includes cell binding data of VHH-Fc chelator conjugates.Binding was not significantly affected with conjugation for all conjugates which were tested, regardless of the linker used or the ultimate CAR of that sample. As shown in Table 8 below, binding was maintained for all purified conjugates. Example 8. Percent Intact Analysis

[0319] The percent intact immunoconjugate was established by HPLC-SEC. 12 μL ofconjugate was added to a glass vial insert in a standard HPLC vial.10 μL of sample was injected onto an Agilent HPLC-SEC with a Wyatt Technology WTC-050S5 SN:0429 BN WBD129 column column and eluted with 1x PBS (100%) for 40 min at a flow rate of 0.5 mL / min. This analysis contributed to results in Tables 6 and 7. Example 9. Endotoxin Level Determination

[0320] Endotoxin test was performed using Wako's Limulus Amebocyte Lysate PyrostarTMES - F Single Test (Cat#: WPESK-0015) according to manufactural protocol. The QC cutoff was set based on the maximum injection dose projected for each animal in the study while following appropriate animal care and FDA guidelines. Results are shown in Table 7.

[0321] Table 7 shows data for purified 126_zu2-compound 3-16WSGR Attorney Docket No.: 60924-730.601 Table 7 r )Example 10. Radiolabeling with In-111

[0322] 40 μg of each of the 4 test articles was diluted to 100 μL with 0.1 M ammoniumacetate buffer in a 500 μL lo-bind Eppendorf tube and 18-25 μL (20-22 MBq) of [111In]InCl3 was added and mixed with a pipette. The reaction mixtures were incubated at 37°C in an incubator for 1 hour. The tubes were then transferred to a 4°C fridge. Results after analysis are shown in Table 8.

[0323] Incorporation of radionuclides was determined by spotting 0.5 μL of sample at theorigin of a 1.5 x 10 cm iTLC strip. The strip was then placed in a 50 mL Falcon tube containing 2 mL of mobile phase (25 mM EDTA in pH 50.1 M sodium acetate buffer) until the solvent had reached the top of the strip. The strip was removed and exposed to a phosphor imaging plate which was then scanned in a Cyclone phosphor imager. Regions of interest were drawn over spots corresponding to the migration of protein-bound and un-bound In-111 and the proportion in each calculated.

[0324] Radioconjugates were also analyzed by SEC-HPLC: A volume corresponding to 0.1-0.2 MBq of the sample was pipetted into a 500 μL lo-bind Eppendorf tube and the radioactivity measured in an ionization chamber. The sample was drawn up into a syringe and injected onto the HPLC system. Samples were eluted with 0.9% w / v saline. The eluate from the system was collected and the radioactivity measured in order to determine the recovery from the column (corrected for activity remaining in the sample tube and the injection syringe). Table 8 – In-111 Radiolabeling and stability data for chelator-conjugatesWSGR Attorney Docket No.: 60924-730.601 Table 8 * Examp e . ad o abe ng w c- 5

[0325] 1000 μg of the test articles was diluted to 500 μL with 0.15 M ammonium acetatebuffer pH 6.0 in a 500 μL lo-bind Eppendorf tube and 9.9 μL (500 kBq) of225Actinium chloride was added and mixed with a pipette (to achieve a final specific activity of 0.5 kBq / ug of protein). The reaction mixture was incubated at 37°C in an incubator for 2 hours. The conjugates was then purified by passing through a PD10 desalting column 8.3 ml (Amersham. Cat# 17- 0851-01) and eluting with the saline formulation buffer indicated in Table 9. If not purified, the samples were diluted with the same formulation buffer. The tubes were then transferred to a 4 °C fridge.

[0326] Incorporation was measured by spotting 0.5 μL of sample at the origin of a 1.5 x 10cm iTLC strip and allowing it to dry for a few minutes. The strip was then placed in a 50 mL Falcon tube containing 2 mL of mobile phase (25 mM EDTA in pH 50.1 M sodium acetate buffer) until the solvent had reached the top of the strip. The strip was removed and allowed to equilibrate for at least 2 hours, after which it was exposed to a phosphor imaging plate which was then scanned in a Cyclone phosphor imager. Regions of interest were drawn over spots corresponding to the migration of protein-bound and un-bound 225Ac and the proportion in each calculated.

[0327] Samples could be assayed by HPLC-SEC: Some data used a BioSEP SEC 5 µms30003007.88 mm column with 20% acetonitrile in PBS elution or a Wyatt 050S55 µm 500 Å 7.8 x 300 mm column with 20% acetonitrile in PBS elution).

[0328] Alternatively, the same analysis is performed using a Tosoh Bioscience TSKgelG3000SW XL (I.D.7.8 mm / Length: 300 mm / Particle size: 5μm / Pore size: 250 Å, Part#: 08541) column combined with a guard column (TSKgel SWXL Guard Column I.D.6.0 mm / Length: 40 mm / Particle size: 7μm, Part#: 08543) using an isocratic elution with a mobile phase consisting of 78 mM KH2PO4, 122 mM K2HPO4, 250 mM KCl, 15% 2-Propanol, pH 7.0, 0.2 μm filtered, over 45 min at a constant flow rate of 0.5 mL / min.WSGR Attorney Docket No.: 60924-730.601

[0329] 50 μL of each sample was drawn up into a Hamilton syringe and injected onto theHPLC system. From 10-30 minutes post injection, 30 second fractions of the eluate (0.25 mL) were collected by hand into counting tubes. The fractions were allowed to reach secular equilibrium for 24 hours and then measured in a gamma counter. A 5 μL sample of each preparation was also counted to enable the recovery from the HPLC system to be calculated. Radiochemical purity was determined by determining the area under the peak for 18.5-22.5 mins and 19.5-23.5 mins, as a percentage of total counts. As shown in Table 17 all chelator-linker combinations showed good labeling efficiency. Table 9 – Ac-225 radiolabeling results Table 9Example 12. Stability of VHH-Fc Radioconjugates

[0330] The stability of the radiolabeled immunoconjugates was tested, both for 225Ac and111In. VHH-Fc chelator-conjugates were radiolabeled (either111In or225Ac) as described above. For stability in PBS, 50 μL of each labelled test article was then added to either 200 μL of PBS (with111In) or 200 µL PBS / ascorbate (with225Ac) and stored at 4°C. For stability in saline, samples were buffer exchanged after elution through a PD10 column as described in Examples 10 and 11, taking 1-1.5 mL of eluant. Aliquots of were taken at different time points and analyzed for radiochemical purity using iTLC and / or HPLC-SEC as described above. The results of these stability experiments are shown in Tables 8 and 9. Example 13. Biodistribution study showing rapid liver clearance of 126_zu2 conjugated to compound 3-16 in naïve mice with a humanized FcRn systemWSGR Attorney Docket No.: 60924-730.601 Objective

[0331] Imaging (e.g., using 111In) provides for the ability to collect pharmacokinetic andbiodistribution data that can be used to perform dosimetry calculations for treatment planning. (See, e.g., Sgouros G, Hobbs RF. “Dosimetry for radiopharmaceutical therapy.” Semin Nucl Med.2014 May;44(3):172-8). Without being bound by theory, a quantitative demonstration of targeting observed with an imaging label is indicative of the ability to target with a radiolabel (e.g., an alpha emitter) capable of causing targeted cell death. This experiment evaluated the biodistribution of 126_zu2 and compound 3-16 immunoconjugate having 111In-labeled test articles by SPECT / CT imaging and PK by peripheral blood collection for gamma counting in human FcRn (Tg32) male mice (mice expressing human FcRn). Methods

[0332] In brief, Tg32 mice, male (n = 5) were injected via the tail vein, then imaged withwhole-body SPECT under anesthesia followed by CT following the experimental design summary below in Table 10. Table 10 – Experimental design summary of Tg32 Biodistribution experiment Injected Volume, Imaging Time n Test ArticleResults

[0333] The 111In-labeled immunoconjugate comprising 126_zu2 and compound 3-16showed rapid clearance from the liver and body. Substantially all111In-label was cleared between 21-144 hours post injection. FIGs.3A-E (at timepoints 1 h, 24 h, 72 h, 144 h, and 240 h, respectively) show imaging results demonstrating rapid clearance from liver and body. Initial accumulation in the heart and liver, followed by accumulation in the bladder before clearance was observed (see FIGs.3A-3E). Table 11 summarizes the qualitative results from the imaging. Table 11 – Qualitative results of Tg32 Biodistribution experiment Table 11WSGR Attorney Docket No.: 60924-730.601 24 (FIG.3B) Reduced presence in liver 72 (FIG.3C) Minimal or substantially no presence detectablea p e . _ u co pou - o p a acoogy Methods

[0334] The final radiochemical purity for all test articles used for in vivo studies was ≥ 95%.Indium-111 (111In) radioactivity was quantified by Single Photon Emission Computed Tomography (SPECT) / Computed Tomography (CT) imaging or gamma counting in blood and tissues. Actinium-225 (225Ac) was quantified by gamma counting based on gamma emissions from221Fr and213Bi measured following secular equilibrium. The uptake or percentage injected dose (%ID) is defined as the tissue radioactivity (MBq) decay-corrected to the injection time, normalized to the activity injected. The activity concentration or percentage injected dose per gram of tissue (%ID / g) is the %ID normalized to the weight of the ex vivo gamma-counted tissue or the volume of the quantified tissue for in vivo SPECT / CT imaging data assuming a tissue density of 1 g / mL. Single dose in vivo pharmacokinetics and biodistribution of111In-126_Zu2(compound 3-16) in naïve human FcRn transgenic (Tg32) mice

[0335] Naïve male human FcRn transgenic (Tg32) mice (n=5) were administered 111In-126_Zu2(compound 3-16) by single IV bolus injection (10 MBq, 20 μg protein, and 0.5 MBq / μg specific activity). In vivo biodistribution was evaluated using SPECT / CT imaging in four mice at 1 hour, 24 hours, 72 hours, 144 hours, and 240 hours post-treatment. In addition, whole blood samples were collected from all five mice at 5 min, 24 hours, 72 hours, 144 hours, and 240 hours for gamma counting.

[0336] 111In-126_Zu2(compound 3-16) cleared rapidly from the blood compartment with 91% of the injected activity cleared in the first 24 hours and an elimination half-life of 34.9 hours (FIG.4). Radionuclide elimination from animals was assessed by SPECT / CT-based determination of %ID of the111In-126_Zu2(compound 3-16) remaining in the whole-body (FIG. 5). Whole-body clearance indicated 45 % of the injected activity cleared in the first 24 hours with 95 % of the injected activity eliminated by 240 hours.

[0337] 111In-126_Zu2(compound 3-16) also showed rapid clearance through tissues withhighest activity concentration in the liver (18.26 ± 0.88 %ID / g), followed by heart (15.41 ± 0.62WSGR Attorney Docket No.: 60924-730.601 %ID / g), lungs (9.78 ± 0.39 %ID / g), and kidneys (8.60 ± 0.28 %ID / g) at 1 hour post-dose administration, declining to below 0.6 %ID / g by 240 hours (FIG.6).111In-126_Zu2(compound 3-16) activity concentration in the bladder increased from 1 hour (4.03 ± 0.50 %ID / g) to 24 hours (10.22 ± 0.52 %ID / g) post injection followed by a rapid decreased by 240 hours post injection (0.17 ± 0.08 %ID / g) (FIG.6). Cumulative excreta assessment following single dose administration of111In-126_zu2(compound 3-16) in naïve human FcRn transgenic (Tg32) mice

[0338] Naïve male human FcRn transgenic (Tg32) mice (n=5) were administered 111In-126_Zu2(compound 3-16) by single IV bolus injection (10 MBq, 20 µg protein, and 0.5 MBq / µg specific activity). Pooled excreta were collected from the group-housed cage at 0-24 hours (day 1), 24-48 hours (day 2), 48-72 hours (day 3), 72-144 hours (day 6), and 144-240 hours (day 10) and measured by a dose calibrator. Feces were then separated from total excreta and both the feces and non-fecal component (urine) were measured individually in the dose calibrator. At the terminal time point (240 hours), the concentration of111In-126_Zu2(compound 3-16) remaining in the kidneys, liver, spleen, and large intestines wall was also evaluated by gamma counting.

[0339] The cumulative radioactivity excreted via urine and feces combined was 44.46 %IDat 1 day post injection with 37.51 %ID excreted in urine and 6.95 %ID excreted in feces (FIG. 7). At the terminal timepoint 10 days post injection, 94.62 % ID111In-126_Zu2(compound 3-16) was excreted from the body with 76.90 % ID excreted in urine and 17.72 %ID excreted in feces indicating predominantly renal elimination (FIG.7). These data are consistent with estimates of radionuclide elimination derived by whole-body SPECT / CT (FIG.5) supporting rapid111In- 126_Zu2(compound 3-16) excretion.

[0340] Gamma counting of the resected excretory organs at 240 hours showed <0.6 %ID / gof injected In-111 activity concentration remaining in all the tissues measured (not shown). Plasma pharmacokinetics of111In-126_zu2(compound 3-16) radioactivity vs antibody concentration in human FcRn transgenic (Tg32) mice

[0341] Naïve male human FcRn transgenic (Tg32) mice (n=15) were administered 111In-126_Zu2(compound 3-16) by single IV bolus injection (3 MBq, 75 µg protein, and 0.04 MBq / µg specific activity). Blood samples collected through cardiac puncture at 5 min and 4 hours, 24 hours, 48 hours, and 144 hours (n = 3 / time point) following injection, were separated into plasma and gamma counted. The antibody component of 126_Zu2(compound 3-16) was quantified in the same plasma samples using a sandwich ELISA. Plasma concentrations for111In-126_Zu2(compound 3-16) and 126_Zu2(compound 3-16) were normalized to theirWSGR Attorney Docket No.: 60924-730.601 respective Cmax (with Cmax = 100 %) and used to compare the systemic exposures between the quantified plasma radioactivity and antibody concentration.

[0342] The systemic exposure of the 111In-126_Zu2(compound 3-16) was comparable whenmeasuring radioactivity or antibody concentration indicating stability in the blood compartment (FIG.8). Single dose ex vivo pharmacokinetics and biodistribution of111In-126_zu2(compound 3-16) and in 1126_Zu2(compound 3-16) (3 MBq, 30 µg protein, and 0.1 MBq / µg specific activity) or225Ac- 126_Zu2(compound 3-16) (15 kBq, 30 µg protein, and 0.5 kBq / µg specific activity) by a single IV injection. Ex vivo biodistribution was evaluated by gamma counting of resected tissues at 1 hour, 24 hours, 72 hours, and 168 hours post-treatment (n=3 / time point). An immunoreactive fraction (IRF) assay was performed to evaluate the binding of111In-126_Zu2(compound 3-16) and225Ac-126_Zu2(compound 3-16) to DLL3 antigen. Immunoreactivity was > 95%.

[0344] Unlike the mice expressing human FcRn, 111In-126_Zu2(compound 13-6) and 225Ac-126_Zu2(compound 13-6) were cleared slower from the blood compartment with longer elimination half-lives (~90 hours) in CD-1 mice as expected with the presence of the endogenous murine FcRn (FIG.9). Blood systemic exposures (consistent Cmax and AUC) established bioequivalence between111In-126_Zu2(compound 13-6) and225Ac- 126_Zu2(compound 13-6).

[0345] 111In-126_Zu2(compound 3-16) and 225Ac-126_Zu2(compound 3-16) showed aconsistent pattern of tissue distribution and tissue exposures (FIGs. 10-11). At the 1 hour timepoint, both111In-126_Zu2(compound 13-6) and225Ac-126_Zu2(compound 13-6) had the highest concentrations in the lungs (8.48 ± 0.40 %ID / g and 10.29 ± 1.92 %ID / g, respectively) followed by liver (7.73 ± 1.18 %ID / g and 10.00 ± 0.29 %ID / g, respectively). Kidney activity concentrations were also similar (6.04 ± 0.62 %ID / g and 7.51 ± 0.38 %ID / g, respectively). By 168 hours, the activity concentrations of both test articles declined below 1.7 %, 1.5 % ID / g, and 3.4 %ID / g in the lungs, liver, and kidneys, respectively. These data demonstrate comparable111In- 126_Zu2(compound 3-16) and225Ac-126_Zu2(compound 3-16) pharmacokinetics and biodistribution and support the use of111In-126_Zu2(compound 3-16) as a surrogate imaging agent. Non-GLP single dose toxicity study of non-labeled (cold) 126_zu2(compound 3-16) in naïve CD- 1 miceWSGR Attorney Docket No.: 60924-730.601

[0346] Naïve male and female CD-1 mice were administered non-labeled (cold)126_Zu2(compound 3-16) at either 6 mg / kg or 30 mg / kg (n = 6 / sex) through a single bolus IV injection. Clinical observations, body weights, food consumption and physical examinations were performed during the course of the study. Organ weights, hematology, coagulation, clinical chemistry, gross and microscopic pathology were assessed 5 days post dose administration. Blood was collected at 5 min and on day 5 following dosing to confirm 126_Zu2(compound 3- 16) exposure.

[0347] A single IV injection of 126_Zu2(compound 3-16) of 6 mg / kg or 30 mg / kg to maleand female CD-1 mice was well tolerated with no test article related findings other than minimal to mild hepatocellular hypertrophy in males only, which was considered adaptive and non- adverse (not shown). Single dose in vivo pharmacokinetics and biodistribution of111In-126_zu2(compound 3-16) in naïve cynomolgus monkeys

[0348] As 126_Zu2 binds both human and cynomolgus monkey DLL3 with high affinity butnot mouse DLL3. Pharmacokinetics and normal tissue biodistribution were evaluated in cynomolgus monkeys, the pharmacologically relevant species. Naïve male and female cynomolgus monkeys (n=1 / sex) were administered111In-126_Zu2(compound 3-16) by single IV injection (male: 286 MBq, female: 173 MBq, 0.3 mg / kg protein, and 0.1 MBq / µg specific activity). In vivo biodistribution was assessed by longitudinal SPECT / CT imaging at 1 hour, 24 hours, 72 hours, 144 hours, and 240 hours post-administration. Whole blood samples were collected at 5 min, 4 hours, 24 hours, 72 hours, and 240 hours post-administration for gamma counting.

[0349] Consistent with the findings in human FcRn transgenic mice, 111In-126_Zu2(compound 3-16) cleared rapidly from the blood compartment with 76 % of the injected activity cleared in the first 24 hours and an elimination half-life of 40 hours (FIG.12).

[0350] Radionuclide elimination from animals was assessed by SPECT / CT-baseddetermination of %ID of the111In-V126_Zu2(compound 3-16) remaining in the whole-body (FIG.13). Whole-body clearance indicated 13-18 % of the injected activity cleared in the first 24 hours, 80 % by 144 hours with 90 % of the injected activity eliminated by 240 hours.

[0351] 111In-126_Zu2(compound 3-16) showed rapid clearance through excretory organs. At1 hour post-dose the highest activity concentration was in the liver (male: 0.14 %ID / g, female: 0.20 % ID / g) with liver peak concentration at 24 hours (male: 0.24 %ID / g, female: 0.30 %ID / g), followed by lungs (male: 0.07 %ID / g, female: 0.14 %ID / g), heart (male: 0.11 %ID / g, female:WSGR Attorney Docket No.: 60924-730.601 0.09 %ID / g) and kidneys (male: 0.06 %ID / g, female: 0.09 %ID / g), all declining below 0.02 %ID / g by 240 hours post-dose (FIG.14). In terms of uptake as a fraction of the total injected activity of111In-126_Zu2(compound 3-16) for excretory tissues, the liver peak of 30 to 45 %ID at 24 hours declined to 3.5 %ID by 240 hours; the kidney peak of 0.76 to 1.1 %ID at 1 hour was down to 0.1 %ID by 240 hours. The pattern of distribution was consistent with findings in human FcRn transgenic mice that showed a generally similar fraction of111In- 126_Zu2(compound 3-16) injected activity in these tissues at 0.3 mg / kg (liver and kidney peak at 1 h of 20.3 %ID and 2.3% ID, respectively, declining to ≤ 0.6 % ID at 240 h, % ID data not shown).

[0352] While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0353] All publications, patent applications, issued patents, and other documents referred toin this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.WSGR Attorney Docket No.: 60924-730.601 SEQUENCES Numbe Annotatio r SEQ n 3WSGR Attorney Docket No.: 60924-730.601 APEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS 1 3WSGR Attorney Docket No.: 60924-730.601 QVQLVESGGGMVQPGGSLRLSCAASGITFSMYSMSWY RQPPGKQRELVAATTTFGSTNYADSVKGRFTISRDNA 29 varWSGR Attorney Docket No.: 60924-730.601 QVQLVESGGGLVQPGGSLRLSCAASGITFSMYSMSWF

Claims

WSGR Attorney Docket No.: 60924-730.601 CLAIMS What is claimed is:

1. An immunoconjugate comprising an antibody that binds DLL3 attached (conjugated) toa compound, wherein: the antibody comprises a VHH variable domain, wherein the VHH variable domain comprises: (a) a heavy chain complementarity determining region 1 (CDRH1)comprising an amino acid sequence of SEQ ID NO: 14, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 15, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 16; (b) a heavy chain complementarity determining region 1 (CDRH1)comprising an amino acid sequence of SEQ ID NO: 17, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 18, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 19; (c) a heavy chain complementarity determining region 1 (CDRH1)comprising an amino acid sequence of SEQ ID NO: 20, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 21, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 22; (d) a heavy chain complementarity determining region 1 (CDRH1)comprising an amino acid sequence of SEQ ID NO: 23, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 24, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 25; or (e) a heavy chain complementarity determining region 1 (CDRH1)comprising an amino acid sequence of SEQ ID NO: 26, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 27, and a heavy chain complementarityWSGR Attorney Docket No.: 60924-730.601 determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 28; and the compound comprises a structure represented by Formula (II), Formula (III), or Formula (IV), or a pharmaceutically acceptable salt thereof:wherein: R1is a chelating moiety or a radionuclide complex thereof; X1is -O-, -S-, -S(=O)-, -S(=O)2-, -NRa-, -C(=O)-, -NRaC(=O)-, - C(=O)NRa-, -(C1-C6alkylene)-X2-, or -(C4-C20polyethylene glycol)-X2-; X2is absent, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, or -C(=O)X4-; each Rais independently selected from hydrogen, and C1-C4alkyl; X4is -NRa-, or -NRaS(=O)2-; L is an optional linker; -NH-R3is the antibody; and v is 1, 2, 3, or 4.

2. The immunoconjugate of claim 1, wherein the compound comprises a structurerepresented by Formula (IIa), or a pharmaceutically acceptable salt thereof:wherein:WSGR Attorney Docket No.: 60924-730.601 -NHCH2CH2CH2CH2-is the side chain of a lysine residue of the antibody R3.

3. An immunoconjugate comprising an antibody that binds DLL3 attached (conjugated) toa compound, wherein: the antibody comprises a VHH variable domain, wherein the VHH variable domain comprises: (a) a heavy chain complementarity determining region 1 (CDRH1)comprising an amino acid sequence of SEQ ID NO: 14, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 15, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 16; (b) a heavy chain complementarity determining region 1 (CDRH1)comprising an amino acid sequence of SEQ ID NO: 17, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 18, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 19; (c) a heavy chain complementarity determining region 1 (CDRH1)comprising an amino acid sequence of SEQ ID NO: 20, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 21, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 22; (d) a heavy chain complementarity determining region 1 (CDRH1)comprising an amino acid sequence of SEQ ID NO: 23, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 24, and a heavy chain complementarity determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 25; or (e) a heavy chain complementarity determining region 1 (CDRH1)comprising an amino acid sequence of SEQ ID NO: 26, a heavy chain complementarity determining region 2 (CDRH2) comprising an amino acid sequence of SEQ ID NO: 27, and a heavy chain complementarityWSGR Attorney Docket No.: 60924-730.601 determining region 3 (CDRH3) comprising an amino acid sequence of SEQ ID NO: 28; and the compound comprises a structure represented by Formula (V), Formula (VI), Formula (VII), or Formula (VIII), or a pharmaceutically acceptable salt thereof:Formula (VI)wherein: R1is a chelating moiety or a radionuclide complex thereof; X1is -O-, -S-, -S(=O)-, -S(=O)2-, -NRa-, -C(=O)-, -NRaC(=O)-, - C(=O)NRa-, -(C1-C6alkylene)-X2-, or -(C4-C20polyethylene glycol)-X2-; X2is absent, -C(=O)-, -NRaC(=O)-, -C(=O)NRa-, or -C(=O)X4-; each Rais independently selected from hydrogen, and C1-C4alkyl; X4is -NRa-, or -NRaS(=O)2-; L is an optional linker; -S-R3is the antibody; and v is 1, 2, 3, or 4.

4. The immunoconjugate of any one of claims 1-3, wherein:WSGR Attorney Docket No.: 60924-730.601 wherein R1is a chelating moiety or a radionuclide complex thereof, wherein the chelating moiety is: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); 1,4,7,10-tetraazacyclododecane-1,4,7 -triacetic acid (DO3A); 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A); α,α',α'',α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTMA); 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM); 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid (DOTPA); 2,2',2''-(10-(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7- triyl)triacetic acid; 6,6'-(((pyridine-2,6-diylbis(methylene))bis((carboxymethyl)azanediyl))- bis(methylene))dipicolinic acid (H4pypa); 6,6',6'',6'''-(((pyridine-2,6- diylbis(methylene))bis(azanetriyl))tetrakis(methylene))-tetrapicolinic acid (H4py4pa); 10-((6-carboxypyridin-2-yl)methyl)-1,4,7,10-tetra-azacyclododecane- 1,4,7-triacetic acid (DO3Apic); or 3,6,9,12-tetrakis(carboxymethyl)-3,6,9,12-tetraazatetradecanedioic acid (TTHA).

5. The immunoconjugate of any one of claims 1-3, wherein:R1is a chelating moiety or a radionuclide complex thereof, wherein the chelating moiety is: .

6. TheR1is a chelating moiety or a radionuclide complex thereof, wherein the chelating moiety is:WSGR Attorney Docket No.: 60924-730.601 ;7. The of claims 1-3, wherein:or a radionuclide complex thereof, wherein the chelating moiety is: .

8. Theof claims 1-7, wherein:X1is absent, -O-, -S-, -NRa-, -C(=O)-, -NRaC(=O)-, or -C(=O)NRa-; or X1is -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, - CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -CH2-X2-, -CH2CH2-X2-, - CH2CH2CH2-X2-, -CH2CH2CH2CH2-X2-, -CH2CH2CH2CH2CH2-X2-, or - CH2CH2CH2CH2CH2CH2-X2-.

9. The immunoconjugate of any one of claims 1-7, wherein:X1is -CH2CH2-or -CH2CH2-X2-; X2is -C(=O)X4-; X4is -NH-, -N(CH3)-, or -N(CH2CH3)-.

10. The immunoconjugate of any one of claims 1-9, wherein:L is -L1-, or -L1-L2-L3-L4-L5-; L1is unsubstituted or substituted C1-C20alkylene, unsubstituted or substituted C1- C20heteroalkylene, C4-C20polyethylene glycol, unsubstituted or substituted C3- C8cycloalkylene, unsubstituted or substituted monocyclic C3- C8heterocycloalkylene, unsubstituted or substituted phenylene, unsubstituted or substituted monocyclic heteroarylene; L2is absent, -C(=O)NR4-(unsubstituted or substituted C1-C10alkylene)-, - NR4C(=O)-(unsubstituted or substituted C1-C10alkylene)-, -C(=O)-(CH2CH2O)m-WSGR Attorney Docket No.: 60924-730.601 (CH2)P-, -C(=O)NR4-(CH2CH2O)n-(CH2)P-, -NR4C(=O)-(CH2CH2O)n-(CH2)P-, or -(CH2CH2O)n-(CH2)P-; each R4is independently selected from hydrogen, and C1-C6alkyl; each m is independently 1, 2, 3, 4, 5, or 6; each p is independently 1, or 2; L3is absent; L4is absent, -C(=O)-( unsubstituted or substituted C1-C6alkylene)-, -C(=O)NR4- (unsubstituted or substituted C1-C6alkylene)-, -NR4C(=O)-(unsubstituted or substituted C1-C6alkylene)-, -C(=O)-(CH2CH2O)n-(CH2)q-, -C(=O)NR4- (CH2CH2O)n-(CH2)q-, -NR4C(=O)-(CH2CH2O)n-(CH2)q-, or -(CH2CH2O)n- (CH2)q-; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2; L5is absent, -C(=O)-(CH2)n-, -C(=O)NR4-(CH2)n-, -NR4C(=O)-(CH2)n-, -C(=O)- (CH2CH2O) (CH2CH2O)(OCH2CH2)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 Rais independently selected from hydrogen, and C1-C4alkyl; wherein heteroalkylene is an alkylene where one carbon atom is replaced with - S(=O)(=NH)-, -S(=O)(=NR5)-, -P(=O)OH-, -NHC(=N-CN)NH-, or -NHC(=N- , -, - , - , - halogen, -OH, -OR5, -CO2H, -NHR5, -C(=O)NHR5, -NHC(=O)R5and substituted C1-C6alkyl, wherein the substituted C1-C6alkyl is substituted with, - OH, -CO2H, -NHR5, -C(=O)NHR5, and -NHC(=O)R5; each R5is independently selected from C1-C10alkyl, C4-C30polyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene.

11. The immunoconjugate of any one of claims 1-10, wherein:L1is unsubstituted or substituted C1-C6alkylene, unsubstituted or substituted C1- C10heteroalkylene, C4-C20polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene.WSGR Attorney Docket No.: 60924-730.60112. The immunoconjugate of any one of claims 1-10, wherein:L5is absent, -NR4C(=O)-(CH2)n-, -C(=O)-(CH2CH2O)n-(CH2)q-, -C(=O)NR4- (CH2CH2O)n-(CH2)q-, or -NR4C(=O)-(CH2CH2O)n-(CH2)q-; each q is independently 1, or 2.

13. The immunoconjugate of any one of claims 1-9, wherein:L1is unsubstituted or substituted C1-C6alkylene, unsubstituted or substituted C1- C10heteroalkylene, C4-C20polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L2is absent, -C(=O)NR4-(unsubstituted or substituted C1-C10alkylene)-, - NR4C(=O)-(unsubstituted or substituted C1-C10alkylene)-, -C(=O)-(CH2CH2O)m- (CH2)P-, -C(=O)NR4-(CH2CH2O)n-(CH2)P-, -NR4C(=O)-(CH2CH2O)n-(CH2)P-, or -(CH2CH2O)n-(CH2)P-; each m is independently 1, 2, 3, 4, 5, or 6; each p is independently 1 or 2; L4is absent; L5is -NR4C(=O)-(CH2)n-, -C(=O)-(CH2CH2O)n-(CH2)q-, -C(=O)NR4- (CH2CH2O)n-(CH2)q-, or -NR4C(=O)-(CH2CH2O)n-(CH2)q-; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2.

14. The immunoconjugate of any one of claims 1-9, wherein:L1is unsubstituted or substituted C1-C6alkylene, unsubstituted or substituted C1- C10heteroalkylene, C4-C20polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L2is absent, -C(=O)NR4-(unsubstituted or substituted C1-C10alkylene)-, - NR4C(=O)-(unsubstituted or substituted C1-C10alkylene)-, -C(=O)-(CH2CH2O)m- (CH2)P-, -C(=O)NR4-(CH2CH2O)n-(CH2)P-, -NR4C(=O)-(CH2CH2O)n-(CH2)P-, or -(CH2CH2O)n-(CH2)P-; each m is independently 1, 2, 3, 4, 5, or 6; each p is independently 1 or 2; L4is absent; L5is absent, -NR4C(=O)-(CH2)n-, -C(=O)-(CH2CH2O)n-(CH2)q-, -C(=O)NR4- (CH2CH2O)n-(CH2)q-, or -NR4C(=O)-(CH2CH2O)n-(CH2)q-; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2.

15. The immunoconjugate of any one of claims 1-9, wherein:WSGR Attorney Docket No.: 60924-730.601 L1is unsubstituted or substituted C1-C6alkylene, unsubstituted or substituted C1- C10heteroalkylene, C4-C20polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L2is absent; L4is absent; L5is absent, -NR4C(=O)-(CH2)n-, -C(=O)-(CH2CH2O)n-(CH2)q-, -C(=O)NR4- (CH2CH2O)n-(CH2)q-, or -NR4C(=O)-(CH2CH2O)n-(CH2)q-; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2.

16. The immunoconjugate of any one of claims 1-14, wherein:L is -L2-L5-; L2is absent, -C(=O)NR4-(unsubstituted or substituted C1-C10alkylene)-, - NR4C(=O)-(unsubstituted or substituted C1-C10alkylene)-, -C(=O)-(CH2CH2O)m- (CH2)P-, -C(=O)NR4-(CH2CH2O)n-(CH2)P-, -NR4C(=O)-(CH2CH2O)n-(CH2)P-, or -(CH2CH2O)n-(CH2)P-; each R4is independently selected from hydrogen, and C1-C6alkyl; each m is independently 1, 2, 3, 4, 5, or 6; each p is independently 1, or 2; L5is absent, -C(=O)-(CH2)n-, -C(=O)NR4-(CH2)n-, -NR4C(=O)-(CH2)n-, -C(=O)- (CH2CH2O)n-(CH2)q-, -C(=O)NR4-(CH2CH2O)n-(CH2)q-, -NR4C(=O)- (CH2CH2O)n-(CH2)q-, -(CH2CH2O)n-(CH2)q-, -C(=O)-(OCH2CH2)n-, or - (OCH2CH2)n-; each R4is independently selected from hydrogen, and C1-C6alkyl; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 0, 1, or 2.

17. The immunoconjugate of any one of claims 1-3, wherein:WSGR Attorney Docket No.: 60924-730.601 X4is -NH-, -N(CH3)-, or -N(CH2CH3)-. L is -L1-, or -L1-L2-L3-L4-L5-; L1is unsubstituted or substituted C1-C6alkylene, unsubstituted or substituted C1- C10heteroalkylene, C4-C20polyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene; L2is absent; L3is absent; L4is absent; L5is -NR4C(=O)-(CH2)n-, -C(=O)-(CH2CH2O)n-(CH2)q-, -C(=O)NR4- (CH2CH2O)n-(CH2)q-, or -NR4C(=O)-(CH2CH2O)n-(CH2)q-; each n is independently 1, 2, 3, 4, 5, or 6; each q is independently 1 or 2.

18. The immunoconjugate of any one of claims 1-3, wherein:R1; or a radionuclide complex thereof; X1isX1is -CH2CH2-X2-; X2is -C(=O)X4-; X4is -NH-, -N(CH3)-, or -N(CH2CH3)-. L is -L2-L5-; L2is -(CH2CH2O)n-(CH2)P-; p is 2; L5is -C(=O)NR4-(CH2)n-, -NR4C(=O)-(CH2)n-, -C(=O)NR4-(CH2CH2O)n- (CH2)q-, or -NR4C(=O)-(CH2CH2O)n-(CH2)q-; R4is selected from hydrogen, and C1-C6alkyl; q is 2; each n is independently 3, 4, 5, or 6.

19. The immunoconjugate of any one of claims 1-3, wherein:WSGR Attorney Docket No.: 60924-730.601 R1; or a radionuclide complex thereof; X1isX4is - , - -, or - -. L is -(CH2CH2O)n-(CH2)2-NR4C(=O)-(CH2CH2O)n-(CH2)2-; R4is selected from hydrogen, and C1-C4alkyl; each n is independently 3, 4, 5, or 6.

20. The immunoconjugate of any one of claims 1-19, whereineach Rais independently selected from C1-C4alkyl; each R4is independently selected from C1-C4alkyl.

21. The immunoconjugate of any one of claims 1-3, wherein:X1-L-is: .

22. The.

23. TheWSGR Attorney Docket No.: 60924-730.601 ,24. The any one adiagnostic or therapeutic radionuclide.

25. The immunoconjugate of any one of claims 1-23, wherein the radionuclide is an Augerelectron-emitting radionuclide, α-emitting radionuclide, β-emitting radionuclide, or γ- emitting radionuclide.

26. The immunoconjugate of any one of claims 1-23, wherein the radionuclide is α-emittingradionuclide.

27. The immunoconjugate of any one of claims 1-23, wherein:the radionuclide is an Auger electron-emitting radionuclide that is 111-indium(111In), 67-gallium (67Ga), 68-gallium (68Ga), 99m-technetium (99mTc), or 195m- platinum (195mPt).; or the radionuclide is an α-emitting radionuclide that is 225-actinium (225Ac), 213-bismuth (213Bi), 223-Radium (223Ra), or 212-lead (212Pb). or the radionuclide is a β-emitting radionuclide that is 90-yttrium (90Y), 177- lutetium (177Lu), 186-rhenium (186Re), 188-rhenium (188Re), 64-copper (64Cu), 67-copper (67Cu), 153-samarium (153Sm), 89-strontium (89Sr), 198-gold (198Au), 169-Erbium (169Er), 165-dysprosium (165Dy), 99m-technetium (99mTc), 89- zirconium (89Zr), or 52-manganese (52Mn); or the radionuclide is a γ-emitting radionuclide that is 60-cobalt (60Co), 103- palldium (103Pd), 137-cesium (137Cs), 169-ytterbium (169Yb), 192-iridium (192Ir), or 226-radium (226Ra).

28. The immunoconjugate of any one of claims 1-23, wherein the radionuclide is suitable forpositron emission tomography (PET) analysis, single-photon emission computerized tomography (SPECT), or magnetic resonance imaging (MRI).

29. The immunoconjugate of any one of claims 1-23, wherein the radionuclide is 225-actinium (225Ac).WSGR Attorney Docket No.: 60924-730.60130. The immunoconjugate of any one of claims 1-29, wherein the antibody further comprisesan Fc domain.

31. The immunoconjugate of any one of claims 30, wherein the Fc domain comprises animmunoglobulin CH2 domain, immunoglobulin CH3 domain, or both an immunoglobulin CH2 and immunoglobulin CH3 domain.

32. The immunoconjugate of claim 31, wherein the Fc domain comprises both theimmunoglobulin CH2 and the immunoglobulin CH3 domain.

33. The immunoconjugate of any one of claims 30-32, wherein the Fc domain is an IgA,IgG1, IgG2, IgG3, or IgG4 isotype.

34. The immunoconjugate of any one of claims 30-33, wherein the Fc domain is an IgG1isotype.

35. The immunoconjugate of any one of claims 30-33, wherein Fc domain is an IgG4isotype.

36. The immunoconjugate of any one of claims 30-35, wherein the Fc domain comprises analteration to one or more amino acid residues that reduces an effector function of the Fc domain or alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn) (relative to wild-type IgG1).

37. The immunoconjugate of any one of claims 30-36, wherein the Fc domain comprises anFalteration to one or more amino acid residues that reduces an effector function of the Fc domain and an alteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) (relative to wild-type IgG1).

38. The immunoconjugate of any one of claims 30-37, wherein the Fc domain comprises analteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) (relative to wild-type IgG1).

39. The immunoconjugate of any one of claims 30-38, wherein the Fc domain comprises analteration to one or more amino acid residues that reduces an effector function of the Fc domain.

40. The immunoconjugate of any one of claim 30-39, wherein the Fc domain comprises analteration to one or more amino acid residues that reduces the effector function, wherein the alteration reduces complement dependent cytotoxicity (CDC), antibody-dependent cell-cytotoxicity (ADCC), antibody-dependent cell-phagocytosis ADCP, or a combination thereof (relative to wild-type IgG1).

41. The immunoconjugate of any one of claims 30-40, wherein the Fc domain comprises analteration to one or more amino acid residues that reduces the effector function of the FcWSGR Attorney Docket No.: 60924-730.601 domain (relative to wild-type IgG1), wherein the alteration comprises L234A, L235E, G237A, A330S, and P331S per EU numbering.

42. The immunoconjugate of any one of claims 30-41, wherein the Fc domain comprises analteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) reduces the serum half-life of the immunoconjugate.

43. The immunoconjugate of any one of claims 30-42, wherein the Fc domain comprises analteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn), wherein the alteration comprises H310A, H435Q, or both H310A and H435Q per EU numbering.

44. The immunoconjugate of any one of claims 30-42, wherein the Fc domain comprises analteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn), wherein the alteration comprises H435Q.

45. The immunoconjugate of any one of claims 30-42, wherein the Fc domain comprises analteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn), wherein the alteration comprises H310A.

46. The immunoconjugate of any one of claims 30-45, wherein the antibody comprises ahinge region connecting the VHH variable domain and the Fc domain.

47. The immunoconjugate of claim 46, wherein the human IgG hinge region comprises theamino acid sequence set forth in SEQ ID NO: 10 or 11.

48. The immunoconjugate of any one of claims 1-47, wherein the VHH variable domaincomprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, 99% or greater sequence identity to any one of SEQ ID NOs: 29-34.

49. The immunoconjugate of any one of claims 1-48, wherein the VHH variable domaincomprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 31.

50. The immunoconjugate of any one of claims 1-49, wherein the VHH variable domaincomprises the amino acid sequence of SEQ ID NO: 31.

51. The immunoconjugate of any one of claims 1-50, wherein the antibody comprises anamino acid sequence having at least 85%, 90%, 95%, 97%, 98%, 99%or greater sequence identity to any one of SEQ ID NOs: 35-39.WSGR Attorney Docket No.: 60924-730.60152. The immunoconjugate of any one of claims 1-51, wherein the antibody comprises anamino acid sequence having at least 85%, 90%, 95%, 97%, 98%, 99%or greater sequence identity to SEQ ID NO: 36.

53. The immunoconjugate of any one of claims 1-52, wherein the antibody comprises theamino acid sequence of SEQ ID NO: 3654. The immunoconjugate of any one of claims 1 to 53, wherein the immunoconjugate bindsDLL3 having an affinity of KDof 10 nanomolar or less.

55. The immunoconjugate of any one of claims 1 to 53, wherein the immunoconjugate bindsDLL3 having an affinity of KDof 5 nanomolar or less.

56. The immunoconjugate of any one of claims 1 to 53, wherein the immunoconjugate bindsDLL3 having an affinity of KDof 2 nanomolar or less.

57. The immunoconjugate of any one of claims 1-56, wherein the molecular weight of theantibody is between 60 and 110 kDa.

58. The immunoconjugate of any one of claims 1-56, wherein the molecular weight of theantibody is between 60 and 90 kDa.

59. The immunoconjugate of any one of claims 1-56, wherein the molecular weight of theantibody is between 70 and 90 kDa.

60. A pharmaceutical composition comprising the immunoconjugate of any one of claims 1-59 and a pharmaceutically acceptable excipient or carrier.

61. The pharmaceutical composition comprising the immunoconjugate of any one of claims1-59 formulated for intravenous administration.

62. A method of making the immunoconjugate of any one of claims 1-59, comprisingloading the immunoconjugate with a radioisotope.

63. A method of treating a cancer or a tumor in an individual comprising administering tothe individual the immunoconjugate of any one of claims 1-59 having the radionuclide complex as R1; thereby treating the cancer or the tumor.

64. The method of claim 63, wherein the individual is a human individual.

65. The method of claim 63 or 64, wherein the cancer or the tumor is a solid cancer ortumor.

66. The method of claim 63 or 64, wherein the cancer or the tumor comprises lung cancer,breast cancer, ovarian cancer, or a neuroendocrine cancer.

67. The method of any one of claims 63-66, comprising administering from 0.5 µCi to 30.0µCi per kilogram to the individual.

68. The method of any one of claims 63-67, comprising administering from 10 m Ci to 75mCi per meter squared of body area to the individual.WSGR Attorney Docket No.: 60924-730.60169. The method of any one of claims 63-68, wherein the cancer or tumor expresses DLL3.

70. A method of killing a cancer cell in an individual comprising administering to theindividual the immunoconjugate of any one of claims 1-59 having the radionuclide complex as R1, thereby killing the cancer cell.

71. The method of claim 70, wherein the individual is a human individual.

72. The method of claim 70 or 71, wherein the cancer cell comprises lung cancer cell, abreast cancer cell, an ovarian cancer cell, or a neuroendocrine cancer cell.

73. The method of any one of claims 70-72, wherein the cancer cell expresses DLL3.

74. A method of delivering a radioisotope to a cancer cell or a tumor cell in an individualcomprising administering to the individual the immunoconjugate of any one of claims 1- 59 having the radionuclide complex as R1, thereby delivering the radioisotope to the cancer cell or the tumor cell.

75. A method of imaging a tumor in an individual comprising administering to the individualthe immunoconjugate of any one of claims 1-59 having the radionuclide complex as R1.

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