Dual modality radionuclide-conjugated antibody-drug conjugates and use thereof

Abstract

Dual modality radionuclide-conjugated antibody-drug conjugates (R-ADCs), a novel therapeutic strategy combining monoclonal antibody targeting with cytotoxic drug payloads and radionuclides, are provided, as well as combinations of different antibodies in which one antibody is linked to the cytotoxic drug and a second antibody targeting the same target is linked to a chelator binding a radionuclide. These approaches utilize the selective binding of antibodies to tumor-associated antigens, enabling targeted delivery of both chemotherapeutic agents and ionizing radiation directly to cancer cells. The integration of radionuclides enhances localized cytotoxicity, resulting in amplified tumor cell destruction through both radiation and chemotherapy. Additionally, the technology facilitates non-invasive imaging for treatment monitoring and dosimetry assessment.

Description

PATENT Atorney Docket No. 081906-1544514-261320PC Client Ref. No. SF2025-122-3-PCT DUAL MODALITY RADIONUCLIDE-CONJUGATED ANTIBODYDRUG CONJUGATES AND USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 767,943, filed March 6, 2025, and U.S. Provisional Application No. 63 / 964,292 filed January 20, 2026, the disclosures of which are each herein incorporated by reference in their entirety for all purposes.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under W81XWH-21-1 -0792, and HT9425-23-1-0139 awarded by the Defense Health Agency, Medical Research and Development Branch, R01 CA271606, and R01 CA289874 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Due to ease of accessibility, tumor cell surface antigens are valuable targets for therapeutic development. The epitope space at the cell surface is highly complex. Relevant antigens may include glycosylated proteins and other post-translationally modified products that may not be readily predicted from studies of genomic copy number or mRNA expression levels (Liu et al. (2004) Cancer Res. 64: 704-710; Kobata and Amano (2005) Immunol. Cell Biol. 83: 429-439; Birkle et al. (2003) Biochimie (Paris) 85: 455-463; Hakomori (2001) Adv. Exp. Med. Biol. 491: 369-402; Hanisch, F. G. (2001) O-Glycosylation of the mucin type. Biol. Chem. 382, 143-1 49; Ugorski and Laskowska (2002) Ac ta Biochim. Pol. 49: 303-311).

[0004] Identification of tumor cell surface epitopes allows the production of antibodies to achieve specific binding to neoplastic cells, an ability that can be utilized in applications such as induction of antibody-dependent cell cytotoxicity (see, e.g., Clynes et al. (2000) Nat. Med.6: 443-446), or inhibition of signaling pathways involved in tumor cell migration, growth, and survival (see, e.g., McWhirter et al. (2006) Proc. Natl. Acad. Sci., USA, 103: 1041-1 046; Fuh et al. (2006) J. Biol. Chem. 281: 6625- 6631). In addition, antibodies targeting1KILPATRICK TOWNSEND 79434972 1internalizing tumor epitopes can be exploited to achieve efficient and specific intracellular delivery of cytotoxins, cytostatic agents, chemotherapeutic drugs or other tumor-modulating agents (see, e.g., Liu et al. (2004) Cancer Res. 64: 704-710; Nielsen et al. (2002) Biochim. Biophys. Acta 1591: 109-118; Pirollo et al. (2006) Hum. Gene Ther. 17: 117-124; Song et al. (2005) / / . Biotechnol. 23:709-717; Liu et al. (2002) J. Mol. Biol. 315: 1063-1073).

[0005] A variety of anti-CD46 antibodies are known, including but not limited to those described in U.S. Patent Nos. 9,593,162; 9,567,402;10,533,056; and Sherbenou DW, etal., JCI Insight. 2018 Sep 6;3(17):el2149.BRIEF SUMMARY

[0006] In some aspects, the disclosure provides an antibody that specifically binds to CD46, wherein the antibody is linked to (i) a cytotoxic effector and (ii) a chelator, wherein a radionuclide is bound to the chelator via coordinate bonding. In some embodiments, the antibody comprises an Fc domain. In some embodiments, the antibody comprises heavy chain CDRs 1, 2 and 3 and light chain CDRs 1, 2, and 3 of any one of YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, or UA8kappa. In some embodiments, the antibody comprises a heavy chain (HC) variable region that comprises three complementarity determining regions (CDRs): HC CDR1, HC CDR2 and HC CDR3 and a light chain (LC) variable region that comprises three CDRs: LC CDR1, LC CDR2, and LC CDR3, wherein said HC CDR1, HC CDR2, HC CDR3 comprise an amino acid sequence of SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82, respectively, and said LC CDR1, LC CDR2, and LC CDR3 comprise an amino acid sequence of SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO: 85, respectively. In some embodiments, the HC comprises SEQ ID NO:86 and the LC comprises SEQ ID NO:87.

[0007] In some embodiments, the cytotoxic effector is linked to the antibody via a cleavable or non-cleavable linker via a cysteine sulfhydryl moiety in the antibody. In some embodiments, the cleavable linker comprises valine-citrulline.

[0008] In some embodiments, the cytotoxic effector is a chemotherapeutic agent. In some embodiments, the cytotoxic effector is a microtubule inhibitor, a DNA-damaging agent, or a polymerase inhibitor. In some embodiments, the cytotoxic effector is selected from the group consisting of an auristatin, Dolastatin-10, synthetic derivatives of the natural product Dolastatin-10, and maytansine or a maytansine derivative. In some embodiments, the cytotoxic effector is selected from the group consisting of Monomethylauristatin F (MMAF),2KILPATRICK TOWNSEND 79434972 1Auristatin E (AE), Monomethylauristatin E (MMAE), vcMMAE, and vcMMAF. In some embodiments, monomethylauristatin E (MMAE) is conjugated to said antibody via a maleimidocaproyl-valine-citrulline-para-amino benzyloxycarbonyl (mc-vc-PAB) linker. In some embodiments, the cytotoxic effector is selected from the group consisting of mertansine (DM1), ravtansine (DM4), calicheamicin, a duocarmycin analog, a pyrrolobenzodiazepine dimer, Exatecan derivative for ADC (DXd), 7-ethyl-10-hydroxy-camptothecin (SN-38), topotecan, or a derivative thereof. In some embodiments, the duocarmycin analog is selected from the group consisting of Duocarmycin Bl (DUMB1), duocarmycin B2 (DUMB2), duocarmycin Cl (DUMC1), duocarmycin C2 (DUMC2), duocarmycin DI (DUMD1), duocarmycin D2 (DUMD2) and DSA.

[0009] In some embodiments, the chelator comprises Macropa, mercaptoacetyltriglycine (MAG3), diethylenetriaminepentaacetic acid (DTP A), Deferoxamine (DFO), DFO*, DFO-Km, DFO-HOPO, l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA) or 1,4,7,10-tetraazacyclododecane- 1,4,7, 10-tetraacetic acid (DOTA) or triethylenetetramine (TETA). In some embodiments, the chelator is linked to the antibody by a linker. In some embodiments, the linker is linked to the antibody via an amine on a lysine side chain of the antibody. In some embodiments, the linker comprises polyethylene glycol (PEG). In some embodiments, the PEG comprises PEG2-10. In some embodiments, the linker comprises PEG2, PEG4, PEGe, or PEGs.

[0010] In some embodiments, the radionuclide emits alpha particles. In some embodiments, the radionuclide is selected from the group consisting of Actinium-225, Thorium-227, Bismuth-213, Astatine-211, Terbium-149, Lead-212 and Bismuth-213.

[0011] In some embodiments, the radionuclide emits beta particles. In some embodiments, the radionuclide is selected from the group consisting of Lutetium- 177, Terbium-161, Yttrium-90 and Lead-212.

[0012] In some embodiments, the radionuclide emits positrons. In some embodiments, the radionuclide is selected from the group consisting of Zirconium-89, Copper-64, Gallium-68, Cerium- 134 and Fluorine- 18.

[0013] Also provided is a pharmaceutical composition comprising a therapeutically-effective amount of the antibody as described above or elsewhere herein.

[0014] Also provided is a composition comprising:(i) a first antibody that binds to CD46, wherein the first antibody is linked to a cytotoxic effector; and3KILPATRICK TOWNSEND 79434972 1(ii) a second antibody that binds to CD46, wherein the second antibody is linked to a chelator, wherein a radionuclide is bound to the chelator via coordinate bonding.

[0015] In some embodiments, the composition is a pharmaceutical composition comprising a therapeutically-effective amount of the first antibody and the second antibody. In some embodiments, the first antibody, or the second antibody, or both the first antibody and the second antibody, comprises heavy chain CDRs 1, 2 and 3 and light chain CDRs 1, 2, and 3 of any one of YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, orUA8kappa. In some embodiments, the first antibody, or the second antibody, or both the first antibody and the second antibody, comprises a heavy chain (HC) variable region that comprises three complementarity determining regions (CDRs): HC CDR1, HC CDR2 and HC CDR3 and a light chain (LC) variable region that comprises three CDRs: LC CDR1, LC CDR2, and LC CDR3, wherein said HC CDR1, HC CDR2, HC CDR3 comprise an amino acid sequence of SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82, respectively, and said LC CDR1, LC CDR2, and LC CDR3 comprise an amino acid sequence of SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO: 85, respectively. In some embodiments, the HC comprises SEQ ID NO:86 and the LC comprises SEQ ID NO:87.

[0016] In some embodiments, the cytotoxic effector is linked to the first antibody via a cleavable or non-cleavable linker via a cysteine sulfhydryl moiety in the first antibody. In some embodiments, the cleavable linker comprises valine-citrulline. In some embodiments, the cytotoxic effector is a chemotherapeutic agent. In some embodiments, the cytotoxic effector is a microtubule inhibitor, a DNA-damaging agent, or a polymerase inhibitor. In some embodiments, the cytotoxic effector is selected from the group consisting of an auristatin, Dolastatin-10, synthetic derivatives of the natural product Dolastatin-10, and maytansine or a maytansine derivative. In some embodiments, the cytotoxic effector is selected from the group consisting of Monomethylauri statin F (MMAF), Auristatin E (AE), Monomethylauristatin E (MMAE), vcMMAE, and vcMMAF. In some embodiments, monomethylauristatin E (MMAE) is conjugated to the first antibody via a maleimidocaproyl-valine-citrulline-para-amino benzyloxycarbonyl (mc-vc-PAB) linker. In some embodiments, the cytotoxic effector is selected from the group consisting of mertansine (DM1), ravtansine (DM4), calicheamicin, a duocarmycin analog, a pyrrolobenzodiazepine dimer, Exatecan derivative for ADC (DXd), 7-ethyl-10-hydroxy-camptothecin (SN-38), topotecan, or a derivative thereof. In some embodiments, the duocarmycin analog is selected from the group consisting of Duocarmycin Bl (DUMB1), duocarmycin B2 (DUMB2), duocarmycin Cl 4KILPATRICK TOWNSEND 79434972 1(DUMC1), duocarmycin C2 (DUMC2), duocarmycin D 1 (DUMD1), duocarmycin D2 (DUMD2) and DSA.

[0017] In some embodiments, the chelator comprises Macropa, mercaptoacetyltriglycine (MAG3), diethylenetriaminepentaacetic acid (DTP A), Deferoxamine (DFO), DFO*, DFO-Km, DFO-HOPO, l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA) or 1,4,7,10-tetraazacyclododecane- 1,4,7, 10-tetraacetic acid (DOTA) or triethylenetetramine (TETA). In some embodiments, the chelator is linked to the second antibody by a linker. In some embodiments, the linker is linked to the second antibody via an amine on a lysine side chain of the second antibody. In some embodiments, the linker comprises polyethylene glycol (PEG). In some embodiments, the PEG comprises PEG2-10. In some embodiments, the linker comprises PEG2, PEGr, PEGe, or PEGs.

[0018] In some embodiments, the radionuclide emits alpha particles. In some embodiments, the radionuclide is selected from the group consisting of Actinium-225, Thorium-227, Bismuth-213, Astatine-211, Terbium-149, Lead-212 and Bismuth-213.

[0019] In some embodiments, the radionuclide emits beta particles. In some embodiments, the radionuclide is selected from the group consisting of Lutetium- 177, Terbium-161, Yttrium-90 and Lead-212.

[0020] In some embodiments, the radionuclide emits positrons. In some embodiments, the radionuclide is selected from the group consisting of Zirconium-89, Copper-64, Gallium-68, Cerium- 134 and Fluorine- 18.

[0021] Also provided is a method of killing a CD46-expressing cancer cell. In some embodiments, the method comprises contacting the antibody as described above or elsewhere herein or the pharmaceutical composition as described above or elsewhere herein or the composition as described above or elsewhere herein to the cancer cell, thereby killing the cancer cell.

[0022] In some embodiments, the cancer cell is in a human and the contacting comprises administering the antibody or pharmaceutically composition or the composition to the human.

[0023] In some embodiments, the cancer is selected from the group consisting of prostate cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer and a solid tumor cancer. In some embodiments, the prostate cancer is a resistant form of prostate cancer. In some embodiments, the prostate cancer is selected from the group consisting of Prostate-Specific Membrane Antigen (PSMA)-low, neuroendocrine, or androgen signaling inhibitor resistant form of prostate cancer. In some embodiments, the cancer is multiple myeloma or a5KILPATRICK TOWNSEND 79434972 1hematological cancer. In some embodiments, the multiple myeloma is relapsing or refractory multiple myeloma.

[0024] Also provided is a method for imaging a tumor in a subject. In some embodiments, the method comprising, administering the antibody as described above or elsewhere herein or the composition as described above or elsewhere herein to a tumor; and detecting emitted signal from the radionuclide. In some embodiments, the radionuclide is selected from the group consisting of Zirconium-89, Cerium-134, Copper-64, Gallium-68, and Fluorine-18. In some embodiments, the emitted signal is detected by a positron emission tomography (PET) scanner, single photon emission computed tomography (SPECT), or magnetic resonance imaging (MRI) system. In some embodiments, the detected signal is used to differentiate tumor tissue from non-tumor tissue, assess tumor progression, or evaluate response to therapy. In some embodiments, the tumor is a cancer selected from the group consisting of prostate cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer and a solid tumor cancer. In some embodiments, the prostate cancer is a resistant form of prostate cancer. In some embodiments, the prostate cancer is selected from the group consisting of Prostate-Specific Membrane Antigen (PSMA)-low, neuroendocrine, or androgen signaling inhibitor resistant form of prostate cancer.DEFINITIONS

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. In this application, the use of the singular includes the plural unless specifically stated otherwise. It is noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

[0026] The term "immunomodulatory imide drug (IMiD)" as used herein refers to a class of drugs containing an imide group. Without being bound by theory, it is believed that IMiDs are useful in the treatment of cancers due to immunomodulatory, antiangiogenic, and antineoplastic properties and as described herein, IMiDs induce CD46 expression in cancer 6KILPATRICK TOWNSEND 79434972 1cells, rendering the cancer cells more susceptible to drugs (e.g., antibodies) that target CD46. The IMiD class of drugs includes, but is not limited to, thalidomide and its analogs. The term "analog" as used herein is a compound having a structure similar to that of another one, but differing from it in respect of a certain component, e.g., the analog can differ in one or more atoms, functional groups, or substructures, which are replaced with other atoms, groups, or substructures. Various IMiDs are known to those skilled in the art, including, for example, thalidomide, lenalidomide, pomalidomide, apremilast, iberdomide and analogs thereof. See, e.g., US Patent Publication No. US 2020 / 0254093. Additional thalidomide analogs are described in, e.g., US Patent Publication No. US2020 / 0325102.

[0027] A “Stat3 inhibitor” inhibits one or more activity of human Stat3. STAT3 activity can include for example, STAT3 phosphorylation, STAT3 dimerization, STAT3 binding to a polynucleotide comprising a STAT3 binding site, STAT3 binding to genomic DNA, activation of a STAT3 responsive gene and STAT3 nuclear translocation. US Patent Publication No. 2017 / 0000884 describes, for example, a non-limiting list of Stat3 inhibitors and methods for measuring their activity.

[0028] As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 pL” means “about 5 pL” and also “5 pL.” Generally, the term “about” includes an amount that would be expected to be within experimental error.

[0029] The terms “antibody” and “immunoglobulin” are used interchangeably herein and are used in the broadest sense and covers fully assembled antibodies, antibody fragments that can bind antigen, for example, Fab, F(ab’)2, Fv, single chain antibodies (scFv), diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, and the like. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0030] The terms “monoclonal antibody” and “mAb” are used interchangeably herein and refer to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies of the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0031] The term “hypervariable region,” as used herein, refers to the amino acid residues of an antibody that are responsible for antigen-binding. The hypervariable region comprises 7KILPATRICK TOWNSEND 79434972 1amino acid residues from a “complementarily determining region” or “CDR” (i.e., residues 24-34 (LI), 50-56 (L2), and 89-97 (L3) in the light-chain variable domain and 31-35 (Hl), 50-65 (H2), and 95-102 (H3) in the heavy-chain variable domain; Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (referred to herein as “Kabat et al”) and / or those residues from a “hypervariable loop” (i.e., residues 26-32 (LI), 50-52 (L2), and 91-96 (L3) in the light-chain variable domain and (Hl), 53-55 (H2), and 96-101 (13) in the heavy chain variable domain; Chothia and Lesk, (1987) J. Mol. Biol., 196:901-917).“Framework” or “FR” residues are those variable domain residues other than the hypervariable region residues, as herein deemed.

[0032] In some instances, the CDRs of an antibody is determined according to (i) the Kabat numbering system Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; or (ii) the Chothia numbering scheme, which will be referred to herein as the "Chothia CDRs" (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; Al-Lazikani et al., 1997, J. Mol. Biol., 273 :927-948; Chothia et al., 1992, J. Mol. Biol., 227:799-817; Tramontano A et al. , 1990, J. Mol. Biol. 215(1): 175-82; and U.S. Patent No. 7,709,226); or (iii) the ImMunoGeneTics (IMGT) numbering system, for example, as described in Lefranc, M.-P., 1999, The Immunologist, 7: 132-136 and Lefranc, M.-P. et al, 1999, Nucleic Acids Res., 27:209-212 ("IMGT CDRs"); or (iv) MacCallum et al, 1996, J. Mol. Biol., 262:732-745. See also, e.g., Martin, A., "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer- Verlag, Berlin (2001).

[0033] With respect to the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which optionally can include one or two additional amino acids, following 35 (referred to in the Kabat numbering scheme as 35 A and 35B) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). As is well known to those of skill in the art, using the Kabat numbering system, the actual linear amino acid sequence of the antibody variable domain can contain fewer or additional amino acids due to a shortening or lengthening of a FR and / or CDR and, as such, an amino acid’s Kabat number is not necessarily the same as its linear amino acid number.8KILPATRICK TOWNSEND 79434972 1

[0034] As used herein, the term “antigen-binding site” refers to the part of the antigen binding molecule that specifically binds to an antigenic determinant. More particularly, the term “antigen binding site” refers the part of an antibody that comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antigen binding molecule may only bind to a particular part of the antigen, which part is termed an epitope. An antigen-binding site may be provided by, for example, one or more variable domains (also called variable regions). Preferably, an antigen-binding site comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0035] By “specific binding” is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antigen binding molecule to bind to a specific antigen can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. Surface Plasmon Resonance (SPR) technique (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of an antigen binding molecule to an unrelated protein is less than about 10% of the binding of the antigen binding molecule to the antigen as measured, e.g. by SPR. In certain embodiments, a molecule that binds to the antigen has a dissociation constant (Kd) of <1 pM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g. 10-7 M or less, e.g. from 10-7M to 10-13M, e.g. from 10-9M to 10-13M).

[0036] As noted above, depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG, IgM, and IgY, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Different isotypes have different effector functions. For example, human IgGl and IgG3 isotypes have ADCC (antibody dependent cell-mediated cytotoxicity) activity. The light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (1), based on the amino acid sequences of their constant domains.9KILPATRICK TOWNSEND 79434972 1

[0037] The term “chimeric antibody,” as used herein refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source (e.g., protein) or species, while the remainder of the heavy and / or light chain is derived from a different source (e.g., protein) or species.

[0038] The term “recombinant human antibody,” as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as a NSO or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in a rearranged form. In some cases, the recombinant human antibodies have been subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.

[0039] The term “valent” as used herein denotes the presence of a specified number of binding sites in an antigen binding molecule. As such, the terms “bivalent”, “tetravalent”, and “hexavalent” denote the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antigen binding molecule. The bispecific antibodies according to the invention are at least “bivalent” and may be “trivalenf ’ or “multivalent” (e.g. “tetravalent” or “hexavalent”). In a particular aspect, the antibodies of the present invention have two or more binding sites and are bispecific. That is, the antibodies may be bispecific even in cases where there are more than two binding sites (i.e. that the antibody is trivalent or multivalent). In particular, the invention relates to bispecific bivalent antibodies, having one binding site for each antigen they specifically bind to.

[0040] The term “monospecific” antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen.

[0041] The terms “individual(s)”, “subject(s)” and “patient(s)” are used interchangeably herein and refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly or a hospice worker).

[0042] The terms “cancer” and “tumor” are used interchangeably herein, encompass all types of oncogenic processes and / or cancerous growths. In embodiments, cancer includes primary 10KILPATRICK TOWNSEND 79434972 1tumors as well as metastatic tissues or malignantly transformed cells, tissues, or organs. In embodiments, cancer encompasses all histopathologies and stages, e.g., stages of invasiveness / severity, of a cancer. In embodiments, cancer includes relapsed and / or resistant cancer.

[0043] 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, the molecules described herein are used to delay development of a disease or to slow the progression of a disease.BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1A-C: Synergistic interaction between [225Ac]Macropa-PEG4-YS5 and YS5-MMAE in prostate cancer cell lines evaluated using ZIP analysis. ZIP synergy score heatmaps for 22Rvl (A) and DU145 (B), and LNCaP (C) cell lines treated with varying concentrations of [225Ac]Macropa-PEG4-YS5 (nCi / mL) and YS5-MMAE (pM). The red areas indicate positive synergy scores (ZIP score >10), confirming synergistic interactions between the two agents. White contours mark the regions of maximal synergy.

[0045] FIG. 2A-B: Cell cycle analysis of 22Rvl cells treated with [225Ac]Macropa-PEG4-YS5, YS5-MMAE, and combination therapy. (A) Representative flow cytometry histograms showing cell cycle distribution (Gi / Go, S, and G2 / M phases) of 22Rvl cells treated with [225Ac]Macropa-PEG4-YS5, YS5-MMAE, or the combination of [225Ac]Macropa-PEG4-YS5 and YS5-MMAE. Propidium iodide staining was used to quantify DNA content, and the percentage of cells in each phase was determined. (B) Quantitative analysis of the cell cycle distribution.

[0046] FIG. 3A-D: DNA damage and apoptosis analysis following treatment with [225Ac]Macropa-PEG4-YS5, YS5-MMAE, and combination therapy. (A) Representative immunofluorescence images showing phospho-H2AX (y-H2AX) foci (green) in the nuclei (blue, D API-stained) of 22Rvl cells after treatment with saline (control), YS5-MMAE (InM), [225Ac]Macropa-PEG4-YS5 (InCi / mL), or the combination of [225Ac]Macropa-PEG4-YS5 and YS5-MMAE. The y-H2AX foci indicate double-strand DNA breaks. Saline-treated 11KILPATRICK TOWNSEND 79434972 1cells showed minimal y-H2AX foci, while YS5-MMAE and [225Ac]Macropa-PEG4-YS5 induced moderate DNA damage. The combination treatment resulted in the highest number of y-H2AX foci, highlighting a synergistic effect of the dual therapy on inducing DNA damage. Scale bar = 10 pm. (B) Quantification of y-H2AX foci per nucleus, presented as violin plots. The combination treatment induced the highest y-H2AX foci counts, significantly greater than all other groups. (C) Representative flow cytometry scatter plots showing cell populations in early apoptosis (Annexin V+ / PI-), late apoptosis (Annexin V+ / PI+), and necrosis (Annexin V- / PI+). Combination treatment resulted in the highest levels of late apoptosis and necrosis compared to monotherapies and saline control. (D) Quantitative analysis of apoptotic populations.

[0047] FIG. 4A-D: Toxicity Analysis of YS5-MMAE, [225Ac]Macropa-PEG4-YS5, and combination therapy in healthy nude mice. (A) Acute toxicity: Body weight measurements over 15 days following a single injection of saline, YS5-MMAE (1.8 mg / kg),[225Ac]Macropa-PEG4-YS5 (0.125 pCi), or the combination of [225Ac]Macropa-PEG4-YS5 and YS5-MMAE. (B) Chronic toxicity: Body weight measurements monitored over 100 days post-injection to assess long-term systemic effects. (C) Complete blood count (CBC) from the chronic toxicity study showing white blood cell (WBC), neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts, as well as red blood cell (RBC) and platelet levels. (D) Serum liver and kidney function markers, including alanine transaminase (ALT), aspartate transaminase (AST), creatinine, albumin, blood urea nitrogen (BUN), alkaline phosphatase (ALP), and total bilirubin, from the chronic toxicity study.

[0048] FIG. 5: Representative histology of major organs collected 15 days after treatment (acute toxicity study). Hematoxylin-and-eosin-stained sections of heart, lung, liver, spleen, kidney, and bone marrow from mice (n = 5 per group) treated with saline, YS5-MMAE (1.8 mg / kg), [225Ac]Macropa-PEG4-YS5 (4.6 kBq), or the combination of YS5-MMAE + [225Ac]Macropa-PEG4-YS5 are shown. Histopathological evaluation demonstrated preserved cellular architecture in the heart, lungs, liver, spleen, and bone marrow across all treatment groups. In the kidneys, while saline and single-agent groups showed no significant abnormalities, mild tubular injury was observed in 3 of 5 mice in the combination treatment group; notably, this did not translate into functional renal impairment.

[0049] FIG. 6: Histological analysis of major organs following treatment with YS5-MMAE, [225Ac]Macropa-PEG4-YS5, and combination therapy in healthy nude mice for chronic toxicity study (120 days post-injections).12KILPATRICK TOWNSEND 79434972 1

[0050] FIG. 7A-C: Therapeutic efficacy of YS5-MMAE, [225Ac]Macropa-PEG4-YS5, and combination therapy in a 22Rvl tumor model. (A) Tumor growth curves of 22Rvl tumorbearing mice treated with saline (control), YS5-MMAE (1.8 mg / kg), [225Ac]Macropa-PEG4-YS5 at 0.06 pCi or 0.125 pCi, and combination therapies of YS5-MMAE (1.8 mg / kg) with [225Ac]Macropa-PEG4-YS5 (0.06 pCi or 0.125 pCi). Tumor volumes were monitored over time, showing that the combination therapies resulted in the greatest tumor suppression compared to monotherapies or control. (B) Kaplan-Meier survival curves for treated and control groups. Mice treated with the combination of YS5-MMAE (1.8 mg / kg) and [225Ac]Macropa-PEG4-YS5 at 0.125 pCi exhibited the longest survival, significantly outperforming all other groups. (C) Normalized body weights of mice during the treatment study. Body weight remained stable across all groups, indicating good tolerability and minimal systemic toxicity associated with the treatments.

[0051] FIG. 8A-C: Combination therapy with [225Ac]Macropa-PEG4-YS5 and YS5-MMAE controls disseminated tumors and extends survival. A. Serial whole-body bioluminescence images of 22Rvl-luc tumor-bearing mice treated once at week 3 with either the combination ([225Ac]Macropa-PEG4-YS5 + YS5-MMAE,), [225Ac]Macropa-PEG4-YS5 alone (4.6 kBq (0.125 pCi)), YS5-MMAE alone (1.8 mg kg'1), or saline. B. Kaplan-Meier survival curves, together with C. body -weight profiles, demonstrate that the combination regimen significantly extends survival without appreciable changes in body weight across the treatment period.

[0052] FIG. 9A-B: Development of macropa conjugated YS5-MMAE. A. Schematic for conjugation of Macropa chelator to CD46 ADC. B. MALDI TOF analysis of the YS5-MMAE and Macropa-PEG4-YS5-MMAE confirming the conjugation of macropa-PEG4. The disulfide bond reduction process used for MMAE conjugation led to the appearance of multiple peaks in the YS5-MMAE spectrum. For quantifying Macropa-PEG4 conjugation, the doubly charged peak at 75 kDa was used. The difference in peak intensity between YS5-MMAE and Macropa-PEG4-YS5-MMAE was divided by the molecular weight of Macropa-PEG4 to estimate the degree of conjugation.

[0053] FIG. 10: Size exclusion chromatography of the Macropa-PEG4 conjugated YS5-MMAE.

[0054] FIG. 11: A saturation binding assay was performed on DU145 cells, showing Kd values of 3.2 and 3.0 nM for YS5-MMAE and Macropa-PEG4-YS5-MMAE.

[0055] FIG. 12A-B: Instant thin layer chromatography (i-TLC) analysis of a dual modality radionuclide-conjugated antibody-drug conjugate (R-ADC) before and after purification. (A) i-TLC of the crude reaction product, analyzed immediately after synthesis (left) and at secular 13KILPATRICK TOWNSEND 79434972 1equilibrium (right). The immediate scan performed for radiolabeling reactions shows two peaks, with 95.67% of activity corresponding to the desired product and 4.33% attributed to free radioisotope. At secular equilibrium (24 h), the purity of the radiolabeled ADC was found to be 100%. (B) i-TLC of the product after purification using centrifugal filters, analyzed immediately after purification (left) and at secular equilibrium (right). The immediate scan shows 81.80% of activity in the desired peak and 18.20% corresponding to free isotope. After reaching secular equilibrium, the purified product demonstrates 99.87% purity in the desired peak, confirming effective purification.

[0056] FIG. 13A-F: Radiosynthesis, characterization, and multimodal evaluation of dualpayload R-ADC demonstrating preserved CD46 binding, high stability, enhanced cytotoxicity, and tumor-selective targeting in vitro and in vivo. A. Magnetic beads-based binding assay showing preserved binding of [225Ac]Macropa-PEG4-YS5-MMAE to CD46. B. The stability of [225Ac]Macropa-PEG4-YS5-MMAE was assessed in serum and PBS over 15 days. The radiolabeled conjugate maintained >95% stability throughout the study, demonstrating excellent radiochemical integrity in serum. C. The colony-forming assay compares the effect of [225Ac]Macropa-PEG4-YS5-M AE with [225Ac]Macropa-PEG4-YS5, showing improved cell killing action following drug conjugation. D. Comparison of YS5-MMAE, Macropa-PEG4-YS5-MMAE, and R-ADC [225Ac]Macropa-PEG4-YS5-MMAE treated at similar dose. The highest cell killing action was observed in R-ADC. E. Colony forming assay to compare the effect of R-ADC prepared at specific activities 6.29 kBq / pg to 0.11 kBq / pg. F. Binding of [225Ac]Macropa-PEG4-YS5-MMAE to CD46-positive 22Rvl cells compared to CD46 knockout cells (22Rvl-CD46-KO), confirming target specificity, (i) The R-ADC [225Ac]Macropa-PEG4-YS5-MMAE at specific activity of 6.3kBq / pg was incubated on 22Rvl and 22Rvl-CD46KO cells for 1 hours, (ii) R-ADC at 0.1 kB / pg was incubated with 22Rvl and 22Rvl-CD46KO cells for 1 hours to study the cell binding.

[0057] FIG. 14A-B: Biodistribution of [225Ac]Macropa-PEG4-YS5-MMAE in 22Rvl tumorbearing mice at day 4 (A) and day 7(B) post-injection comparing low (0.1 kBq / pg) and high (6.3 kBq / pg) specific activity formulations (n=4).

[0058] FIG. 15: Biodistribution study of the [225Ac]Macropa-PEG4-YS5-MMAE 10 days post-injections in nude mice bearing 22Rvl tumors, using a low specific activity formulation.

[0059] FIG. 16A-C: Analytical confirmation of compositional and functional stability of [225Ac]Macropa-PEG4-YS5-MMAE following PBS storage for 7 days. A. Repeated DAR assessments (Days 0, 4, and 7) showed minimal variation, with values consistently between 2.2 and 2.8, indicating that the MMAE payload remained attached. B. Day-714KILPATRICK TOWNSEND 79434972 1immunoreactivity testing using CD46 magnetic beads demonstrated preserved target engagement, whereas binding was diminished in blocked controls and in bead preparations lacking CD46. C. Cytotoxicity profiles in 22Rvl cells were comparable for Day-0 and Day-7 samples, indicating that the stored conjugate maintained therapeutic potency.

[0060] FIG. 17A-B: iTLC showing radiolabeling of [134Ce]Macropa-PEG4-YS5-MMAE (A), and purity following the PD10 purification process (B).

[0061] FIG. 18A-G: Tumor growth curves of individual mice treated with radiolabeled and non-radiolabeled conjugates. Mice with prostate cancer tumors (22Rvl) were used in the treatment study. Tumor volumes of individual mice were monitored over time following treatment with different agents. Tumor growth is plotted as volume (mm3) versus days posttreatment. Mice treated with saline (A) exhibit rapid growth, while YS5-MMAE (B) and a Macropa-PEG4-YS5-MMAE (C) at 1.8 mg / mL dose showed modest tumor growth inhibition observed, suggesting some therapeutic efficacy of MMAE-conjugated antibody. The radiolabeled antibody without MMAE [225Ac]Macropa-PEG4-YS5 at 0.06 (D) and 0.125 pCi (E), shows increased tumor growth suppression, highlighting the effect of Ac-225-mediated a-particle therapy. The synergistic effects of Ac-225 and MMAE yield improved tumor suppression compared to radiolabeled antibody without MMAE. The improved tumor suppression was observed at 0.06 (F) and 0.125 pCi (G) R-ADC. [225Ac]Macropa-PEG4-YS5-MMAE (0.125 pCi, 1.8 mg / kg) showed Highest tumor suppression, indicating enhanced therapeutic efficacy with higher activity and dual mechanisms of action (radiation and cytotoxic drug).

[0062] FIG. 19A-B: Body weights and median survival of the prostate cancer tumor-bearing mice treated with radiolabeled and non-radiolabeled conjugates. A. Body weight measurements: body weight changes were monitored over time to assess systemic toxicity. B.Kaplan Meier plots showing median survival for treatment groups (n=9). Median survival: Saline = 24 days, YS5-MMAE (1.8 mg / mL) = 27 days, Macropa-PEG4-YS5-MMAE (1.8 mg / mL) = 27 days. [225Ac]Macropa-PEG4-YS5 (0.06 pCi) = 30 days, [225Ac]Macropa-PEG4-YS5 (0.125 pCi) = 64 days, [225Ac]Macropa-PEG4-YS5-MMAE (0.06 pCi, 1.8 mg / kg) =68 days. [225Ac]Macropa-PEG4-YS5-MMAE (0.125 pCi , 1.8 mg / kg) = undefined / long-term survival benefits.

[0063] FIG. 20: PET imaging of the relapsed tumors using [89Zr]DFO-YS5 confirming the CD46 expression.

[0064] FIG. 21A-D: Therapeutic efficacy and tolerability of [225Ac]Macropa-PEG4-YS5-MMAE in prostate cancer tumor models. A. Tumor volume measurements over time in PC-315KILPATRICK TOWNSEND 79434972 1xenografts treated with saline (control) or 0.125 pCi of [225Ac]Macropa-PEG4-YS5-MMAE. Tumor growth was significantly inhibited in the treatment group compared to controls. Body weights of treated mice remained stable, indicating good tolerability of the treatment without significant systemic toxicity. B. Tumor volume measurements in DU145 xenografts showing effective tumor growth suppression in the treatment group compared to controls. Body weight measurements remained stable throughout the study period. C. Tumor volume measurements in C4-2B xenografts reveal strong tumor suppression in response to 0.125 pCi of [225Ac]Macropa-PEG4-YS5-MMAE. D The tumor volumes and body weight measurement for the adenocarcinoma LTL-484 subcutaneous tumor models in mice treated with saline or [225Ac]Macropa-PEG4-YS5-MMAE.

[0065] FIG. 22A-E: Toxicity evaluation of [225Ac]Macropa-PEG4-YS5-MMAE in athymic nude mice. A-C. Complete blood count (CBC) parameters were measured longitudinally after a single intravenous injection of R-ADC (4.6 kBq, 1.8 mg / kg) or saline control. Violin plots display white blood cell (WBC, A), red blood cell (RBC, B), and platelet (PLT, C) counts across multiple time points (n=4). D. Serum biochemistry markers for liver (ALT, AST, ALP), kidney (BUN, creatinine), and total bilirubin levels were assessed on days 40 and 94 post-injection. E. Representative H&E-stained tissue sections from major organs (spleen, liver, kidneys, heart, lungs, and bone) of R- ADC-treated (bottom row) mice collected on day 40 and day 94. Histopathological evaluation demonstrated preserved cellular architecture in the heart, lungs, liver, spleen, and bone marrow. In contrast, kidney sections from the Day 94 cohort exhibited features of mild, non-progressive radiation nephropathy, specifically focal tubular dilation and proteinaceous casts, corresponding to cumulative injury scores of 2-4.

[0066] FIG.23: PET / CT images of 22Rvl tumor-bearing mice at 24 hours, 48 hours, 96 hours, and 168 hours post-injection of [134Ce]Macropa-PEG4-YS5-MMAE.

[0067] FIG. 24: PET imaging and biodistribution of [134Ce]Macropa-PEG4-YS5 in 22Rvl tumor-bearing mice. PET / CT images of 22Rvl tumor-bearing mice at 24 hours (day 1), 48 hours (day 2), 96 hours (day 4), and 168 hours (day 7) post-injection of [134Ce]Macropa-PEG4-YS5.

[0068] FIG. 25: Biodistribution of [134Ce]Macropa-PEG4-YS5 and [134Ce]Macropa-PEG4-YS5-MMAE in 22Rvl tumor-bearing mice at 168 hours post-injection (n=4). %IA / g values are shown for major organs, including the liver, kidneys, spleen, and tumor.

[0069] FIG. 26: Magnetic beads assay showing binding of [89Zr]DFO*-YS5-M AE confirming retention of immunoreactivity.16KILPATRICK TOWNSEND 79434972 1

[0070] FIG. 27A-B: pPET / CT imaging and biodistribution with [89Zr]DFO-YS5-MMAE in 22Rvl tumor-bearing mice. A. Axial, coronal, and maximum intensity projection (MIP) PET / CT images of mice (M1-M4) at 24, 48, 96, and 168 hours post-injection of [89Zr]DFO*-YS5-MMAE. The images show sustained and specific uptake in 22Rvl tumors over time. Color scale represents % injected dose per gram of tissue (%ID / g), indicating consistent tumor retention up to 7 days post-injection. B. Biodistribution of [89Zr]DFO-YS5-MMAE in 22Rvl tumor-bearing mice at 168 hours post-injection (n=4).

[0071] FIG. 28A-B: PET / CT imaging and biodistribution with [89Zr]DFO-YS5-MMAE in DU145 tumor-bearing mice. A. Axial, coronal, and MIP PET / CT images of mice (M1-M3) at 24, 48, 96, and 168 hours post-injection show sustained tumor uptake of [89Zr]DFO*-YS5-MMAE. Color scale indicates %ID / g. B. Organ distribution of [89Zr]DFO*-YS5-MMAE in DU145 bearing mice, at 168 h post injections.

[0072] FIG. 29A-B: PET / CT imaging and biodistribution with [89Zr]DFO-YS5 in 22Rvl tumor-bearing mice. A. Axial, coronal, and MIP PET / CT images of mice (M1-M3) at 24, 48, 96, and 168 hours post-injection show tumor uptake of [89Zr]DFO-YS5. B. Organ distribution of [89Zr]DFO-YS5 in 22Rvl bearing mice, at 168 h post-injections (n=3).

[0073] FIG. 30A-C : In vitro and in vivo efficacy evaluation of [225Ac]Macropa-PEG4-YS5-MMAE in MM.1 S Multiple Myeloma (MM) cell line and MM disseminated model A. Binding affinity study indicates high binding of [225Ac]Macropa-PEG4-YS5-MMAE with high and low specific activity as compare to [225Ac]Macropa-PEG4-YS5 B. Colony formation assay with low specific activity of [225Ac]Macropa-PEG4-YS5-MMAE demonstrate efficient cell killing (less proliferation) compared to control wells. C. In vitro cell killing assay with high specific activity of [225Ac]Macropa-PEG4-YS5-MMAE and low specific activity of [225Ac]Macropa-PEG4-YS5-MMAE demonstrates higher toxicity as compared to [225Ac]Macropa-PEG4-YS5.

[0074] FIG. 31A-E: CD46-targeted alpha therapy and ADC combination strategies show enhanced efficacy and tolerability in multiple myeloma models. A. Bioluminescence imaging of metastatic MM.ls-Luc NSG xenografts treated with saline, [225Ac]Ac-Macropa-PEG4-YS5 (2.3 kBq), YS5 MMAE (1.8 mg / kg), or [225Ac]Ac-Macropa-PEG4-YS5 (2.3 kBq) + YS5 MMAE (1.8 mg / kg). B. In vivo BLI imaging in MM. IS model shows marked tumor suppression and prolonged survival with [225Ac]Macropa-PEG4-YS5-MMAE. C. Kaplan-Meier plots showing survival of the animals from MM. IS model treatment study. D. BLI imaging confirming tumor control from [225Ac]Macropa-PEG4-YS5 treatment in RPMI model. E. Improved survival of the animals bearing MM. Is tumor models treated with [225Ac]Macropa-PEG4-YS5-MMAE.17KILPATRICK TOWNSEND 79434972 1

[0075] FIG. 32A-B: Survival analysis (A) and the body weight measurements (B) from the combination treatment study in MM. Is-Luc disseminated tumors.

[0076] FIG. 33: Body weight measurements for the MM. IS disseminated tumor-bearing animals treated with [225Ac]Macropa-PEG4-YS5-MMAE.

[0077] FIG.34: Body weight measurements for the RPMI disseminated tumor bearing animals treated with [225Ac]Macropa-PEG4-YS5-MMAE.DETAILED DESCRIPTION

[0078] The inventors have discovered that cancer cell killing can be greatly improved by delivery of both a cytotoxin and a radionuclide to CD46-expressing cancer cells. Delivery of the cytotoxin and the chelator can be delivered by two separate anti-CD46 antibodies, but optimally one antibody linked to both the toxin and a chelator binding the radionuclide can be used for particularly efficient killing. Depending on the radionuclide used, one can also monitor the antibody and cells bound by the antibody by detecting the radionuclide.

[0079] In some embodiments, the anti-CD46 antibody is an internalizing antibody, meaning that the antibodies are internalized by tumor cells, for example via the macropinocytosis pathway. In some embodiments, the antibody is an internalizing antibody, meaning that the antibodies are internalized by tumor cells, for example via the macropinocytosis pathway. For example, the antibodies can be internalized by the tumor-selective macropinocytosis pathway, without the need of crosslinking, and localize to the lysosomes, which makes them well suited for use as antibody drug conjugates (ADCs) and other targeted therapeutics that utilize intracellular payload release. A large number of anti-CD46 antibodies are known, including but not limited to those described in U.S. Patent Nos. 9,593,162; 9,567,402 and 10,533,056.

[0080] In some embodiments, the anti-CD46 specifically bind CD46, in particular domains 1 and / or 2 of CD46, and are internalized by CD46 positive cancer cells in situ, e.g., when the cancer cell is in the tissue microenvironment.

[0081] The antibodies designated herein as YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and UA8kappa (see, e.g., Table 1) are exemplary anti-CD46 antibodies. In certain embodiments, antibodies that comprise VL CDR1 and / or VL CDR2, and / or VL CDR3, and / or VH CDR1 and / or VH CDR2, and / or VH CDR3 of one or more of these antibodies are contemplated. In certain embodiments, antibodies that comprise the VH domain and / or the VL domain of one or more of these antibodies are contemplated. Also contemplated are 18KILPATRICK TOWNSEND 79434972 1antibodies that compete for binding at CD46 with one or more of as YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8kappa.

[0082] The amino acid sequences of the VH and VL domains of YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8kappa antibodies are shown in Table 1.

[0083] Table 1. Anti-CD46 antibody sequences. YS5 and YS5F differ by one amino acid in VH CDR1 (L vs. F). YS5 and YS5vlD have identical VH but one amino acid difference in the VL CDR2 (N vs. D). 3G7HY, 3G7NY, 3G7RY (aka 3G8), and 3G7 have one residue difference in VH CDR3, but entirely different VLs. YS6 and 3G7 have identical VH but different VL.19KILPATRICK TOWNSEND 79434972 120KILPATRICK TOWNSEND 79434972 1

[0084] In various embodiments the antibodies comprise the three VH CDRs and / or the three VL CDRs of antibodies 3051.1, G12FC3, M6c42b, 4F3YW, M40prl46, UA20, UA8, 585II41, 585II41.1, 585II56, 3076, 3051, M49R, RCI-14, II79 4, II79 3, T5II-4B.1, T5IL 4B.2, RCI-11, RCI-20, CI-11 A, CI-14A, or S95-2 that are described in PCT / US2008 / 076704 (WO 2009 / 039192) or the mPA7 antibody. The amino acid sequences of the VH and VL chains of these antibodies and the CDRs comprising these domains are shown in PCT / US2008 / 076704 and the amino acid sequences of these domains are reproduced below in Table 2.

[0085] Table 2. Additional antibodies. The sequence shown below are scFv antibodies (the VL and VH regions are joined by a (Gly4Ser)3 (SEQ ID NO:43) linker, however it will be recognized that other antibody forms comprising the CDRs (or the VH and / or VL domains) are possible.21KILPATRICK TOWNSEND 79434972 122KILPATRICK TOWNSEND 79434972 123KILPATRICK TOWNSEND 79434972 1

[0086] Using the amino acid sequences provided for the YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and UA8kappa antibodies, numerous antibody forms can be prepared, e.g., as described below. Such forms include, but are not limited to a substantially intact (e.g., full-length) immunoglobulin (e.g., an IgA, IgE, IgG, and the like), an antibody fragment (e.g., Fv, Fab, (Fab')2, (Fab')3, a minibody, and the like), a single chain antibody (e.g., scFv), a diabody, a unibody, an affibody, and the like.

[0087] It will be recognized, that where the antibodies are single chain antibodies, the VH and VL domains comprising such antibody can be joined directly together or by a peptide linker. Illustrative peptide linkers include, but are not limited to GGGGS GGGGS GGGGS (SEQ ID NO: 67), GGGGS GGGGS (SEQ ID NO: 68), GGGGS (SEQ ID NO: 69), GS GGGGS GGGGS GGS GGGGS (SEQ ID NO: 70), SGGGGS (SEQ ID NO:71), GGGS (SEQ ID NO:72), VPGV (SEQ ID NO:73), VPGVG (SEQ ID NO:74), GVPGVG (SEQ ID NO: 75), GVG VP GVG (SEQ ID NO: 76), VP GVG VP GVG (SEQ ID NO: 77), GGSSRSS (SEQ ID NO:78), and GGSSRSSSSGGGGSGGGG (SEQ ID NO:79), and the like.

[0088] As indicated above, in various embodiments, the antibody binds (e.g., specifically binds CD46 (e.g., domains 1 and / or 2)). Typical antibodies contemplated herein will specifically bind prostate cancer cells including, but not limited to, cells of a cell line selected from the group consisting of DU145 cells, PC3 cells, and LnCaP cells. In certain embodiments the antibody binds to a prostate tumor cell with an affinity greater than (KD less than) about 5 nM when measured on live prostate tumor cells by FACS. In certain embodiments the affinity is greater than (KD less than) about 1 nM, or at about 100 pM, or about 50 pM, or about 10 pM, or about 1 pM.24KILPATRICK TOWNSEND 79434972 1

[0089] Using the sequence information provided herein antibodies comprising one or more of the CDRs comprising, e.g, YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and UA8kappa, or antibodies comprising the VH and / or VL domain(s) of these antibodies can readily be prepared using standard methods (e.g. chemical synthesis methods and / or recombinant expression methods) well known to those of skill in the art, e.g., as described below.

[0090] In addition, other CD46-specific antibodies can be identified by screening for antibodies that bind to the same epitope (e.g. that compete with one or more of YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8kappa antibodies for binding to CD446 and / or to a cell expressing or overexpressing CD46, e.g., a prostate cancer cell) and / or by modification of the YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and / or UA8kappa antibodies identified herein to produce libraries of modified antibody and then rescreening antibodies in the library for improved binding to and / or internalization into cells expressing or overexpressing CD46, e.g., CD46-expressing cancer cells.

[0091] In some embodiments, the antibody is a recombinant antibody (or antigen binding fragment thereof) that specifically binds CD46. In some embodiments, the antibody is a recombinant antibody (or antigen binding fragment thereof) that specifically binds to an antigen such as a tumor cell surface antigen. In some embodiments, the antibody is a recombinant antibody (or antigen binding fragment thereof) that specifically binds Her2, Trop-2, Nectin-4, Folate receptor alpha, Tissue factor, C-Met, PSMA, EGFR, CEACAM5, CD30, CD79b, CD22, CD33, CD19, BCMA, or DLL3. In some embodiments, the antibody or antigen binding fragment or variant thereof is a monoclonal antibody. In some embodiments, the antibody or antigen binding fragment or variant thereof is a human antibody, a murine antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody comprises or consists of a functional fragment of a full-length antibody (e.g., an antigen binding fragment of a full-length antibody) such as a monovalent Fab, a bivalent Fab’2, a single-chain variable fragment (scFv), or a functional fragment or variant thereof. In some embodiments, the recombinant antibody (or antigen binding fragment thereof) comprises an immunoglobulin variable heavy chain domain (VH). In some embodiments, the recombinant antibody (or antigen binding fragment thereof) comprises an25KILPATRICK TOWNSEND 79434972 1immunoglobulin variable light chain domain (VL). In some embodiments, the recombinant antibody (or antigen binding fragment thereof) comprises a VH and a VL.

[0092] In some embodiments, the antibody is trastuzumab or ado-trastuzumab and specifically binds HER2. In some embodiments, the antibody is Sacituzumab or Datopotamab and specifically binds Trop-2. In some embodiments, the antibody is Enfortumab and specifically binds Nectin-4. In some embodiments, the antibody is Mirvetuximab and specifically binds Folate receptor alpha. In some embodiments, the antibody is Tisotumab and specifically binds Tissue factor. In some embodiments, the antibody is Telisotuzumab and specifically binds C-Met. In some embodiments, the antibody is J591 or humanized J591 and specifically binds PSMA. In some embodiments, the antibody is Cetuximab and specifically binds EGFR. In some embodiments, the antibody is an anti-CEACAM5 antibody (e.g., the anti-CEACAM5 antibody present in the M9140 (Precemtabart tocentecan (Precem-TcT)*) ADC) and specifically binds CEACAM5. In some embodiments, the antibody is Brentuximab and specifically binds CD30. In some embodiments, the antibody is Polatuzumab and specifically binds CD79b. In some embodiments, the antibody is Inotuzumab and specifically binds CD22. In some embodiments, the antibody is Gemtuzumab and specifically binds CD33. In some embodiments, the antibody is Loncastuximab and specifically binds CD 19. In some embodiments, the antibody is Belantamab and specifically binds BCMA. In some embodiments, the antibody is Tarlatamab and specifically binds DLL3.

[0093] In some embodiments, the antibody (or antigen binding fragment thereof) comprises an Fc region. In some embodiments, the antibody (or antigen binding fragment thereof) is a full-length antibody. In some embodiments, the antibody (or antigen binding fragment thereof) comprises a first light chain that comprises a light chain variable region and a light chain constant region; a first heavy chain that comprises a heavy chain variable region and a heavy chain constant region; a second light chain that comprises a light chain variable region and a light chain constant region; and a second heavy chain that comprises a heavy chain variable region and a heavy chain constant region. In some embodiments, the first and second light chains have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the first and second light chains bind the same epitope. In some embodiments, the first and second heavy chains have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the first and second heavy chains bind the same epitope.26KILPATRICK TOWNSEND 79434972 1

[0094] In some embodiments, the antibody (or antigen binding fragment thereof) is derived from non-human (e.g. rabbit or mouse) antibodies. In some instances, the humanized form of the non-human antibody contains a minimal non-human sequence to maintain original antigenic specificity. In some cases, the humanized antibodies are human immunoglobulins (acceptor antibody), wherein the CDRs of the acceptor antibody are replaced by residues of the CDRs of a non-human immunoglobulin (donor antibody), such as rat, rabbit, or mouse donor having the desired specificity, affinity, avidity, binding kinetics, and / or capacity. In some instances, one or more framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues of the donor antibody.

[0095] In some embodiments, the CD46 binding antibody comprises an immunoglobulin variable heavy chain domain (VH) that comprises at least one, two, or three complementarity determining regions (CDRs) disclosed in Table 1, 2, or 3 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0096] In some embodiments, the CD46 binding antibody comprises an immunoglobulin variable light chain domain (VL) that comprises at least one, two, or three complementarity determining regions (CDRs) disclosed in Table 1, 2 or 4 a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0097] In some embodiments, the CD46 binding antibody comprises a VH that comprises at least one, two, or three complementarity determining regions (CDRs) disclosed in Table 1, 2, or 3 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity); and a VL that comprises at least one, two, or three complementarity determining regions (CDRs) disclosed in Table 1, 2, or 4 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity). For example, the CD46 binding antibody can comprise a VH that comprises at least one, two, or three complementarity determining regions (CDRs) disclosed in Table 3 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity); and a VL that comprises at least one, two, or three complementarity determining regions (CDRs) disclosed in Table 4 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0098] In some embodiments, the CD46 binding antibody comprises a VH that comprises a CDR1 of SEQ ID NO: 80, a CDR2 of SEQ ID NO: 81, and a CDR3 of SEQ ID NO: 82.27KILPATRICK TOWNSEND 79434972 1

[0099] In some embodiments, the CD46 binding antibody comprises a VL that comprises a CDR1 of SEQ ID NO: 83, a CDR2 of SEQ ID NO: 84, and a CDR3 of SEQ ID NO: 85.

[0100] In some embodiments, the CD46 binding antibody comprises a VH that comprises a CDR1 of SEQ ID NO: 80, a CDR2 of SEQ ID NO: 81, and a CDR3 of SEQ ID NO: 82; and a VL that comprises a CDR1 of SEQ ID NO: 83, a CDR2 of SEQ ID NO: 84, and a CDR3 of SEQ ID NO: 85.

[0101] Table 3. VH CDR amino acid sequences of anti-CD46 antibodies as defined by Kabat et al.Note “YS5FL” and “YS5” refer to the same antibody, which differs at some positions from “YS5F”.

[0102] Table 4. VL CDR amino acid sequences of anti-CD46 antibodies as defined by Kabat et al.

[0103] YS5FL has been found to bind specifically to the surface of LnCap-C4-2B, LnCap-C4, DU145, PC3-luc, and Hs27 prostate cancer cells, but not to non-tumor BPH1 cells. Likewise, YS5FL binds specifically to the surface of RPMI8226, MM. IS, MM.1R, and INA6 multiple myeloma cells.

[0104] In some embodiments, a CDR described herein comprises one, two, or three amino acid modifications. In some embodiments, said modification is a substitution, addition, or deletion. In some embodiments, a CDR described herein comprises one, two, or three conservative amino acid substitutions. In some embodiments, the one, two, or three amino acid modifications does not substantially modify binding to human CD46. In some embodiments, the one, two, or three amino acid modifications modifies binding to human CD46. In some embodiments, a VH-CDR3 and / or VL CDR3 comprises an amino acid substitution that modifies binding to human CD46, immunogenicity, or some other feature. In some embodiments, the amino acid substitution is an alanine (A).28KILPATRICK TOWNSEND 79434972 1

[0105] In some embodiments, the CD46 binding antibody comprises a VH that comprises an amino acid sequence disclosed in Table 5 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0106] In some embodiments, the CD46 binding antibody comprises a VL that comprises an amino acid sequence disclosed in Table 6 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0107] In some embodiments, the CD46 binding antibody comprises a VH that comprises an amino acid sequence disclosed in Table 5 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity); and a VL that comprises an amino acid sequence disclosed in Table 6 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0108] In some embodiments, the CD46 binding antibody comprises a VH that comprises an amino acid sequence of SEQ ID NO: 86, or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0109] In some embodiments, the CD46 binding antibody comprises a VL that comprises an amino acid sequence of SEQ ID NO: 87, or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0110] In some embodiments, the CD46 binding antibody comprises a VH that comprises an amino acid sequence of SEQ ID NO: 86, or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity); and a VL that comprises an amino acid sequence of SEQ ID NO: 87, or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).[OHl] Table 5. Amino acid sequence of the anti-CD46 variable heavy chain binding domains.

[0112] Table 6. Amino acid sequence of the anti-CD46 variable light chain binding domains.29KILPATRICK TOWNSEND 79434972 1

[0113] In some embodiments, the CD46 binding antibody comprises a heavy chain that comprises an amino acid sequence disclosed in Table 7 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0114] In some embodiments, the CD46 binding antibody comprises a light chain that comprises an amino acid sequence disclosed in Table 8 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0115] In some embodiments, the CD46 binding antibody comprises a heavy chain that comprises an amino acid sequence disclosed in Table 7 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity); and a light chain that comprises an amino acid sequence disclosed in Table 8 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0116] In some embodiments, CD46 binding antibody comprises a heavy chain that comprises an amino acid sequence of SEQ ID NO: 88, or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0117] In some embodiments, the CD46 binding antibody comprises a light chain that comprises an amino acid sequence of SEQ ID NO: 89, or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0118] In some embodiments, the CD46 binding antibody comprises a heavy chain that comprises an amino acid sequence of SEQ ID NO: 88, or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity); and a light chain that comprises an amino acid sequence of SEQ ID NO: 89, or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0119] Table 7. Amino acid sequence of the anti-CD46 heavy chain.30KILPATRICK TOWNSEND 79434972 1

[0120] Table 8. Amino acid sequence of the anti-CD46 light chain.

[0121] In some embodiments, the anti-CD46 antibody disclosed herein comprises an immunoglobulin constant region (e.g., an Fc region). Exemplary Fc regions can be chosen from the heavy chain constant regions of IgGl, IgG2, IgG3 or IgG4; more particularly, the heavy chain constant region of human IgGl or IgG4. In some embodiments, the immunoglobulin constant region (e.g., the Fc region) is altered, e.g., mutated, to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function.

[0122] In some embodiments, the antibody disclosed herein comprises an immunoglobulin constant region (e.g., an Fc region). Exemplary Fc regions can be chosen from the heavy chain constant regions of IgGl, IgG2, IgG3 or IgG4; more particularly, the heavy chain constant region of human IgGl or IgG4. In some embodiments, the immunoglobulin constant region (e.g., the Fc region) is altered, e.g., mutated, to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function. In some embodiments, the antibody is an immunoglobin, a single-chain variable fragment (scFv), or a nanobody.31KILPATRICK TOWNSEND 79434972 1

[0123] In some embodiments, antibodies (and antigen binding fragment thereof) are produced using any method known in the art to be useful for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression techniques.

[0124] In some embodiments, an antibody (or antigen binding fragment thereof) is expressed recombinantly. In some embodiments, the nucleic acid encoding the antibody (or antigen binding fragment thereof) is assembled from chemically synthesized oligonucleotides. In some embodiments, a nucleic acid molecule encoding an antibody is generated from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence.

[0125] In some embodiments, an antibody (or antigen binding fragment thereof) is made by immunizing an animal, such as a mouse, to generate polyclonal or monoclonal antibodies.

[0126] In some embodiments, an expression vector comprising the nucleotide sequence of an antibody or the nucleotide sequence of an antibody is transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In some embodiments, the expression of the antibody is regulated by a constitutive, an inducible or a tissue, specific promoter.

[0127] A variety of host-expression vector systems can be utilized to express an antibody (or antigen binding fragment thereof) described herein. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing an antibody or its binding fragment coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing an antibody or its binding fragment coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an antibody or its binding fragment coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing an antibody or its binding fragment coding sequences; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5K promoter).32KILPATRICK TOWNSEND 79434972 1

[0128] For long-term, high-yield production of recombinant proteins, stable expression may be preferred. In some embodiments, cell lines that stably express an antibody are made.Following the introduction of the foreign DNA, engineered cells are then allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. A selectable marker in the recombinant plasmid may be used to confer resistance to the selection.

[0129] In some embodiments, any method known in the art for purification of an antibody can be used, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

[0130] As noted above, the methods and compositions involve delivery of an effector agent (e.g., a toxin) that is linked to an anti-CD46 antibody, e.g., an anti-CD46 antibody as described herein. In some cases, an effector agent is linked to a first anti-CD46 antibody and a chelator binding a radionuclide is linked to a second anti-CD46 antibody. However, in many embodiments, the effector agent and the chelator binding the radionuclide and both linked to the same anti-CD46 antibody. Thus, it should be understood that any of the antibodies described herein linked to an effector agent can be further also (a) be linked to any chelator binding a radionuclide as described herein or (b) be combined and / or administered with a second anti-CD46 antibody linked to any chelator binding a radionuclide as described herein. In some embodiments, the effector agent is an anti-cancer agent (or prodrug thereof). In some embodiments, the effector agent is a chemotherapeutic agent (or prodrug thereof). In some embodiments, the effector agent is a microtubule inhibitor (or prodrug thereof), a DNA-damaging agent (or prodrug thereof), or a polymerase inhibitor (or prodrug thereof).

[0131] As noted above, the methods and compositions involve delivery of an effector agent (e.g., a toxin) that is linked to an antibody. In some cases, an effector agent is linked to a first antibody and a chelator binding a radionuclide is linked to a second antibody. However, in many embodiments, the effector agent and the chelator binding the radionuclide and both linked to the same antibody. Thus, it should be understood that any of the antibodies described herein linked to an effector agent can be further also (a) be linked to any chelator binding a radionuclide as described herein or (b) be combined and / or administered with a second antibody linked to any chelator binding a radionuclide as described herein. In some embodiments, the effector agent is an anti-cancer agent (or prodrug thereof). In some embodiments, the effector agent is a chemotherapeutic agent (or prodrug thereof). In some embodiments, the effector agent is a microtubule inhibitor (or prodrug thereof), a DNA- 33KILPATRICK TOWNSEND 79434972 1damaging agent (or prodrug thereof), or a polymerase inhibitor (or prodrug thereof). In some embodiments, an antibody linked to an effector agent (e.g., a toxin or cytotoxic effector) is selected from the group consisting of Trastuzumab deruxtecan-nxki (Enhertu), Ado-trastuzumab emtansine (Kadcyla), Sacituzumab govitecan-hziy (Trodelvy), Datopotamab deruxtecan (Dato-DXd), Enfortumab vedotin-ejfv (Padcev), Mirvetuximab soravtansine-gynx (Elahere), Tisotumab vedotin-tftv (Tivdak), Telisotuzumab vedotin (Teliso-V), M9140, Brentuximab vedotin (Adcetris), Polatuzumab vedotin-piiq (Polivy), Inotuzumab ozogamicin (Besponsa), Gemtuzumab ozogamicin (Mylotarg), Loncastuximab tesirine-lpyl (Zynlonta), and Belantamab mafodotin-blmf (Blenrep).

[0132] In some embodiments, the effector agent is a microtubule inhibitor (or prodrug thereof). In some embodiments, the microtubule inhibitor is an auristatin (or a derivative thereof), dolastatin-10 (or a derivative thereof), or maytansine (or a derivative thereof). In some embodiments, the microtubule inhibitor is monomethylauristatin F (MMAF), auristatin E (AE), monomethylauristatin E (MMAE), valine-citrulline MMAE (vcMMAE), valinealanine MMAE, valine-citrulline MMAF (vcMMAF) or valine-alanine MMAF. In some embodiments, the microtubule inhibitor is monomethylauristatin E (MMAE).

[0133] In some embodiments, the effector agent comprises or consists of a compound of Formula A:(Formula A)Molecular formula: C39H67N5O734KILPATRICK TOWNSEND 79434972 1

[0134] In some embodiments, the effector agent is a maytansinoid. In some embodiments, the maytansinoid is mertansine (DM1) or ravtansine (DM4).

[0135] In some embodiments, the effector agent is a Calicheamicin or a Calicheamicin derivative.

[0136] In some embodiments, the effector agent is Duocarmycin or a Duocarmycin analog. Exemplary duocarmycin analogs are described in, e.g., Morcos etal., Cancers (Basel) 2024 Sep 27;16(19):3293. In some embodiments, the duocarmycin analog is selected from Duocarmycin Bl (DUMB1), duocarmycin B2 (DUMB2), duocarmycin Cl (DUMC1), duocarmycin C2 (DUMC2), duocarmycin DI (DUMD1), duocarmycin D2 (DUMD2) and DSA.

[0137] In some embodiments, the effector agent is a pyrrolobenzodiazepine (PBD) dimer. See, e.g., Hartley, Expert Opin Biol Ther. 2021 Jul;21(7):931-943; and Staben et al., J. Med. Chem. 2020, 63, 17, 9603-9622.

[0138] In some embodiments, the effector agent is a Camptothecin or a derivative thereof. Exemplary derivatives can include but are not limited to Exatecan derivative for ADC (DXd), 7-ethyl-10-hydroxy-camptothecin (SN-38), topotecan and CPT2, a camptothecin analog (see, e.g., Lyski et al, Mol Cancer Ther 2021;20:329-39).

[0139] In some embodiments, the effector agent is conjugated to the anti-CD46 antibody. In some embodiments, the effector agent is attached to the anti-CD46 antibody via a linker. In some embodiments, the linker is a peptide linker, a small molecule linker, or a linker that comprises a peptide and a small molecule. Exemplary peptide linkers include, but are not limited to, peptide linkers comprising glycine, serine, or glycine and serine.

[0140] In some embodiments, the effector agent is conjugated to the antibody. In some embodiments, the effector agent is attached to the antibody via a linker. In some embodiments, the linker is a peptide linker, a small molecule linker, or a linker that comprises a peptide and a small molecule. Exemplary peptide linkers include, but are not limited to, peptide linkers comprising glycine, serine, or glycine and serine.

[0141] In some embodiments, the linker is cleavable. In some embodiments, the linker is cleaved only upon internalization into a cell. In some embodiments, the cleavable linker is only cleavable upon internalization into a cancer cell. In some embodiments, the cleavable portion of a linker is a peptide (e.g., a dipeptide, e.g., ValCit). In some embodiments, the cleavable linker is cleavable by cathepsin. In some embodiments, the linker comprises maleimide. In some embodiments, the linker comprises caproic acid. In some embodiments,35KILPATRICK TOWNSEND 79434972 1the linker comprises maleimide and caproic acid. In some embodiments, the linker comprises maleimide, caproic acid, and a cleavable dipeptide.

[0142] In some embodiments, the linker comprises or consists of is a maleimidocaproyl-valinecitrulline- para-amino benzyloxycarbonyl (mc-vc-PAB).

[0143] In some embodiments, the linker comprises or consists of a compound of Formula B:(Formula B)

[0144] In some embodiments, an effector moiety is attached to a cysteine residue of the anti-CD46 antibody. In some embodiments, an anti-CD46 antibody is partially reduced prior to conjugation to an effector moiety such that 1-4 interchain disulfide bonds are reduced while intrachain disulfide bonds are not reduced. Partial reduction exposes pairs of cysteine residues, rendering them accessible to conjugation to adducts such as but not limited to mc-vc-PAB-MMAE. In some embodiments, the following interchain cysteine pairs of YS5FL are exposed: C219 of the first heavy chain and C214 of the first light chain; C219 of the second heavy chain and C214 of the second light chain; C225 of the first heavy chain and C225 of the second light chain; and C228 of the first heavy chain and C228 of the second light chain. In some embodiments, an effector such as mc-vc-PAB-MMAE is conjugated to 0, 1, 2, 3, or 4 pairs of cysteine residues on YS5FL.

[0145] In some embodiments, the ratio of effector agents to anti-CD46 antibodies is 1 : 1, 2: 1, 3:1, 4:1, 5:1, 6:1, 7:1 or 8:1. In some embodiments, the ratio of effector agents to anti-CD46 antibodies is 2:1, 4:1, 6:1, or 8:1. In some embodiments, the ratio of effector agents to anti-CD46 antibodies is about 4:1. In some embodiments, the average ratio of effector agents to36KILPATRICK TOWNSEND 79434972 1anti-CD46 antibodies is about 3.7:1. In some embodiments, the ratio of effector agents to anti-CD46 antibodies is about 4: 1 and each effector agent is the same.

[0146] In some embodiments, the ratio of effector agents to antibodies is 1 : 1, 2: 1, 3 : 1, 4: 1, 5:1, 6:1, 7:1 or 8:1. In some embodiments, the ratio of effector agents to antibodies is 2:1, 4: 1, 6: 1, or 8: 1. In some embodiments, the ratio of effector agents to antibodies is about 4: 1. In some embodiments, the average ratio of effector agents to antibodies is about 3.7:1. In some embodiments, the ratio of effector agents to antibodies is about 4: 1 and each effector agent is the same.

[0147] An exemplary immunoconjugate provided herein comprises an anti-CD46 YS5FL antibody linked to a monomethyl auristatin E (MMAE) effector agent via a maleimidocaproyl-valine-citrullinepara- amino benzyloxycarbonyl (mc-vc-PAB), and optionally linked to a chelator binding a radionuclide. In some embodiments, the ratio of MMAE to YSFL antibody is about 4:1.

[0148] In some embodiments, the immunoconjugate comprises the antibody conjugate below in Formula C, wherein the comprises heavy chain of SEQ ID NO: 9; and a light chain of SEQ ID NO: 10, and is further linked to a chelator as described herein. This immunoconjugate would comprise the YS5FL antibody (or other antibody described herein) attached to MMAE through a mc-vc-PAB linker (depicted) and optionally also linked to a chelator binding a radionuclide (not depicted). In some embodiments, the optional chelator is Macropa. In some embodiments, the radionuclide is225Ac.(Formula C)

[0149] In some embodiments, an anti-CD46 immunoconjugate described herein is manufactured by a process comprising reduction or partial reduction of disulfide bonds of an immunoglobulin. In some embodiments, an anti-CD46 immunoconjugate described herein is manufactured by a process comprising reduction or partial reduction of interchain disulfide bonds of an immunoglobulin. In some embodiments, the reducing agent is dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In some embodiments, an effector-linker complex comprising a maleimide reactive group is conjugated to pairs of reduced cysteines37KILPATRICK TOWNSEND 79434972 1of an immunoglobulin. In some embodiments, the effector-linker complex is mc-vc-PAB-MMAE.

[0150] In some embodiments, an effector-linker complex is conjugated at C219, C225, or C228 of a YS5FL heavy chain (SEQ ID NO: 9) or C214 of a YS5FL light chain (SEQ ID NO: 10), or any combination thereof. In some embodiments, the effector-linker complexes are conjugated to C219 of a YS5FL heavy chain and C214 of a YS5FL light chain. In some embodiments, an anti-CD46 immunoconjugate comprises two YS5FL heavy chains and two YS5FL light chains and effector-linker complexes are conjugated to C219 of a YS5FL first heavy chain, C214 of a first YS5FL light chain, C219 of a YS5FL second heavy chain, and C214 of a second YS5FL light chain. In some embodiments, an anti-CD46 immunoconjugate comprises two YS5FL heavy chains and an effector-linker complex is conjugated to C225 of a first YS5FL heavy chain and C225 of a second YS5FL heavy chain. In some embodiments, an anti-CD46 immunoconjugate comprises two YS5FL heavy chains and an effector-linker complex is conjugated to C228 of a first YS5FL heavy chain and C228 of a second YS5FL heavy chain. In some embodiments, an immunoconjugate comprises two, four, six, or eight effectors and the effectors are conjugated to any one, two, three, or four, respectively, of the following pairs of cysteines: C219 of HC1 and C214 of LC1; C219 of HC2 and C214 of LC2; C225 of HC1 and C225 of HC2; and C228 of HC1 and C228 of HC2.

[0151] In some embodiments, purified YS5FL mAb (e.g., 10 mg / ml) can adjusted to a pH of 6.8 with sodium phosphate buffer and then treated with TCEP (e.g., TCEP / mAb ratio of 2.1) for two hours at 22°C. Reduced mAb can be reacted with mc-vc-PAB-MMAE (e.g., drug / mAb ratio of 6) in 9% dimethylacetamide for 15 minutes. In some embodiments, the mAb can reduced a second time for one hour, conjugated a second time for 60 min, and the reaction can quenched, e.g., by lowering the pH to 5.0 with IM acetic acid, yielding a immunoconjugate, for example with a drug to antibody ratio of about 3.7, as determined by hydrophobic interaction chromatography.

[0152] An anti-CD46 antibody provided herein is linked to a chelator that binds a radionuclide. As noted herein, in some embodiments, one anti-CD46 antibody is linked to (i) a cytotoxic effector and (ii) a chelator binding a radionuclide. In other embodiments, a first anti-CD46 antibody is linked to a cytotoxic effector and a second anti-CD46 antibody is linked to a chelator binding a radionuclide. In either option, the radionuclide can be used to kill cancer cells and, in some embodiments, can also be used to monitor treatment with the antibody or antibodies. For example, the radionuclide conjugate can allow for real-time38KILPATRICK TOWNSEND 79434972 1imaging and dosimetry assessment, which in some embodiments allows for patient stratification, treatment planning, and response monitoring.

[0153] An antibody provided herein is linked to a chelator that binds a radionuclide. As noted herein, in some embodiments, one antibody is linked to (i) a cytotoxic effector and (ii) a chelator binding a radionuclide. In other embodiments, a first antibody is linked to a cytotoxic effector and a second antibody is linked to a chelator binding a radionuclide. In either option, the radionuclide can be used to kill cancer cells and, in some embodiments, can also be used to monitor treatment with the antibody or antibodies. For example, the radionuclide conjugate can allow for real-time imaging and dosimetry assessment, which in some embodiments allows for patient stratification, treatment planning, and response monitoring.

[0154] The chelator linked to the antibody can be selected for its ability to bind a desired radionuclide or for other manufacturing criteria. Exemplary chelators can include but are not limited to Macropa (see, e.g., Blei etal., Inorg. Chem. 2023, 62, 50, 20699-20709), mercaptoacetyltriglycine, also known as MAGs (see, e.g., Wang, Nat Protoc.2006; 1(3): 1477-80), diethylenetriaminepentaacetic acid also known as DTPA (see, e.g., Tominaga, et al., Toxicol Lett. 2021 Oct 10:350:283-291), Deferoxamine (DFO), DFO-Km (see, e.g., Salith et al., Inorg. Chem. 2023, 62, 50, 20806-20819), DFO* (see, e.g., Guarrochena, et al., ChemMedChem Volume 19, Issue 3 (2024)), DFO-HOPO (see, e.g., Allott, et al., Chem. Commun., 2017,53, 8529-8532), l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA) (see, e.g., Lee, et al., Diagnostics (Basel) 2023 Aug 11;13(16):2649) or triethylenetetramine (TETA).

[0155] Linkage of the chelators to the antibody can be achieved as described above for the effector. Optionally different reactivity is used to link the effector and the chelator. For example, in some embodiments, reduced cysteines on the antibody can be used to link the effector, whereas an amine or carboxyl group (e.g., a lysine) on the antibody can be used to link the chelator to the antibody, or vice versa. In some embodiments, the linker comprises or consists of polyethylene glycol (PEG). PEGs can be of any length as desired. In some embodiments, the PEG2-10 is used as a linker. Exemplary PEG linkers can be for example, PEG2, PEG4, PEGe, PEGs or PEG10. In some embodiments, the PEG linker is linked to a lysine on the antibody.

[0156] The ratio of chelator molecules to antibody can be selected as desired. In some embodiments the ratio of chelator to antibody is 0.5-2, e.g., in some embodiments, 08.-1.39KILPATRICK TOWNSEND 79434972 1

[0157] A variety of radionuclides can be bound to the chelator as desired. Indeed, the selection of radionucleotide may dictate the selection of chelator because different chelators may be selective for binding to certain radionuclides. In some embodiments, the radionuclide can emit alpha particles, beta particles, and / or positrons. Criteria for selecting a radionuclide can depend, for example, on therapeutic or imaging purposes. Exemplary radionuclides are described below.

[0158] Exemplary Alpha-emitting radionuclides include, for example, Actinium-225, Thorium-227, Bismuth-213, Astatine-211, Terbium-149, and Bismuth-213. Alpha particles have a high linear energy transfer (LET), causing double-stranded DNA breaks and leading to potent cytotoxicity. Their short path length (50-100 pm) minimizes off-target damage to healthy tissue. Exemplary beta-emitting radionuclides include, for example, Lutetium- 177, Terbium-161, and Yttrium-90. Exemplary alpha / beta dual-emitting radionuclides include for example, lead-212 (212Pb) (via decay into alpha-emitting Bismuth-212 and Polonium-212). Exemplary imaging radionuclides can include, for example, Zirconium-89, Gallium-68, Cerium- 134, Fluorine- 18 and Copper-64.

[0159] In some embodiments, the methods and compositions described herein include usage of one or more anti-CD46 antibody (e.g., linked to an effector, a chelator linked to a radionuclide, or both) for imaging or detection, or both, of a tumor in a subject. In some embodiments, a method as described herein may comprise administering one or more anti-CD46 antibody (e.g., linked to an effector, a chelator linked to a radionuclide, or both) to a tumor and detecting emitted signal from the radionuclide. In some embodiments, a method as described herein may comprise administering one or more anti-CD46 antibody (e.g., linked to an effector, a chelator linked to a radionuclide, or both) to a subject and detecting emitted signal from the radionuclide. The one or more anti-CD46 antibody may be conjugated to a radionuclide selected from the group consisting of Zirconium-89, Cerium-134, Copper-64, Gallium-68, and Fluorine-18.

[0160] In some embodiments, the methods and compositions described herein include usage of one or more antibodies (e.g., linked to an effector, a chelator linked to a radionuclide, or both) for imaging or detection, or both, of a tumor in a subject. In some embodiments, a method as described herein may comprise administering one or more antibodies (e.g., linked to an effector, a chelator linked to a radionuclide, or both) to a tumor and detecting emitted signal from the radionuclide. In some embodiments, a method as described herein may comprise administering one or more antibodies (e.g., linked to an effector, a chelator linked to a radionuclide, or both) to a subject and detecting emitted signal from the radionuclide.40KILPATRICK TOWNSEND 79434972 1The one or more antibodies may be conjugated to a radionuclide selected from the group consisting of Zirconium-89, Cerium-134, Copper-64, Gallium-68, and Fluorine-18.

[0161] In some embodiments, the emitted signal from the radionuclide is detected using a positron emission tomography (PET) scanner, single photon emission computed tomography (SPECT), or magnetic resonance imaging (MRI) system. Imaging may be conducted at one or more time points after administration, such as 24, 48, 96, or 168 hours, to visualize the tumor, assess tumor uptake, and monitor biodistribution or pharmacokinetics of the antibody conjugate.

[0162] In some embodiments, the detected signal is used to differentiate tumor tissue from non-tumor tissue, assess tumor progression, or evaluate response to therapy. Quantitative imaging metrics such as percentage of injected dose per gram (%ID / g) or standardized uptake value (SUV) may be used to compare tumor signal to background signal and track changes over time.

[0163] In some embodiments, the tumor is a cancer selected from the group consisting of prostate cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, and other solid tumor cancers. In some embodiments, the method is used for imaging prostate cancer, including resistant forms such as PSMA-low, neuroendocrine, or androgen signaling inhibitor resistant prostate cancer.

[0164] In some embodiments, the antibody conjugate is administered at a dose ranging from about 0.01 mg / kg to about 10 mg / kg body weight, with the radionuclide conjugate at a specific activity ranging from about 0.01 pCi / pg to about 10 pCi / pg. In some embodiments, pharmaceutical compositions may be formulated at a concentration of about 0.1 mg / mL to about 100 mg / mL, suitable for intravenous administration.

[0165] In some embodiments, the imaging method enables early detection of cancer, monitoring recurrence or metastasis, and guiding therapeutic decisions based on quantitative imaging results.

[0166] In some embodiments, the methods and compositions described herein include usage of one or more anti-CD46 antibody (e.g., linked to an effector, a chelator linked to a radionuclide, or both) in combination with an immunomodulatory imide drug (IMiD) or Signal Transducer And Activator of Transcription 3 (Stat3) inhibitor. This combination can result in significant improvement of the efficacy of the anti-CD46 antibody. Without intending to limit the scope of the invention it is believed that IMiDs and Stat3 inhibitors induce expression of CD46 in cancer cells thereby allowing for improved efficacy of the anti-CD46 antibody, which can be linked to an effector molecule such as a cytotoxin.41KILPATRICK TOWNSEND 79434972 1

[0167] IMiDs used in the methods and compositions described herein can be selected, for example, from those known in the art. In one embodiment, the IMiD includes thalidomide or analogs thereof. Thalidomide is registered under the trade name Thalidomid™ (Celgene Corp). Thalidomide and analogs thereof, and methods of making the same, are known, for example, as described in U.S. Pat. Nos. 6,045,501; 7,230,012; 7,435,745.

[0168] In another embodiment, the IMiD includes pomalidomide or analogs thereof.Pomalidomide is registered under the trade name Pomalyst™ (Celgene Corp). Pomalidomide and analogs thereof, and methods of making the same, are known, for example, as described in U.S. Pat. Nos. 5,635,517; 6,316,471; 6,476,052; 8,158,653; 8,198,262; 8,673,939;8,735,428; and 8,828,427.

[0169] In another embodiment, the IMiD includes lenalidomide or analogs thereof.Lenalidomide is registered under the trade name Revlimid™ (Celgene Corp). Lenalidomide and analogs thereof, and methods of making the same are known, for example, as described in U.S. Pat. Nos. 5,635,517; 6,555,554; 7,119,106; 7,465,800; 7,855,217; 8,288,415; and 8,530,498.

[0170] In another embodiment, the IMiD is apremilast or analogs thereof. Apremilast is registered under the trade name Otezla™ (Celgene Corp). Apremilast and analogs thereof, and methods of making the same, are known to those skilled in the art, for example, those described in U.S. Pat. Nos. 6,020,358; 7,427,638; and 7,893,101.

[0171] In another embodiment, the IMiD is iberdomide or analogs thereof. Iberdomide structure is provided in e.g., US 20200330445.

[0172] Stat3 inhibitors used in the methods and compositions described herein can be selected, for example, from those known in the art. Exemplary Stat3 inhibitors include but are not limited to N-(l', 2-Dihydroxy-l,2'-binaphthalen-4'-yl)-4-methoxybenzenesulfonamide (C188-9), STAT3 Inhibitory, 6-Nitrobenzo[b]thiophene 1,1-dioxide (Stattic), (1E,6E)-1,7-Bis(4-hydroxy-3-methoxyphenyl)-l,6-heptadiene-3, 5-dione (curcumin), N-Hexyl-2-(l-naphthalenyl)-5-[[4-(phosphonooxy)phenyl]methyl]-4- -oxazolecarboxamide (S3LM2001), 8-hydroxy-3-methyl-3,4-dihydrotetraphene-l,7, 12(2H)-trione (STA-21), 2-Hydroxy-4-[[2-[[(4-methylphenyl)sulfonyl]oxy]acetyl]amino]benzoic acid (S3I-201), Cepharanthine, Cucurbitacin I, Cucumis sativus L, Niclosamide, Cryptotanshinone, SD 1008, Stat3 Inhibitor III, WP1066, Nifuroxazide, Stat3 Inhibitor VI, S3I-201, STA-21, Kahweol, STAT3 Inhibitor IX, Cpdl88; STAT3 Inhibitor VI, S31-201; STAT3 Inhibitor VII Ethyl-l-(4-cyano-2,3,5,6-tetrafluorophenyl)-6,7,8-trifluoro-4-oxo-l,4-dihydroquinoline-3-carboxylate; STAT3 Inhibitor VIII, 5,15-DPP, STAT3 Inhibitor X, HJB; STAT3 Inhibitor XII, SPI; STAT342KILPATRICK TOWNSEND 79434972 1Inhibitor XI, STX-0119; STAT3 Inhibitor XIV, LLL12; FLLL32; FLLL62; Napabucasin (BBI608); DSP-0337 (prodrug of napabucasin); OPB-51602; OPB-31121; OPB-111077; Pyrimethamine; WP1066 or derivatives or analogues or salts thereof.

[0173] In some embodiments, one or more selective glucocorticoid receptor agonist or modulator (SEGRAM) are also administered to the human. Selective glucocorticoid receptor agonists (SEGRAs) are historically and typically steroidal in structure while selective glucocorticoid receptor modulators (SEGRMs) are typically nonsteroidal. The latter class is able to modulate the activity of a GR agonist and / or may not classically bind the glucocorticoid receptor ligand-binding pocket. See, e.g., Sundahl et al, Pharmacology & Therapy, Volume 152, August 2015, Pages 28-41. The combined abbreviation of selective glucocorticoid receptor agonist or modulator is SEGRAM. Exemplary SEGRAs include, for example, dexamethasone, prednisone, and cortisol. Exemplary SEGRMs include, for example, mapracorat, fosdagrocorat (PF-04171327), and dagrocorat.

[0174] Pharmaceutically acceptable forms of iMiD, Stat3 inhibitor or SEGRAM compounds, such as free base, salts, polymorphs, solvates, solutions, isomers, amorphous, crystalline, co crystalline, solid solution, prodrugs, analogs, derivatives, and metabolites are contemplated for use in the methods described herein. The compound may be in the form of a pharmaceutically acceptable salt, such as an acid addition salt or a base salt, or a solvate thereof, including a hydrate thereof. Suitable acid addition salts are formed from acids which form non-toxic salts and examples are the hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, nitrate, phosphate, hydrogen phosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, succinate, saccharate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate and pamoate salts.

[0175] In some embodiments, an agent that induces expression of CD46, wherein the agent is an immunomodulatory imide drug (IMiD) and optionally a glucocorticoid receptor agonist or modulator (SEGRAM), is administered to a human having a cancer as described herein for a first period of time sufficient to result in increased expression of CD46 in cancer cells, followed by administration of an anti-CD46 antibody or immunoconjugate described herein for a second period of time after the first period of time. See, e.g., U.S. Patent Publication No. 2024 / 0101699.

[0176] Also provides are methods of treating a CD46-expressing cancer in a human by administering to the human, optionally at the site of a tumor, an anti-CD46 antibody linked to (i) a cytotoxic effector and (ii) a chelator binding a radionuclide, or alternatively43KILPATRICK TOWNSEND 79434972 1administering a first anti-CD46 antibody linked to a cytotoxic effector and a second anti-CD46 antibody linked to a chelator binding a radionuclide.

[0177] Also provides are methods of treating an antigen-expressing cancer in a human by administering to the human, optionally at the site of a tumor, an antibody linked to (i) a cytotoxic effector and (ii) a chelator binding a radionuclide, or alternatively administering a first antibody linked to a cytotoxic effector and a second antibody linked to a chelator binding a radionuclide.

[0178] In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is castration resistant prostate cancer. In some embodiments, the cancer is metastatic prostate cancer. In some embodiments, the prostate cancer is a resistant form of prostate cancer. In some embodiments, the prostate cancer is Prostate-Specific Membrane Antigen (PSMA)-low, neuroendocrine, or androgen signaling inhibitor resistant form of prostate cancer.

[0179] In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is relapsing multiple myeloma. In some embodiments, the cancer is remitting multiple myeloma. In some embodiments, the cancer is relapsing or remitting multiple myeloma. In some embodiments, the cancer is refractory multiple myeloma. In some embodiments, the cancer is relapsing or refractory multiple myeloma.

[0180] In some embodiments, the cancer is colon cancer, bladder cancer, lung cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, ovarian cancer, lymphoma (including but not limited to Hodgkin's lymphoma), acute myeloid leukemia (AML), or metastatic renal cell carcinoma (mRCC).

[0181] Pharmaceutical compositions as described herein (e.g., anti-CD46 antibodies linked to an effector, a chelator bound to a radionuclide, or both) can be prepared in accordance with methods well known and routinely practiced in the art. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions described herein. Applicable methods for formulating the antibodies and determining appropriate dosing and scheduling can be found, for example, in Remington: The Science and Practice of Pharmacy, 21stEd., University of the Sciences in Philadelphia, Eds., Lippincott Williams & Wilkins (2005); and in Martindale: The Complete Drug Reference, Sweetman, 2005, London: Pharmaceutical Press., and in Martindale, Martindale: The Extra Pharmacopoeia, 31st Edition., 1996, Amer Pharmaceutical Assn, and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978, each of which are hereby incorporated 44KILPATRICK TOWNSEND 79434972 1herein by reference. Pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or efficacious dose of the antibody or other compounds descried herein is employed in the pharmaceutical compositions. The antibodies can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the desired response (e.g., a therapeutic response). In determining a therapeutically or prophylactically effective dose, a low dose can be administered and then incrementally increased until a desired response is achieved with minimal or no undesired side effects. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0182] Actual dosage levels of the active ingredients in the pharmaceutical compositions can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.

[0183] A therapeutically effective amount of the antibodies and compounds will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The dosages for administration can range from, for example, about 1 ng to about 10,000 mg, about 5 ng to about 9,500 mg, about 10 ng to about 9,000 mg, about 20 ng to about 8,500 mg, about 30 ng to about 7,500 mg, about 40 ng to about 7,000 mg, about 50 ng to about 6,500 mg, about 100 ng to about 6,000 mg, about 200 ng to about 5,500 mg, about 300 ng to about 5,000 mg,45KILPATRICK TOWNSEND 79434972 1about 400 ng to about 4,500 mg, about 500 ng to about 4,000 mg, about 1 pg to about 3,500 mg, about 5 pg to about 3,000 mg, about 10 pg to about 2,600 mg, about 20 pg to about 2,575 mg, about 30 pg to about 2,550 mg, about 40 pg to about 2,500 mg, about 50 pg to about 2,475 mg, about 100 pg to about 2,450 mg, about 200 pg to about 2,425 mg, about 300 pg to about 2,000, about 400 pg to about 1,175 mg, about 500 pg to about 1,150 mg, about 0.5 mg to about 1,125 mg about 1 mg to about 1,100 mg, about 1.25 mg to about 1,075 mg, about 1.5 mg to about 1,050 mg, about 2.0 mg to about 1,025 mg, about 2.5 mg to about 1,000 mg, about 3.0 mg to about 975 mg, about 3.5 mg to about 950 mg, about 4.0 mg to about 925 mg, about 4.5 mg to about 900 mg, about 5 mg to about 875 mg, about 10 mg to about 850 mg, about 20 mg to about 825 mg, about 30 mg to about 800 mg, about 40 mg to about 775 mg, about 50 mg to about 750 mg, about 100 mg to about 725 mg, about 200 mg to about 700 mg, about 300 mg to about 675 mg, about 400 mg to about 650 mg, about 500 mg, or about 525 mg to about 625 mg, e.g., 1 to 10 mg / kg, 1.8 to 2.7 mg / kg of an anti-CD46 antibody described herein and / or antigen binding portion thereof, and / or immunoconjugate thereof as described herein. The dosages for administration can range from, for example, about 1 ng to about 10,000 mg, about 5 ng to about 9,500 mg, about 10 ng to about 9,000 mg, about 20 ng to about 8,500 mg, about 30 ng to about 7,500 mg, about 40 ng to about 7,000 mg, about 50 ng to about 6,500 mg, about 100 ng to about 6,000 mg, about 200 ng to about 5,500 mg, about 300 ng to about 5,000 mg, about 400 ng to about 4,500 mg, about 500 ng to about 4,000 mg, about 1 pg to about 3,500 mg, about 5 pg to about 3,000 mg, about 10 pg to about 2,600 mg, about 20 pg to about 2,575 mg, about 30 pg to about 2,550 mg, about 40 pg to about 2,500 mg, about 50 pg to about 2,475 mg, about 100 pg to about 2,450 mg, about 200 pg to about 2,425 mg, about 300 pg to about 2,000, about 400 pg to about 1,175 mg, about 500 pg to about 1,150 mg, about 0.5 mg to about 1,125 mg about 1 mg to about 1,100 mg, about 1.25 mg to about 1,075 mg, about 1.5 mg to about 1,050 mg, about 2.0 mg to about 1,025 mg, about 2.5 mg to about 1,000 mg, about 3.0 mg to about 975 mg, about 3.5 mg to about 950 mg, about 4.0 mg to about 925 mg, about 4.5 mg to about 900 mg, about 5 mg to about 875 mg, about 10 mg to about 850 mg, about 20 mg to about 825 mg, about 30 mg to about 800 mg, about 40 mg to about 775 mg, about 50 mg to about 750 mg, about 100 mg to about 725 mg, about 200 mg to about 700 mg, about 300 mg to about 675 mg, about 400 mg to about 650 mg, about 500 mg, or about 525 mg to about 625 mg, e.g., 1 to 10 mg / kg, 1.8 to 2.7 mg / kg of an antibody described herein and / or antigen binding portion thereof, and / or immunoconjugate thereof as described herein. Dosage regiments may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or 46KILPATRICK TOWNSEND 79434972 1detrimental effects ( / .<?., side effects) of an antibody or antigen binding portion thereof are minimized and / or outweighed by the beneficial effects. Exemplary dosages for IMiDs can be determined as known in the art. Exemplary IMiD dosages can be, for example, 1-10 mg per dose per day. Exemplary SEGRAM (e.g., dexamethasone) dosages can be, for example, 0.1-60 (e.g., 0.1-20 or 20 or 40 mg) mg per dose per day.

[0184] Compositions described herein can be formulated as a pharmaceutically acceptable salt, i.e., a biologically compatible salt of a disclosed compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, and the like. Pharmaceutically acceptable acid addition salts are those salts that retain the biological effectiveness of the free bases while formed by acid partners that are not biologically or otherwise undesirable, e.g., inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, as well as organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. Pharmaceutically acceptable base addition salts include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Exemplary salts are ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. See, for example, S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977; 66:1-19, which is incorporated herein by reference. For therapeutic use, salts of the compounds are those wherein the counter-ion is pharmaceutically acceptable. However, salts of acids and bases which are non- 47KILPATRICK TOWNSEND 79434972 1pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0185] Pharmaceutical compositions as described herein can be administered by a variety of methods known in the art. The route and / or mode of administration vary depending upon the desired results. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target. The pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, intranasal, inhalational, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, e.g., antibody, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.EXEMPLARY EMBODIMENTS

[0186] The following description provides exemplary embodiments of the invention, including various features, elements, and possible implementations. It is understood that these embodiments are merely illustrative and do not limit the scope of the invention.Modifications, substitutions, and equivalents may be apparent to those skilled in the art without departing from the scope of the invention.

[0187] 1. An antibody that specifically binds to CD46, wherein the antibody is linked to (i) a cytotoxic effector and (ii) a chelator, wherein a radionuclide is bound to the chelator via coordinate bonding. 2. The antibody of embodiment 1, wherein the antibody comprises an Fc domain. 3. The antibody of any one of embodiments 1-2, wherein the antibody comprises heavy chain CDRs 1, 2 and 3 and light chain CDRs 1, 2, and 3 of any one of YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, orUA8kappa. 4. The antibody of any one of embodiments 1-2, wherein the antibody comprises a heavy chain (HC) variable region that comprises three complementarity determining regions (CDRs): HC CDR1, HC CDR2 and HC CDR3 and a light chain (LC) variable region that comprises three CDRs: LC CDR1, LC CDR2, and LC CDR3, wherein said HC CDR1, HC CDR2, HC CDR3 comprise an amino acid sequence of SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82, respectively, and said LC CDR1, LC CDR2, and LC CDR3 comprise an amino acid sequence of SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO: 85, respectively. 5. The antibody of embodiment 4, wherein the HC comprises SEQ ID NO:86 and the LC comprises SEQ ID NO:87. 6. The 48KILPATRICK TOWNSEND 79434972 1antibody of any one of embodiments 1-5, wherein the cytotoxic effector is linked to the antibody via a cleavable or non-cleavable linker via a cysteine sulfhydryl moiety in the antibody. 7. The antibody of embodiment 6, wherein the cleavable linker comprises valinecitrulline. 8. The antibody of any one of embodiments 1-7, wherein the cytotoxic effector is a chemotherapeutic agent. 9. The antibody of any one of embodiments 1-8, wherein the cytotoxic effector is a microtubule inhibitor, a DNA-damaging agent, or a polymerase inhibitor. 10. The antibody of any one of embodiments 1-8, wherein the cytotoxic effector is selected from the group consisting of an auristatin, Dolastatin-10, synthetic derivatives of the natural product Dolastatin-10, and maytansine or a maytansine derivative. 11. The antibody of any one of embodiments 1-8, wherein the cytotoxic effector is selected from the group consisting of Monomethylauristatin F (MMAF), Auristatin E (AE), Monomethylauristatin E (MMAE), vcMMAE, and vcMMAF. 12. The antibody of embodiment 11, wherein monomethylauristatin E (MMAE) is conjugated to said antibody via a maleimidocaproyl-valine-citrulline-para-amino benzyloxy carbonyl (mc-vc-PAB) linker. 13. The antibody of any one of embodiments 1-8, wherein the cytotoxic effector is selected from the group consisting of mertansine (DM1), ravtansine (DM4), calicheamicin, a duocarmycin analog, apyrrol obenzodiazepine dimer, Exatecan derivative for ADC (DXd), 7-ethyl-10-hydroxy-camptothecin (SN-38), topotecan, or a derivative thereof. 14. The antibody of embodiment 13, wherein the duocarmycin analog is selected from the group consisting of Duocarmycin Bl (DUMB1), duocarmycin B2 (DUMB2), duocarmycin Cl (DUMC1), duocarmycin C2 (DUMC2), duocarmycin DI (DUMD1), duocarmycin D2 (DUMD2) and DSA. 15. The antibody of any one of embodiments 1-14, wherein the chelator comprises Macropa, mercaptoacetyltriglycine (MAGs), diethylenetriaminepentaacetic acid (DTP A), Deferoxamine (DFO), DFO*, DFO-Km, DFO-HOPO, l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA) or triethylenetetramine (TETA). 16. The antibody of any one of embodiments 1-15, wherein the chelator is linked to the antibody by a linker. 17. The antibody of embodiment 16, wherein the linker is linked to the antibody via an amine on a lysine side chain of the antibody. 18. The antibody of embodiment 16 or 17, wherein the linker comprises polyethylene glycol (PEG). 19. The antibody of embodiment 18, wherein the PEG comprises PEG2-10. 20. The antibody of embodiment 18, wherein the linker comprises PEG2, PEG4, PEGe, or PEGs. 21. The antibody of any of embodiments 1-20, wherein the radionuclide emits alpha particles. 22. The antibody of embodiment 21, wherein the radionuclide is selected from the group consisting of Actinium-225, Thorium-227, Bismuth-213, Astatine-211, Terbium-149, Lead- 49KILPATRICK TOWNSEND 79434972 1212 and Bismuth-213. 23. The antibody of any of embodiments 1-20, wherein the radionuclide emits beta particles. 24. The antibody of embodiment 23, wherein the radionuclide is selected from the group consisting of Lutetium- 177, Terbium-161, Yttrium-90 and Lead-212. 25. The antibody of any of embodiments 1-20, wherein the radionuclide emits positrons. 26. The antibody of embodiment 25, wherein the radionuclide is selected from the group consisting of Zirconium-89, Copper-64, Gallium-68, Cerium-134 and Fluorine-18. 27. A pharmaceutical composition comprising a therapeutically-effective amount of the antibody of any one of embodiments 1-26. 28. A composition comprising: a first antibody that binds to CD46, wherein the first antibody is linked to a cytotoxic effector; and a second antibody that binds to CD46, wherein the second antibody is linked to a chelator, wherein a radionuclide is bound to the chelator via coordinate bonding. 29. The composition of embodiment 28, wherein the composition is a pharmaceutical composition comprising a therapeutically-effective amount of the first antibody and the second antibody. 30. The composition of embodiment 28 or 29, wherein the first antibody, or the second antibody, or both the first antibody and the second antibody, comprises heavy chain CDRs 1, 2 and 3 and light chain CDRs 1, 2, and 3 of any one of YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, or UA8kappa. 31. The composition of any one of embodiments 28-30, wherein the first antibody, or the second antibody, or both the first antibody and the second antibody, comprises a heavy chain (HC) variable region that comprises three complementarity determining regions (CDRs): HC CDR1, HC CDR2 and HC CDR3 and a light chain (LC) variable region that comprises three CDRs: LC CDR1, LC CDR2, and LC CDR3, wherein said HC CDR1, HC CDR2, HC CDR3 comprise an amino acid sequence of SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82, respectively, and said LC CDR1, LC CDR2, and LC CDR3 comprise an amino acid sequence of SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO: 85, respectively. 32. The composition of embodiment 31, wherein the HC comprises SEQ ID NO:86 and the LC comprises SEQ ID NO:87. 33. The composition of any one of embodiments 28-32, wherein the cytotoxic effector is linked to the first antibody via a cleavable or non-cleavable linker via a cysteine sulfhydryl moiety in the first antibody. 34. The composition of embodiment 33, wherein the cleavable linker comprises valine-citrulline.35. The composition of any one of embodiments 28-34, wherein the cytotoxic effector is a chemotherapeutic agent. 36. The composition of any one of embodiments 28-35, wherein the cytotoxic effector is a microtubule inhibitor, a DNA-damaging agent, or a polymerase inhibitor. 37. The composition of any one of embodiments 28-35, wherein the cytotoxic 50KILPATRICK TOWNSEND 79434972 1effector is selected from the group consisting of an auristatin, Dolastatin-10, synthetic derivatives of the natural product Dolastatin-10, and maytansine or a maytansine derivative.38. The composition of any one of embodiments 28-35, wherein the cytotoxic effector is selected from the group consisting of Monomethylauri statin F (MMAF), Auristatin E (AE), Monomethylauristatin E (MMAE), vcMMAE, and vcMMAF. 39. The composition of embodiment 38, wherein monomethylauristatin E (MMAE) is conjugated to the first antibody via a maleimidocaproyl-valine-citrulline-para-amino benzyloxycarbonyl (mc-vc-PAB) linker.40. The composition of any one of embodiments 28-35, wherein the cytotoxic effector is selected from the group consisting of mertansine (DM1), ravtansine (DM4), calicheamicin, a duocarmycin analog, a pyrrolobenzodiazepine dimer, Exatecan derivative for ADC (DXd), 7-ethyl-10-hydroxy-camptothecin (SN-38), topotecan, or a derivative thereof. 41. The composition of embodiment 40, wherein the duocarmycin analog is selected from the group consisting of Duocarmycin Bl (DUMB1), duocarmycin B2 (DUMB2), duocarmycin Cl (DUMC1), duocarmycin C2 (DUMC2), duocarmycin D 1 (DUMD1), duocarmycin D2 (DUMD2) and DS A. 42. The composition of any one of embodiments 28-41, wherein the chelator comprises Macropa, mercaptoacetyltriglycine (MAGs), diethylenetriaminepentaacetic acid (DTP A), Deferoxamine (DFO), DFO*, DFO-Km, DFO-HOPO, l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA) or 1,4,7,10-tetraazacyclododecane- 1,4,7, 10-tetraacetic acid (DOTA) or triethylenetetramine. 43. The composition of any one of embodiments 28-41, wherein the chelator is linked to the second antibody by a linker. 44. The composition of embodiment 43, wherein the linker is linked to the second antibody via an amine on a lysine side chain of the second antibody. 45. The composition of embodiment 43 or 44, wherein the linker comprises polyethylene glycol (PEG). 46. The composition of embodiment 45, wherein the PEG comprises PEG2-10. 47. The composition of embodiment 45, wherein the linker comprises PEG2, PEG4, PEGe, or PEGs.48. The composition of any of embodiments 28-47, wherein the radionuclide emits alpha particles. 49. The composition of embodiment 48, wherein the radionuclide is selected from the group consisting of Actinium-225, Thorium-227, Bismuth-213, Astatine-211, Terbium-149, Lead-212 and Bismuth-213. 50. The composition of any of embodiments 28-47, wherein the radionuclide emits beta particles. 51. The composition of embodiment 50, wherein the radionuclide is selected from the group consisting of Lutetium- 177, Terbium-161, Yttrium-90 and Lead-212. 52. The composition of any of embodiments 28-47, wherein the radionuclide emits positrons. 53. The composition of embodiment 52, wherein the radionuclide is selected from the group consisting of Zirconium-89, Copper-64, Gallium-68, Cerium-134 and51KILPATRICK TOWNSEND 79434972 1Fluorine-18. 54. A method of killing a CD46-expressing cancer cell, the method comprising contacting the antibody of any one of embodiments 1-26 or the pharmaceutical composition of embodiment 27 or the composition of embodiments 28-53 to the cancer cell, thereby killing the cancer cell. 55. The method of embodiment 54, wherein the cancer cell is in a human and the contacting comprises administering the antibody or the pharmaceutical composition or the composition to the human. 56. The method of embodiments 54 or 55, wherein the cancer is selected from the group consisting of prostate cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer and a solid tumor cancer. 57. The method of embodiment 56, wherein the prostate cancer is a resistant form of prostate cancer. 58. The method of embodiments 56 or 57, wherein the prostate cancer is selected from the group consisting of Prostate-Specific Membrane Antigen (PSMA)-low, neuroendocrine, or androgen signaling inhibitor resistant form of prostate cancer. 59. The method of embodiments 54 or 55, wherein the cancer is multiple myeloma or a hematological cancer.60. The method of embodiment 59, wherein the multiple myeloma is relapsing or refractory multiple myeloma. 61. A method for imaging a tumor in a subject, the method comprising, administering the antibody of any one of embodiments 1-26 or the composition of embodiments 28-53 to a tumor; and detecting emitted signal from the radionuclide. 62. The method of embodiment 61, wherein the radionuclide is selected from the group consisting of Zirconium-89, Cerium-134, Copper-64, Gallium-68, and Fluorine-18. 63. The method of embodiments 61 or 62, wherein the emitted signal is detected by a positron emission tomography (PET) scanner, single photon emission computed tomography (SPECT), or magnetic resonance imaging (MRI) system. 64. The method of any one of embodiments 61-63, wherein the detected signal is used to differentiate tumor tissue from non-tumor tissue, assess tumor progression, or evaluate response to therapy. 65. The method of any one of embodiments 61-64, wherein the tumor is a cancer selected from the group consisting of prostate cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer and a solid tumor cancer. 66. The method of embodiment 65, wherein the prostate cancer is a resistant form of prostate cancer. 67. The method of embodiments 65 or 66, wherein the prostate cancer is selected from the group consisting of Prostate-Specific Membrane Antigen (PSMA)-low, neuroendocrine, or androgen signaling inhibitor resistant form of prostate cancer.

[0188] 1. An antibody, wherein the antibody is linked to (i) a cytotoxic effector and (ii) a chelator, wherein a radionuclide is bound to the chelator via coordinate bonding. 2. The antibody of embodiment 1, wherein the antibody binds to a tumor cell surface antigen. 3. The 52KILPATRICK TOWNSEND 79434972 1antibody of embodiment 2, wherein the tumor cell surface antigen is selected from the group consisting of: Her2, Trop-2, Nectin-4, Folate receptor alpha, Tissue factor, C-Met, PSMA, EGFR, CEACAM5, CD30, CD79b, CD22, CD33, CD19, BCMA, and DLL3. 4. The antibody of any one of embodiments 1-3, wherein the antibody is selected from the group consisting of: trastuzumab, ado-trastuzumab, Sacituzumab, Datopotamab, Enfortumab, Mirvetuximab, Tisotumab, Telisotuzumab, J591, humanized J591, Cetuximab, Brentuximab, Polatuzumab, Inotuzumab, Gemtuzumab, Loncastuximab, Belantamab, and Tarlatamab. 5. The antibody of embodiment 4, wherein the antibody linked to a cytotoxic effector is selected from the group consisting of: Trastuzumab deruxtecan-nxki (Enhertu), Ado-trastuzumab emtansine (Kadcyla), Sacituzumab govitecan-hziy (Trodelvy), Datopotamab deruxtecan (Dato-DXd), Enfortumab vedotin-ejfv (Padcev), Mirvetuximab soravtansine-gynx (Elahere), Tisotumab vedotin-tftv (Tivdak), Telisotuzumab vedotin (Teliso-V), M9140, Brentuximab vedotin (Adcetris), Polatuzumab vedotin-piiq (Polivy), Inotuzumab ozogamicin (Besponsa), Gemtuzumab ozogamicin (Mylotarg), Loncastuximab tesirine-lpyl (Zynlonta), and Belantamab mafodotin-blmf (Blenrep). 6. The antibody of any one of embodiments 1-5, wherein the cytotoxic effector is linked to the antibody via a cleavable or non-cleavable linker via a cysteine sulfhydryl moiety in the antibody. 7. The antibody of embodiment 6, wherein the cleavable linker comprises valine-citrulline. 8. The antibody of any one of embodiments 1-7, wherein the cytotoxic effector is a chemotherapeutic agent. 9. The antibody of any one of embodiments 1-8, wherein the cytotoxic effector is a microtubule inhibitor, a DNA-damaging agent, or a polymerase inhibitor. 10. The antibody of any one of embodiments 1-8, wherein the cytotoxic effector is selected from the group consisting of an auristatin, Dolastatin-10, synthetic derivatives of the natural product Dolastatin-10, and maytansine or a maytansine derivative. 11. The antibody of any one of embodiments 1-8, wherein the cytotoxic effector is selected from the group consisting of Monomethylauristatin F (MMAF), Auristatin E (AE), Monomethylauristatin E (MMAE), vcMMAE, and vcMMAF.12. The antibody of embodiment 11, wherein monomethylauristatin E (MMAE) is conjugated to said antibody via a maleimidocaproyl-valine-citrulline-para-amino benzyloxy carbonyl (mc-vc-PAB) linker. 13. The antibody of any one of embodiments 1-8, wherein the cytotoxic effector is selected from the group consisting of mertansine (DM1), ravtansine (DM4), calicheamicin, a duocarmycin analog, a pyrrolobenzodiazepine dimer, Exatecan derivative for ADC (DXd), 7-ethyl-10-hydroxy-camptothecin (SN-38), topotecan, or a derivative thereof. 14. The antibody of embodiment 13, wherein the duocarmycin analog is selected from the group consisting of Duocarmycin Bl (DUMB1), duocarmycin B2 (DUMB2),53KILPATRICK TOWNSEND 79434972 1duocarmycin Cl (DUMC1), duocarmycin C2 (DUMC2), duocarmycin DI (DUMD1), duocarmycin D2 (DUMD2) and DSA. 15. The antibody of any one of embodiments 1-14, wherein the chelator comprises Macropa, mercaptoacetyltriglycine (MAG3), diethylenetriaminepentaacetic acid (DTP A), Deferoxamine (DFO), DFO*, DFO-Km, DFO-HOPO, l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane- 1,4,7, 10-tetraacetic acid (DOTA) or triethylenetetramine (TETA). 16. The antibody of any one of embodiments 1-15, wherein the chelator is linked to the antibody by a linker. 17. The antibody of embodiment 16, wherein the linker is linked to the antibody via an amine on a lysine side chain of the antibody. 18. The antibody of embodiment 16 or 17, wherein the linker comprises polyethylene glycol (PEG). 19. The antibody of embodiment 18, wherein the PEG comprises PEG2-10. 20. The antibody of embodiment 18, wherein the linker comprises PEG2, PEG4, PEGe, or PEGs. 21. The antibody of any of embodiments 1-20, wherein the radionuclide emits alpha particles. 22. The antibody of embodiment 21, wherein the radionuclide is selected from the group consisting of Actinium-225, Thorium-227, Bismuth-213, Astatine-211, Terbium-149, Lead-212 and Bismuth-213. 23. The antibody of any of embodiments 1-20, wherein the radionuclide emits beta particles.24. The antibody of embodiment 23, wherein the radionuclide is selected from the group consisting of Lutetium- 177, Terbium-161, Yttrium-90 and Lead-212. . The antibody of any of embodiments 1-20, wherein the radionuclide emits positrons. 26. The antibody of embodiment 25, wherein the radionuclide is selected from the group consisting of Zirconium-89, Copper-64, Gallium-68, Cerium-134 and Fluorine-18. 27. A pharmaceutical composition comprising a therapeutically-effective amount of the antibody of any one of embodiments 1-26. 28. A composition comprising: a first antibody, wherein the first antibody is linked to a cytotoxic effector; and a second antibody, wherein the second antibody is linked to a chelator, wherein a radionuclide is bound to the chelator via coordinate bonding. 29. The composition of embodiment 28, wherein the composition is a pharmaceutical composition comprising a therapeutically-effective amount of the first antibody and the second antibody. 30. The composition of embodiment 28 or 29, wherein the first antibody, or the second antibody, or both the first antibody and the second antibody, bind to a tumor cell surface antigen. 31. The composition of embodiment 30, wherein the tumor cell surface antigen is selected from the group consisting of: Her2, Trop-2, Nectin-4, Folate receptor alpha, Tissue factor, C-Met, PSMA, EGFR, CEACAM5, CD30, CD79b, CD22, CD33, CD19, BCMA, and DLL3. 32. The composition of any one of embodiments 28-31, wherein the first antibody or the second antibody are each selected from the group consisting of trastuzumab, ado-trastuzumab,54KILPATRICK TOWNSEND 79434972 1Sacituzumab, Datopotamab, Enfortumab, Mirvetuximab, Tisotumab, Telisotuzumab, J591, humanized J591, Cetuximab, Brentuximab, Polatuzumab, Inotuzumab, Gemtuzumab, Loncastuximab, Belantamab, Tarlatamab. 33. The composition of any one of embodiments 28-32, wherein the cytotoxic effector is linked to the first antibody via a cleavable or non-cleavable linker via a cysteine sulfhydryl moiety in the first antibody. 34. The composition of embodiment 33, wherein the cleavable linker comprises valine-citrulline. 35. The composition of any one of embodiments 28-34, wherein the cytotoxic effector is a chemotherapeutic agent. 36. The composition of any one of embodiments 28-35, wherein the cytotoxic effector is a microtubule inhibitor, a DNA-damaging agent, or a polymerase inhibitor. 37. The composition of any one of embodiments 28-35, wherein the cytotoxic effector is selected from the group consisting of an auristatin, Dolastatin-10, synthetic derivatives of the natural product Dolastatin-10, and maytansine or a maytansine derivative.38. The composition of any one of embodiments 28-35, wherein the cytotoxic effector is selected from the group consisting of Monomethylauri statin F (MMAF), Auristatin E (AE), Monomethylauristatin E (MMAE), vcMMAE, and vcMMAF. 39. The composition of embodiment 38, wherein monomethylauristatin E (MMAE) is conjugated to the first antibody via a maleimidocaproyl-valine-citrulline-para-amino benzyloxycarbonyl (mc-vc-PAB) linker.40. The composition of any one of embodiments 28-35, wherein the cytotoxic effector is selected from the group consisting of mertansine (DM1), ravtansine (DM4), calicheamicin, a duocarmycin analog, a pyrrolobenzodiazepine dimer, Exatecan derivative for ADC (DXd), 7-ethyl-10-hydroxy-camptothecin (SN-38), topotecan, or a derivative thereof. 41. The composition of embodiment 40, wherein the duocarmycin analog is selected from the group consisting of Duocarmycin Bl (DUMB1), duocarmycin B2 (DUMB2), duocarmycin Cl (DUMC1), duocarmycin C2 (DUMC2), duocarmycin D 1 (DUMD1), duocarmycin D2 (DUMD2) and DSA. 42. The composition of any one of embodiments 28-36, wherein the first antibody linked to the cytotoxic effector is selected from the group consisting of:Trastuzumab deruxtecan-nxki (Enhertu), Ado-trastuzumab emtansine (Kadcyla), Sacituzumab govitecan-hziy (Trodelvy), Datopotamab deruxtecan (Dato-DXd), Enfortumab vedotin-ejfv (Padcev), Mirvetuximab soravtansine-gynx (Elahere), Tisotumab vedotin-tftv (Tivdak), Telisotuzumab vedotin (Teliso-V), M9140, Brentuximab vedotin (Adcetris), Polatuzumab vedotin-piiq (Polivy), Inotuzumab ozogamicin (Besponsa), Gemtuzumab ozogamicin (Mylotarg), Loncastuximab tesirine-lpyl (Zynlonta), and Belantamab mafodotin-blmf (Blenrep). 43. The composition of any one of embodiments 28-42, wherein the chelator comprises Macropa, mercaptoacetyltriglycine (MAGs), diethylenetriaminepentaacetic acid 55KILPATRICK TOWNSEND 79434972 1(DTP A), Deferoxamine (DFO), DFO*, DFO-Km, DFO-HOPO, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) or l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA) or triethylenetetramine. 44. The composition of any one of embodiments 28-43, wherein the chelator is linked to the second antibody by a linker. 45. The composition of embodiment 44, wherein the linker is linked to the second antibody via an amine on a lysine side chain of the second antibody. 46. The composition of embodiment 44 or 45, wherein the linker comprises polyethylene glycol (PEG). 47. The composition of embodiment 46, wherein the PEG comprises PEG2-10. 48. The composition of embodiment 46, wherein the linker comprises PEG2, PEG4, PEGe, or PEGs. 49. The composition of any of embodiments 28-48, wherein the radionuclide emits alpha particles. 50. The composition of embodiment 49, wherein the radionuclide is selected from the group consisting of Actinium-225, Thorium-227, Bismuth-213, Astatine-211, Terbium-149, Lead-212 and Bismuth-213. 51. The composition of any of embodiments 28-48, wherein the radionuclide emits beta particles. 52. The composition of embodiment 51, wherein the radionuclide is selected from the group consisting of Lutetium- 177, Terbium-161, Yttrium-90 and Lead-212. 53. The composition of any of embodiments 28-48, wherein the radionuclide emits positrons. 54. The composition of embodiment 53, wherein the radionuclide is selected from the group consisting of Zirconium-89, Copper-64, Gallium-68, Cerium-134 and Fluorine-18. 55. A method of killing an antigenexpressing cancer cell, the method comprising contacting the antibody of any one of embodiments 1-26 or the pharmaceutical composition of embodiment 27 or the composition of embodiments 28-54 to the cancer cell, thereby killing the cancer cell. 56. The method of embodiment 55, wherein the cancer cell is in a human and the contacting comprises administering the antibody or the pharmaceutical composition or the composition to the human. 57. The method of embodiments 55 or 56, wherein the cancer is selected from the group consisting of prostate cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer and a solid tumor cancer. 58. The method of embodiment 57, wherein the prostate cancer is a resistant form of prostate cancer. 59. The method of embodiments 57 or 58, wherein the prostate cancer is selected from the group consisting of Prostate-Specific Membrane Antigen (PSMA)-low, neuroendocrine, or androgen signaling inhibitor resistant form of prostate cancer. 60. The method of embodiments 55 or 56, wherein the cancer is multiple myeloma or a hematological cancer. 61. The method of embodiment 60, wherein the multiple myeloma is relapsing or refractory multiple myeloma. 62. A method for imaging a tumor in a subject, the method comprising, administering the antibody of any one of embodiments 1-26 or the composition of embodiments 28-54 to a tumor; and detecting 56KILPATRICK TOWNSEND 79434972 1emitted signal from the radionuclide. 63. The method of embodiment 62, wherein the radionuclide is selected from the group consisting of Zirconium-89, Cerium-134, Copper-64, Gallium-68, and Fluorine-18. 64. The method of embodiments 62 or 63, wherein the emitted signal is detected by a positron emission tomography (PET) scanner, single photon emission computed tomography (SPECT), or magnetic resonance imaging (MRI) system. 65. The method of any one of embodiments 62-64, wherein the detected signal is used to differentiate tumor tissue from non-tumor tissue, assess tumor progression, or evaluate response to therapy. 66. The method of any one of embodiments 62-65, wherein the tumor is a cancer selected from the group consisting of prostate cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer and a solid tumor cancer. 67. The method of embodiment 66, wherein the prostate cancer is a resistant form of prostate cancer. 68. The method of embodiments 66 or 67, wherein the prostate cancer is selected from the group consisting of Prostate-Specific Membrane Antigen (PSMA)-low, neuroendocrine, or androgen signaling inhibitor resistant form of prostate cancer.EXAMPLESExample 1Combination therapy of [225Ac]Macropa-PEG4-YS5 and YS5-MMAE in vitro

[0189] Human prostate cancer cell lines 22Rvl, DU145, and LNCaP were cultured in in RPMI-1640 medium. Cells were seeded into 96-well plates at a density of 2,000 cells per well and allowed to adhere overnight. The following treatments were applied in separate experiments:1. Monotherapy Treatments:o [225Ac]Macropa-PEG4-YS5o YS5-MMAE2. Combination Therapy:o A combination of225Ac-YS5 and MMAE at varying dose levels.

[0190] For combination treatments, a matrix-based design was used, where serial dilutions of both agents were combined in varying proportions to cover a wide range of concentrations. Each condition was tested in triplicate wells. Plates were incubated for 72 hours at 37°C. At the end of the treatment period, cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega), which quantifies ATP levels as a marker of metabolically active cells. Briefly, 100 pL of CellTiter-Glo reagent was added to each well,57KILPATRICK TOWNSEND 79434972 1followed by incubation at room temperature for 10 minutes to stabilize the luminescent signal. Luminescence was measured using a microplate reader.

[0191] Dose-response curves were generated for both225Ac-Macropa-PEG4-YS5 and YS5-MMAE, and % of cell viability was calculated. The combination treatment data were analyzed using the Zero Interaction Potency (ZIP) model to evaluate synergy. The ZIP model compares the observed combination effect to the expected additive effect of the two drugs, with a synergy score calculated for each combination. A positive synergy score (>10 ZIP score) indicates a synergistic interaction, while a negative score suggests antagonism. The synergy analysis was performed using the SynergyFinder tool. All three prostate cancer cell lines (22Rvl, DU145, and LNCaP) exhibited areas with ZIP synergy scores greater than 10, confirming the presence of synergistic interactions between [225Ac]Macropa-PEG4-YS5 and YS5-MMAE in combination therapy (FIG. 1 A-C). ZIP analysis supports the potential of combining a-particle therapy and cytotoxic drug mechanisms for enhanced therapeutic efficacy.Cell cycle analysis:

[0192] The cell cycle analysis was performed to evaluate the effects of [225Ac]Macropa-PEG4-YS5, YS5-MMAE, and their combination on cell cycle progression and to identify potential mechanisms of action, such as cell cycle arrest. This assay provides insights into whether the treatments induce G2 / M arrest or other alterations that contribute to their antiproliferative effects.

[0193] 22Rvl prostate cancer cells were seeded into 6-well plates at a density of 2 * 105cells per well and allowed to adhere overnight. The cells were divided into four treatment groups: control (Untreated), [225Ac]Macropa-PEG4-YS5 (InCi / mL), YS5-MMAE (1 nM), and the combination of [225Ac]Macropa-PEG4-YS5 (lnCi / mL)+ YS5-MMAE (1 nM). Treatments were continued for 96 hours. At the end of the treatment period, cells were harvested by trypsinization, washed with cold phosphate-buffered saline (PBS), and fixed in 70% ice-cold ethanol overnight at -20°C. Fixed cells were washed with PBS to remove excess ethanol and incubated with PBS containing 50 pg / mL RNase A at 37°C for 30 minutes to degrade RNA. Following RNAse treatment, cells were stained with 50 pg / mL propidium iodide (PI) in the dark at room temperature for 15 minutes. Stained cells were analyzed using a flow cytometer equipped with a 488 nm laser, with PI fluorescence detected in the detector. The percentage of cells in the Gi / Go, S, and G2 / M phases based on DNA content histograms.58KILPATRICK TOWNSEND 79434972 1

[0194] Untreated cells exhibited a typical cell cycle distribution, with an average of 52.48 ± 2.65% of cells in the Gi / Go phase, 27.07 ± 6.14% in the S phase, and 20.45 ± 3.04% in the G2 / M phase (FIG. 2A, B). This reflects the normal growth pattern of untreated 22Rvl cells. Treatment with [225Ac]Macropa-PEG4-YS5 caused a reduction in the proportion of cells in the G1 / G0 phase (52.43 ± 0.84%) and S phase (18.31 ± 1.92%) while increasing the G2 / M phase population to 28.60 ± 2.35% (FIG. 2A, B). This shift indicates radiation-induced cell cycle arrest at G2 / M due to DNA damage caused by a-particle emission. YS5-MMAE, a microtubule-disrupting agent, significantly decreased the percentage of cells in the G1 / G0 phase (25.86 ± 7.35%) and increased the G2 / M phase population (48.88 ± 8.28%), consistent with its mechanism of action that arrests cells during mitosis (FIG. 2A, B). The S phase population remained relatively stable (26.59 ± 1.78%). The combination treatment ([225Ac]Macropa-PEG4-YS5 + YS5-MMAE) further enhanced the G2 / M phase arrest, with 49.73 ± 3.59% of cells in G2 / M, representing the highest among all treatment groups. The proportion of cells in the G1 / G0 phase was further reduced (24.40 ± 4.24%), and the S phase population remained similar to that seen with YS5-MMAE monotherapy (25.87 ± 1.09%) (FIG. 2A, B). These results highlight the synergistic interaction between the radiotherapy and cytotoxic drug, causing pronounced cell cycle disruption.

[0195] [225Ac]Macropa-PEG4-YS5 and YS5-MMAE induced distinct cell cycle alterations consistent with their respective mechanisms of action. The combination treatment caused the most significant G2 / M phase arrest, suggesting enhanced efficacy due to the complementary effects of radiation-induced DNA damage and microtubule disruption.DNA damage and apoptosis assay:

[0196] The y-H2AX staining assay was performed to assess DNA damage induced by the different treatments. y-H2AX is a sensitive marker of DNA double-strand breaks, which are a key mechanism of action for a-particle radiation emitted by [225Ac]Macropa-PEG4-YS5. This assay allowed quantification and visualization of treatment-induced DNA damage, providing insight into the efficacy of the radiolabeled and combination therapies at the molecular level.

[0197] The Annexin V / PI apoptosis assay was conducted to evaluate the extent of cell death and the mode of cell death (early apoptosis, late apoptosis, or necrosis) induced by the treatments. Apoptosis is a major mechanism through which therapeutic agents exert their anti-cancer effects, and combining a-particle radiation with the cytotoxic agent MMAE was hypothesized to enhance cell death. These assays together provided complementary data on the mechanisms underlying the therapeutic effects of the tested agents.59KILPATRICK TOWNSEND 79434972 1DNA damage study

[0198] 22Rvl cells were seeded on glass coverslips in 6-well plates and allowed to adhere overnight. The cells were treated with YS5-MMAE (InM), [225Ac]Macropa-PEG4-YS5 (InCi / mL), or a combination of [225Ac]Macropa-PEG4-YS5 and YS5-MMAE for 96 hours. After treatment, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 5% bovine serum albumin (BSA) for 30 minutes. Cells were incubated with anti-phospho-H2AX (y-H2AX) antibody (1:500 dilution) overnight at 4°C, followed by a secondary antibody conjugated to Alexa Fluor 488 (1 : 1000 dilution) for 1 hour at room temperature. Nuclei were counterstained with DAPI, and coverslips were mounted using an antifade mounting medium. Fluorescent images were captured using a confocal microscope, and y-H2AX foci were quantified using image analysis software.

[0199] The DNA damage induced by different treatments was assessed by quantifying y-H2AX foci per nucleus, a marker of double-strand DNA breaks. Untreated control cells exhibited a low baseline level of foci, with an average of 0-2 foci per cell, reflecting minimal endogenous DNA damage (FIG.3A, B). Cells treated with the antibody-drug conjugate (ADC) YS5-MMAE demonstrated an increase in y-H2AX foci, with 3-7 foci per cell on average, indicating moderate DNA damage caused by the cytotoxic drug. Treatment with [225Ac]Macropa-PEG4-YS5 significantly increased the number of y-H2AX foci per nucleus compared to both the control and ADC groups. On average, cells treated with [225Ac]Macropa-PEG4-YS5 displayed 5-14 foci per nucleus, suggesting extensive DNA double-strand breaks induced by a-particle-mediated radiation (FIG. 3A, B). The combination treatment of [225Ac]Macropa-PEG4-YS5 and YS5-MMAE produced the highest levels of DNA damage, with 10-21 foci per cell observed across the majority of nuclei (FIG.3A, B). This synergistic effect highlights the enhanced DNA-damaging capability of the combination treatment, leveraging both a-particle-induced radiation and the cytotoxic action ofMMAE.Quantification of apoptotic cells

[0200] 22Rvl cells were seeded in 6-well plates at a density of 2 * 105cells per well and allowed to adhere overnight. Cells were treated YS5-MMAE, [225Ac]Macropa-PEG4-YS5, or a combination of [225Ac]Macropa-PEG4-YS5 and YS5-MMAE for 96 hours. After treatment, both adherent and floating cells were collected by trypsinization and centrifugation at 300 g for 5 minutes. Cells were stained 5 pL of Annexin V-FITC and 5 pL of propidium iodide (PI,60KILPATRICK TOWNSEND 79434972 150 pg / mL) for 15 minutes in the dark at room temperature, and the samples were analyzed by flow cytometry.

[0201] The untreated (control) group displayed negligible apoptosis, with an average of 2.09% of cells in the early apoptotic phase, 1.02% in the late apoptotic phase, and 1.00% classified as dead cells (FIG. 3C, 3D). Treatment with [225Ac]Macropa-PEG4-YS5 induced a significant increase in apoptosis, with an average of 20.43% of cells in the early apoptotic phase, 8.53% in the late apoptotic phase, and 3.59% classified as dead cells, indicating the substantial impact of a-particle radiation on cell death (FIG.3C, 3D). YS5-MMAE treatment also increased apoptosis, with an average of 16.73% of cells in the early apoptotic phase, 6.24% in the late apoptotic phase, and 2.85% classified as dead cells, consistent with the cytotoxic effects of the MMAE payload (FIG. 3C, 3D). The combination treatment produced the most dramatic effect, with minimal early apoptosis (1.24%) but a higher proportion of cells undergoing late apoptosis (7.78%) and a striking increase in dead cells (44.33%), suggesting enhanced cell-killing efficacy through synergistic mechanisms of action.Acute and chronic toxicity analysis of monotherapy and combination therapy

[0202] Acute Toxicity Study: Healthy nude mice were randomized into four treatment groups (n=5 per group): saline (control), YS5-MMAE (1.8 mg / kg), [225Ac]Macropa-PEG4-YS5 (0.125 pCi), and the combination of [225Ac]Macropa-PEG4-YS5 and YS5-MMAE.Treatments were administered as a single intravenous injection. Body weights were recorded every 2-3 days for 15 days post-injection to monitor acute systemic effects.

[0203] Chronic Toxicity Study: The same treatment groups were used for chronic toxicity evaluation, with body weights recorded every 3-7 days over a period of 100 days. At the end of the study, blood samples were collected via cardiac puncture under anesthesia for complete blood count (CBC) and serum chemistry analyses. Organs were harvested for histopathological analysis. Blood samples were analyzed for WBC, neutrophils, lymphocytes, monocytes, eosinophils, basophils, RBCs, and platelet counts using an automated hematology analyzer. Liver and kidney function analysis: Serum samples were analyzed for ALT, AST, creatinine, albumin, BUN, ALP, and total bilirubin using a clinical chemistry analyzer to evaluate liver and kidney function.

[0204] Results: Acute Toxicity :Body weight measurements over 15 days (FIG. 4A) showed no significant weight loss in any treatment group compared to the saline control. All groups maintained stable weights, suggesting minimal acute systemic toxicity following single-dose administration.61KILPATRICK TOWNSEND 79434972 1

[0205] Chronic Toxicity: Over 100 days, body weight measurements (FIG. 4B) remained stable across all treatment groups, with no significant differences between saline, YS5-MMAE, [225Ac]Macropa-PEG4-YS5, and combination therapy. This indicates good tolerability and minimal chronic systemic toxicity.

[0206] Complete Blood Count (CBC): CBC data (FIG. 4C) demonstrated that WBC, neutrophil, lymphocyte, monocyte, eosinophil, basophil, RBC, and platelet counts were within the normal range for all groups. No significant hematological abnormalities were observed, confirming minimal hematotoxicity.

[0207] Liver and Kidney Function: Serum analysis (FIG. 4D) revealed no significant changes in ALT, AST, creatinine, albumin, BUN, ALP, or total bilirubin levels among the treatment groups compared to the control. All values were within normal physiological ranges.

[0208] Histopathology provided additional evidence of tolerability. In the acute cohort, all major organs were unremarkable, with the exception of three of five combination-treated kidneys, which displayed mild tubular injury that did not translate into functional impairment, as reflected by stable BUN and creatinine values and continued weight gain (FIG. 5). The chronic study, provided in FIG. 6, representative histology of major organs collected 120 days after treatment (chronic study), hematoxylin-and-eosin sections of heart, lung, liver, spleen, kidney, and bone marrow from mice receiving saline, YS5-MMAE (1.8 mg / kg),[225Ac]Macropa-PEG4-YS5 (4.6 kBq), or the combination of YS5-MMAE + [225Ac]Macropa-PEG4-YS5 are shown (n = 5 per group). In the chronic cohort (FIG. 6), only minimal renal injury (cumulative score of 1-2) was noted across all treatment groups (saline, YS5-MMAE, [225Ac]Macropa-PEG4-YS5, and combination). Rare focal perivascular inflammation was observed in the lungs (1 / 5 animals) from the saline, YS5-MMAE, and [225Ac]Macropa-PEG4-YS5 groups, but was absent in the combination group. Additionally, focal periductal chronic inflammation was detected in 1 / 5 animals from the YS5-MMAE and combination groups. These lesions were limited, non-progressive, and unaccompanied by any functional abnormalities (FIG. 6). Collectively, these findings indicate that the combination of [225Ac]Macropa-PEG4-YS5 and YS5-MMAE did not induce additional toxicity despite enhanced in vitro therapeutic efficacy, supporting further therapeutic development.Combination therapy of [225Ac]Macropa-PEG4-YS5 and YS5-MMAE

[0209] Male nude mice (6-8 weeks old) were subcutaneously implanted with 22Rvl prostate cancer cells (2.5 * 106cells per mouse) mixed with Matrigel (1 : 1 ratio) in the right flank. Tumors were allowed to grow until they reached an average volume of -100 mm3before treatment initiation. Mice were randomly divided into six groups (n=5 per group): (1) saline 62KILPATRICK TOWNSEND 79434972 1(control), (2) YS5-MMAE (1.8 mg / kg), (3) [225Ac]Macropa-PEG4-YS5 (0.06 pCi), (4)[225Ac]Macropa-PEG4-YS5 (0.125 pCi), (5) combination of YS5-MMAE (1.8 mg / kg) with [225Ac]Macropa-PEG4-YS5 (0.06 pCi), and (6) combination of YS5-MMAE (1.8 mg / kg) with [225Ac]Macropa-PEG4-YS5 (0.125 pCi). Treatments were administered as a single intravenous injection in 100 pL of saline. Tumor volumes were measured three times weekly using calipers. Body weights were recorded weekly to monitor systemic toxicity. This study aimed to evaluate the therapeutic efficacy and tolerability of these treatments in a prostate cancer model.

[0210] Results: The results from FIG. 7A and FIG. 7B collectively demonstrate the therapeutic efficacy of the tested treatment regimens in controlling tumor growth and improving survival in mice bearing prostate cancer tumors. In FIG. 7A, mice treated with saline exhibited the most rapid tumor growth, with volumes reaching the highest levels among all groups, indicating the aggressive nature of the tumor in the absence of treatment. The YS5-MMAE group showed moderate tumor growth suppression compared to the saline group, but tumor volumes were still significantly larger than those observed in groups treated with radiolabeled antibodies. The dose of YS5-MMAE (1.8 mg / kg) is a subtherapeutic dose, intentionally selected to combine it with radiolabeled antibody. Treatment with [225Ac]Macropa-PEG4-YS5 (0.06 pCi) alone led to significant tumor suppression relative to both the saline and YS5-MMAE groups, with further tumor reduction observed in the 0.125 pCi radiolabeled antibody group, indicating a dose-dependent effect. Combination therapies demonstrated the greatest therapeutic efficacy, with the combination of YS5-MMAE and [225Ac]Macropa-PEG4-YS5 (0.06 pCi) showing marked tumor volume reduction compared to monotherapies. Notably, the combination of YS5-MMAE and [225Ac]Macropa-PEG4-YS5 (0.125 pCi) resulted in the most significant tumor suppression, with some mice achieving complete tumor regression by the end of the study.

[0211] In FIG. 7B, survival analysis corroborates the tumor volume results, revealing clear differences in median survival among the treatment groups. Mice treated with saline exhibited the shortest median survival of 21 days, while YS5-MMAE monotherapy extended survival slightly to 22.5 days. Radiolabeled antibody treatments improved survival significantly, with median survival of 32.5 days for the 0.06 pCi group and 46 days for the 0.125 pCi group. The combination therapies further enhanced survival outcomes, with the YS5-MMAE and [225Ac]Macropa-PEG4-YS5 (0.06 pCi) group achieving a median survival of 46 days, comparable to the higher-dose radiolabeled monotherapy. The combination of YS5-MMAE and [225Ac]Macropa-PEG4-YS5 (0.125 pCi) resulted in the greatest survival 63KILPATRICK TOWNSEND 79434972 1benefit, with a median survival of 84.5 days and one censored subject due to long-term survival.FIG. 7C: Body weight analysis revealed a slight decrease in body weight across all treatment groups at early time points post-treatment, likely due to the acute effects of therapy.However, body weights were gradually regained over the course of the study, and no significant long-term weight loss was observed in any group, indicating good tolerability of the treatments. The combination therapy tested on disseminated tumor models of prostate cancer also showed a promising antitumor response. Bioluminescence imaging (FIG. 8A) and survival curve (FIG. 8B), confirms the effective treatment of tumors using combination of YS5-MMAE and [225Ac]Macropa-PEG4-YS5.R-ADC Combination and Radiolabeling to Produce [225Ac]Macropa-PEG4-YS5-MMAE

[0212] From the combination therapy of [225Ac]Macropa-PEG4-YS5 and YS5-MMAE, it was hypothesized that a similar strategy could be employed in a single molecular species, combining Actinium-225 and MMAE on one IgG molecule (FIG. 9A). YS5 with mc-vc-pab-MMAE was conjugated following partial reduction to generate the ADC with 3.4 to 3.6 MMAE molecules per antibody molecule. Subsequent conjugation with Macropa-PEG4-TFP resulted in 0.91 chelators per Macropa-PEG4-YS5-MMAE (FIG. 9B).

[0213] Size exclusion chromatography showed no aggregation of Macropa-PEG4-YS5-MMAE (FIG. 10). To evaluate changes in antigen-binding potential following Macropa-PEG4 conjugation, a flow cytometry -based binding assay was performed. The dissociation constant (Ka) values for YS5-MMAE and Macropa-PEG4-YS5-MMAE were determined to be 3.2 nM and 3.0 nM, respectively (FIG. 11).

[0214] Subsequently,225Ac radiolabeling of the Macropa-PEG4-YS5-MMAE was performed, with the final product obtained in >95% radiochemical purity (FIG. 12A-B). The isolated yield was 68% (n = 3), with a specific activity of 6.3 kBq / pg for [225Ac]Macropa-PEG4-YS5-MMAE (R-ADC). For purity assessment, small aliquots were spotted onto iTLC-SG strips and developed using 10 mM EDTA buffer (pH 5.5). Radioactivity was quantified at the origin versus the solvent front after secular equilibrium (5 to 8 hrs) to determine radiochemical purity, with acceptable purity defined as greater than 95%. iTLC assessment is shown in FIG. 12A-B, of the dual modality radionuclide-conjugated antibody-drug conjugate (R-ADC) before and after purification. FIG. 12 A: i-TLC of the crude reaction product, analyzed immediately after synthesis (left) and at secular equilibrium (right). The immediate scan performed for radiolabeling reactions shows two peaks, with 95.67% of activity64KILPATRICK TOWNSEND 79434972 1corresponding to the desired product and 4.33% attributed to free radioisotope. At secular equilibrium (24 h), the purity of the radiolabeled ADC was found to be 100%. FIG. 12B: i-TLC of the product after purification using centrifugal filters, analyzed immediately after purification (left) and at secular equilibrium (right). The immediate scan shows 81.80% of activity in the desired peak and 18.20% corresponding to free isotope. After reaching secular equilibrium, the purified product demonstrates 99.87% purity in the desired peak, confirming effective purification.Magnetic Beads Binding Assay

[0215] Magnetic beads coated with CD46 protein were incubated with [225Ac]Macropa-PEG4-YS5-MMAE to measure binding affinity. Blocking studies were performed by adding an excess of unlabeled YS5 antibody, and binding was compared to beads without CD46.

[0216] (FIG. 13A) Magnetic Beads Assay: [225Ac]Macropa-PEG4-YS5-MMAE exhibited high binding affinity (84.3±8.2 %) to CD46-coated beads. Blocking with excess unlabeled YS5 antibody reduced binding to 29 ±3.5%, confirming specificity. Binding further decreased (-20.0 ±1.3%) when CD46 was absent, indicating dependence on CD46 for targeting. The R-ADC demonstrated strong binding to CD46-coated magnetic beads (84.3±8.2%), significantly reduced by excess YS5 or in non-antigen-treated beads.[225Ac]Macropa-PEG4-YS5-MMAE Stability Studies in Serum and PBS

[0217] [225Ac]Macropa-PEG4-YS5-MMAE was incubated in human serum and PBS buffer at 37°C. Samples were collected at different time points (4 hours, Day 1, 2, 4, 7, 10, and 15) and analyzed by instant thin-layer chromatography (i-TLC) to assess radiochemical stability.

[0218] (FIG. 13B) Stability in Serum: [225Ac]Macropa-PEG4-YS5-MMAE maintained >95% radiochemical stability in human serum over 15 days, demonstrating excellent in vitro integrity under physiological conditions. No aggregation was seen in the purified product by size exclusion HPLC (supporting figure SI 5).

[0219] (FIG. 13B) Stability in PBS: Stability in PBS buffer was similarly high, with >95% radiochemical purity maintained for 15 days, indicating robustness of the conjugate under buffer conditions.

[0220] The stabilities of [225Ac]Macropa-PEG4-YS5-MMAE in serum and PBS are shown overlain in FIG. 13B.65KILPATRICK TOWNSEND 79434972 1Colony-Forming Assay

[0221] 22Rvl cells (200 per well) were seeded in 6-well plates and treated with radiolabeled or non-radiolab eled conjugates at various concentrations for 96 hours. Post-treatment, the cells were washed and incubated for two weeks to allow colony formation. Colonies were then fixed, stained, and quantified. Dose-response curves were generated, and ICso values were calculated to evaluate the cytotoxicity of each treatment.

[0222] FIG. 13C: Comparison of Radiolabeled Conjugates: [225Ac]Macropa-PEG4-YS5-MMAE exhibited significantly higher cytotoxicity (ICso = 0.21 ± 0.03 kBq / mL) compared to [225Ac]Macropa-PEG4-YS5 (ICso = 1.02 ± 0.09 kBq / mL), highlighting the enhanced efficacy of the MMAE payload (FIG. 13C).

[0223] FIG. 13D Cytotoxicity of Radiolabeled and Non-Radiolabeled Conjugates: YS5-MMAE, Macropa-PEG4-YS5-MMAE, and [225Ac]Macropa-PEG4-YS5-MMAE were compared for their ability to inhibit colony formation. The treatment given at the same molar activities resulted in a decrease in cell viability. The [225Ac]Macropa-PEG4-YS5-MMAE showed potent killing action with lowest IC50 concentration (4.6 pM), compared to YS5-MMAE (192.8 pM) and Macropa-PEG4-YS5-MMAE (424.6 pM). Also shown in units of log(Bq / mL) in FIG. 13D, R-ADC [225Ac]Macropa-PEG4-YS5-MMAE again showed the strongest effect, with an ICso of 4.7 ± 0.7 pM compared to 197.0 ± 44.2 pM for YS5-MMAE and 398.0 ± 40.7 pM for Macropa-PEG4-YS5-MMAE (FIG. 13D).

[0224] FIG. 13E: Cell killing comparison of specific activities: The radiolabeled ADC [225Ac]Macropa-PEG4-YS5-MMAE was added to cells to study colony forming assay. Here molecules with two specific activities (0.12 pCi / pg and 0.043 pCi / pg) were tested. The treatment of these two agents at the same radioactivity dose was given to cells. ICso values for the high-specific activity (0.12 pCi / pg) and low-specific activity (0.043 pCi / pg) conjugates were 4.6 pM and 13.7 pM, respectively, demonstrating a higher potency with increased specific activity.Cell Binding Assay

[0225] The binding specificity of [225Ac]Macropa-PEG4-YS5-MMAE was assessed using 22Rvl prostate cancer cells. 22Rvl cells (1 million) were incubated with [225Ac]Macropa-PEG4-YS5-MMAE (100 ng) for 2 hours at 37°C in CO2 incubator. Blocking was performed by adding 100-fold molar excess of unlabeled YS5 antibody along with [225Ac]Macropa-PEG4-YS5-MMAE. CD46 knockout (KO) cells were also tested for specificity.66KILPATRICK TOWNSEND 79434972 1

[0226] (FIG. 13F) Cell Binding Assay: Incubation with 22Rvl cells showed 4% binding of [225Ac]Macropa-PEG4-YS5-MMAE. Blocking with a 100-fold molar excess of cold YS5 reduced binding to 0.14%, and CD46 KO cells exhibited negligible binding (0.01%), confirming target specificity.

[0227] [225Ac]Macropa-PEG4-YS5-MMAE demonstrated high stability in both serum and PBS, maintaining radiochemical purity over 15 days, a key attribute for in vivo applications. The magnetic beads and cell binding assays confirm the specificity of the conjugate for CD46, with minimal off-target binding. Blocking experiments and CD46 KO validation further highlight the CD46-targeting mechanism.

[0228] These results establish the stability and targeting specificity of [225Ac]Macropa-PEG4-YS5-MMAE, supporting its potential as a targeted radiopharmaceutical for theranostic applications in CD46-expressing cancers.Further Modulation of Specific Activity Enhances in vitro Cytotoxicity while Maintaining in vivo Tumor Targeting of [225Ac]Macropa-PEG4-YS5-MMAE

[0229] Radiolabeling conditions were adjusted by varying both the antibody mass and the input activity of225Ac so that the specific activity of the resulting R-ADC could be precisely controlled (Table 9).Table 9: Effect of Antibody Mass and225Ac Activity on Radiolabeling Yield and Specific Activity of [225Ac]Macropa-PEG4-YS5-MMAE.

[0230] When 120 pg of Macropa-PEG4-YS5-M AE was reacted with 1110 kBq of225Ac, the purified product contained 755 kBq of radioactivity, corresponding to a specific activity of 6.3 kBq / pg. Using 80 pg with 740 kBq gave 370 kBq (4.4 kBq / pg, 50% recovery), while 148 kBq produced 129.5 kBq (1.6 kBq / pg, 87.5% recovery). Labeling 100 pg with 15.2 kBq 67KILPATRICK TOWNSEND 79434972 1gave 11.2 kBq (0.11 kBq / pg, 74% recovery). These results show that either altering the starting antibody-to-radionuclide ratio or supplementing the reaction mixture with cold Macropa-PEG4-YS5-MMAE provides straightforward ways to tailor the specific activity of the final conjugate.

[0231] To assess the impact of ADC mass, high-specific-activity [225Ac]Macropa-PEG4-YS5-MMAE (6.3 kBq / pg) was mixed with increasing amounts of cold Macropa-PEG4-YS5-MMAE to reduce specific activity. Colony-forming assays showed that lower specific activity enhanced cytotoxicity (FIG. 13E). The ICso for R-ADC with 6.3 kBq / pg specific activity was 5.8 ± 0.9 Bq / mL, and progressively decreased to 1.8 ± 0.6, 1.1 ± 0.4, 0.8 ± 0.3, and 0.2 ± 0.1 Bq / mL at 3.44, 1.14, 0.37, and 0.11 kBq / pg, respectively. These results indicate that increasing total ADC mass improves in vitro cell killing.

[0232] Subsequently, the immunoreactivity of [225Ac]Macropa-PEG4-YS5-MMAE and the effect of specific activity on cell binding in 22Rvl and CD46-knockout cells (22Rvl-CD46-KO) following a 1-hour incubation was evaluated (FIG. 13F). At a 340 Bq dose with 6.3 kBq / pg specific activity, binding to 22Rvl cells was 4.0 ± 0.3%. A 100-fold excess of unlabeled YS5 reduced binding to 0.14 ± 0.06%, and CD46-KO cells showed negligible uptake (0.01 ± 0.01%). Using the same dose at lower specific activity (0.1 kBq / pg) decreased binding to 1.15 ± 0.2%, consistent with partial competitive saturation by excess cold conjugate (FIG. 13F).

[0233] Since excess cold Macropa-PEG4-YS5-MMAE enhanced in vitro cytotoxicity but reduced uptake, it was evaluated whether this mass effect alters in vivo distribution. 22Rvl subcutaneous tumor-bearing mice received either (i) [225Ac]Macropa-PEG4-YS5-MMAE at 4.6 kBq (6.3 kBq / pg) or (ii) the same activity with 43 pg of additional cold conjugate (0.1 kBq / pg) (FIG. 14A-B). Biodistribution analysis on days 4 and 7 after injection (FIG. 14A-B), Table 10, and Table 11) showed no significant difference in tumor uptake: 10.2 ± 2.0 vs.11.1 ± 2.8 %IA / g (day 4) and 8.7 ± 2.8 vs. 7.7 ± 2.3 %IA / g (day 7). Notably, tumor uptake increased to 14.8 ± 15.3 %IA / g on day 10 with the low-specific-activity formulation (FIG.15, Table 12), confirming that added antibody mass did not impair tumor targeting in vivo.Table 10: Biodistribution comparison of high specific activity (6.3 kBq / pg) and low specific activity (0.1 kBq / pg) [225Ac]Macropa-PEG4-YS5-MMAE in mice (n = 4) at day 4 postinjection.68KILPATRICK TOWNSEND 79434972 1Table 11: Biodistribution comparison of high specific activity (6.3 kBq / pg) and low specific activity (0.1 kBq / pg) [225Ac]Macropa-PEG4-YS5-MMAE in mice (n = 3) at day 7 postinjection.69KILPATRICK TOWNSEND 79434972 1Table 12: Biodistribution profile of low specific activity (0.1 kBq / pg) [225Ac]Macropa-PEG4-YS5-MMAE 22Rvl bearing mice (n = 3) at day 10 post-injection.70KILPATRICK TOWNSEND 79434972 1Prostate cancer tumor model treatment and monitoring

[0234] Male nude mice (7 weeks old) were inoculated subcutaneously with 22Rvl prostate cancer cells (2.5 million) mixed with Matrigel (1 : 1) to form tumors. Tumors were allowed to grow to approximately 100 mm3before treatment. Mice were randomized into groups and treated with various agents:Group A: Saline (control).Group B: YS5-MMAE (1.8 mg / mL).Group C: Macropa-PEG4-YS5-MMAE (1.8 mg / mL).Group D: [225Ac]Macropa-PEG4-YS5 (0.06 pCi).Group E: [225Ac]Macropa-PEG4-YS5 (0.125 pCi).Group F: [225Ac]Macropa-PEG4-YS5-MMAE (0.06 pCi, 1.8 mg / kg).Group G: [225Ac]Macropa-PEG4-YS5-MMAE (0.125 pCi, 1.8 mg / kg).

[0235] The radiolabeled ADC treatment groups received either 0.06 or 0.125 pCi dose, along with the cold macropa-PEG4-YS5-MMAE to maintain 1.8 mg / kg dose of ADC. Tumor volumes were measured twice weekly and plotted as volume (mm3) versus days posttreatment.

[0236] Tumor Growth Curves: Rapid tumor growth was observed in control mice (FIG. 18A), indicating no therapeutic effect. YS5-MMAE (FIG. 18B) and Macropa-PEG4-YS5-MMAE (FIG. 18C) showed modest tumor growth inhibition, demonstrating limited efficacy of MMAE-conjugated antibodies alone. [225Ac]Macropa-PEG4-YS5, radiolabeled antibody without MMAE demonstrated tumor suppression at both 0.06 pCi (FIG. 18D) and 0.125 pCi (FIG. 18E), highlighting the therapeutic effect of Ac-225-mediated a-particle therapy. R-ADC [225Ac]Macropa-PEG4-YS5-MMAE with MMAE exhibited superior tumor suppression compared to radiolabeled antibodies without MMAE. Tumor suppression was greater at 0.125 pCi (FIG. 18G) than 0.06 pCi (FIG. 18F), demonstrating dose-dependent efficacy.[225Ac]Macropa-PEG4-YS5-MMAE (0.125 pCi, 1.8 mg / kg) (FIG. 18G) showed the highest level of tumor suppression, confirming the synergistic effects of Ac-225 and MMAE.71KILPATRICK TOWNSEND 79434972 1

[0237] This study demonstrates the enhanced therapeutic potential of the dual-modality R-ADC [225Ac]Macropa-PEG4-YS5-MMAE compared to single-modality agents. Radiolabeled ADCs with Ac-225 alone suppressed tumor growth effectively but did not match the efficacy of the combination of Ac-225 and MMAE. The highest dose of [225Ac]Macropa-PEG4-YS5-MMAE (0.125 pCi, 1.8 mg / kg) demonstrated the greatest tumor suppression, confirming the importance of combining radiation and cytotoxic drug mechanisms.Body weights and Kaplan Meier curves

[0238] The body weight of the mice in all treatment groups, including those receiving radiolabeled conjugates, remained stable throughout the study, with no significant weight loss observed (FIG. 19A). This indicates minimal systemic toxicity and excellent tolerability of the treatments.

[0239] Analysis of tumor volume and survival highlighted the therapeutic efficacy of both radiolabeled and non-radiolab eled agents. The control group treated with saline exhibited rapid tumor progression, with a median survival of only 24 days (FIG. 19B). Treatment with the non-radiolab eled agents YS5-MMAE and Macropa-PEG4-YS5-MMAE, both at a dose of 1.8 mg / mL, showed modest tumor growth suppression. These treatments extended the median survival slightly to 27 days, indicating limited therapeutic efficacy (FIG. 19B).

[0240] In contrast, radiolabeled antibodies without MMAE demonstrated greater tumor control and survival benefits in a dose-dependent manner. Treatment with [225Ac]Macropa-PEG4-YS5 at 0.06 pCi reduced tumor growth compared to the non-radiolab eled groups and extended the median survival to 30 days (FIG. 19B). Increasing the dose to 0.125 pCi further enhanced tumor suppression, with this group achieving a median survival of 64 days, highlighting the significant impact of Ac-225-mediated a-particle therapy.

[0241] The radiolabeled ADC [225Ac]Macropa-PEG4-YS5-MMAE, which combines the effects of radiation and the cytotoxic MMAE payload, exhibited the most potent therapeutic effects. At 0.06 pCi (1.8 mg / kg), the R-ADC demonstrated superior tumor suppression compared to radiolabeled antibodies without MMAE, resulting in a median survival of 68 days (FIG. 19B). At a higher dose of 0.125 pCi (1.8 mg / kg), [225Ac]Macropa-PEG4-YS5-MMAE showed the greatest tumor suppression and prolonged survival, with the median survival remaining undefined due to long-term survival benefits in this group.

[0242] The tumor volume data from the previous figure corroborates these findings, as tumors in this group were effectively controlled or eradicated over the study duration.72KILPATRICK TOWNSEND 79434972 1

[0243] These results demonstrate the superior efficacy of [225Ac]Macropa-PEG4-YS5-MMAE compared to non-radiolab eled agents and radiolabeled antibodies without MMAE. The data highlight the synergistic effects of radiation and the MMAE payload, providing strong evidence for the potential of this dual-modality therapeutic agent in the treatment of prostate cancer.PET Imaging with [134Ce]Macropa-PEG4-YS5-MMAE and [89Zr]Macropa-PEG4-YS5-MMAE

[0244] To assess imaging potential, two approaches were employed: direct radiolabeling of the Macropa-conjugated ADC with134Ce, and DFO*-based labeling of the ADC with89Zr.[134Ce]Macropa-PEG4-YS5-MMAE was radiolabeled with high purity confirmed by iTLC (FIG. 17A-B)

[0245] Male nude mice bearing subcutaneous 22Rvl prostate cancer tumors were injected intravenously with [134Ce]Macropa-PEG4-YS5-MMAE (3.5 pCi / pg, 60% yield, >99% purity, FIG. 23). PET imaging was performed at multiple time points post-injection (24 h, 48 h, 96 h, and 168 h) to evaluate the biodistribution and pharmacokinetics of the radiolabeled agent. At the endpoint (day 7 post-injection), mice were sacrificed, and major organs, including the tumor, were collected. The activity of [134Ce]Macropa-PEG4-YS5-MMAE in each organ was quantified using a gamma counter and expressed as the percentage of injected dose per gram of tissue (%ID / g).

[0246] The PET imaging results show high and specific uptake of [134Ce]Macropa-PEG4-YS5-MMAE in the 22Rvl tumors at all time points (FIG. 23). Tumor uptake was prominent at 24 hours and remained high through 168 hours post-injection, indicating sustained retention and effective targeting of the tumor. Minimal off-target accumulation was observed in non-tumor tissues, demonstrating favorable pharmacokinetics and targeting specificity. The ex vivo biodistribution study (FIG. 25), provided in Table 13, confirmed the imaging results. The tumor showed significant retention of [134Ce]Macropa-PEG4-YS5-MMAE, with an activity of 18.2 ± 8.2 %ID / g, indicating efficient targeting. Among non-tumor tissues, the liver and spleen exhibited the highest uptake, which is consistent with the expected clearance pathway for antibody-based agents. Other organs, including the kidneys, lungs, heart, and muscle, showed negligible activity, further supporting the favorable biodistribution of the radiolabeled ADC.73KILPATRICK TOWNSEND 79434972 1Table 13: Biodistribution of the [134Ce]Macropa-PEG4-YS5 and [134Ce]Macropa-PEG4-YS5-MMAE in mice bearing 22RV1 subcutaneous xenografts (168 h post-injections, n=5). Values are in % injected activity per gram

[0247] The molecular imaging and biodistribution studies demonstrate the robust tumortargeting capability and favorable pharmacokinetic profile of [134Ce]Macropa-PEG4-YS5-MMAE in a subcutaneous 22Rvl prostate cancer model. The high and sustained tumor uptake observed in PET images across multiple time points reflects the stability of the radiolabeled conjugate and its effective binding to CD46, a target expressed in 22Rvl tumors. Ex vivo biodistribution data further corroborate the imaging results, confirming significant tumor retention and clearance predominantly through the liver and spleen, consistent with74KILPATRICK TOWNSEND 79434972 1antibody-based agent pharmacology. Together, these results support the potential utility of [134Ce]Macropa-PEG4-YS5-MMAE as a theranostic agent, combining imaging and therapeutic capabilities for prostate cancer. Notably, tumor and liver uptake were similar for drug- and non-drug-conjugated Macropa-YS5 ([134Ce]Macropa-PEG4-YS5), indicating that MMAE attachment did not compromise targeting (FIG. 24 and FIG. 25).

[0248] [89Zr]DFO* -YS5-MMAE was used for immunoPET. The ADC was conjugated with DFO*, and89Zr was performed. Binding of [89Zr]DFO*-YS5-MMAE to CD46-positive 22Rvl cells was 76%, and dropped to 5.5% with excess cold YS5 (FIG. 26). PET imaging revealed progressive tumor accumulation: 18.4 ± 7.6 %IA / g at 24 h, reaching 37.7 ± 6.6 %IA / g at 168 h (FIG. 27A, 27B, Table 14). DU145 models showed similarly consistent uptake (FIG. 28A-B, Table 15), confirming that CD46 targeting is preserved in both imaging formats and tumor contexts. Uptake of the [89Zr]DFO*-YS5-MMAE in the 22Rvl tumor model followed a similar uptake pattern to the PET imaging agent [89Zr]DFO-YS5 (FIG. 29A-B, Table 16).Table 14: Biodistribution of the [89Zr]DFO*-MMAE in mice bearing 22RV1 subcutaneous xenografts (168 h post injections, n=4).75KILPATRICK TOWNSEND 79434972 1Table 15: Biodistribution of the [89Zr]DFO*-MMAE in mice bearing DU145 subcutaneous xenografts (168 h post injections, n=4).Table 16: Biodistribution of the [89Zr]DFO*-MMAE in mice bearing DU145 subcutaneous xenografts (168 h post injections, n=4).76KILPATRICK TOWNSEND 79434972 1Treatment of prostate cancer tumor models: PC3, DU145, and C4-2B

[0249] The therapeutic efficacy of the R-ADC was tested in additional three tumor models of prostate cancer: PC3, DU145, and C4-2B. Male nude mice (7 weeks old) were subcutaneously injected with 2.5 million cells of (PC-3, DU145, or C4-2B) prostate cancer mixed with Matrigel (1 : 1) to establish xenograft tumors. Tumors were allowed to grow to -100 mm3before treatment.

[0250] Mice in each tumor model were randomized into two groups:Control Group: Saline-treated.Treatment Group: 0.125 pCi of [225Ac]Macropa-PEG4-YS5-MMAE administered intravenously. This treatment group also received the antibody-drug conjugate dose at 1.8 mg / kg dose.

[0251] Tumor volumes were measured twice weekly using calipers and calculated as (length x width2) / 2. Body weights were monitored concurrently to assess systemic toxicity and tolerability.

[0252] PC-3 Tumor Model (FIG. 21A): Treatment with 0.125 pCi of [225Ac]Macropa-PEG4-YS5-MMAE resulted in significant tumor growth inhibition compared to the saline control group. By day 47, tumors in the saline group grew rapidly, reaching an average volume of 1,083.8 ±577 mm3, whereas the treated group showed minimal tumor progression (77.2 ± 67.0 mm3). Body weight measurements remained stable in the treated group, indicating good tolerability and the absence of significant systemic toxicity.

[0253] DU145 Tumor Model (FIG. 21B): InDU145 xenografts, treatment with 0.125 pCi of [225Ac]Macropa-PEG4-YS5-MMAE effectively suppressed tumor growth. Tumors in the saline group showed progressive growth, reaching an average volume of 294.2 ±143 mm3by day 47, whereas tumors in the treated group exhibited significant regression and minimal77KILPATRICK TOWNSEND 79434972 1progression (57.6 ±21 mm3). Body weights in the treated group remained stable, further supporting the tolerability of the treatment.

[0254] C4-2B Tumor Model (FIG. 21C): Treatment with 0.125 pCi of [225Ac]Macropa-PEG4-YS5-MMAE demonstrated strong tumor suppression in C4-2B xenografts. Tumors in the saline-treated mice grew rapidly, with volumes exceeding 922.3 ± 458.5 mm3by day 47, while the treated group maintained significantly smaller tumor volumes throughout the study (84.1 ± 64.3). Body weights in the treatment group remained consistent, confirming the safety and tolerability of the radiolabeled ADC.

[0255] The data from all three tumor models (PC-3, DU145, and C4-2B) demonstrate the robust therapeutic efficacy of [225Ac]Macropa-PEG4-YS5-M AE in suppressing tumor growth. The observed tumor inhibition highlights the potent anti-tumor effects of this dualmodality radiolabeled ADC, which combines a-particle-mediated radiation and the cytotoxic activity of MMAE. The stability of body weights across all models indicates minimal systemic toxicity, a critical factor for therapeutic agents. The consistent results across diverse prostate cancer models suggest that [225Ac]Macropa-PEG4-YS5-MMAE is broadly effective in prostate cancers with varying levels of androgen receptor sensitivity and aggressiveness. These findings strongly support the potential clinical translation of [225Ac]Macropa-PEG4-YS5-MMAE for the treatment of advanced prostate cancer.Treatment of prostate cancer tumor models xenograft LTL-484 PDX

[0256] R-ADC (at 4.6 kBq, 1.8 mg / kg) treatment efficacy was further examined in several prostate-cancer patient-derived adenocarcinoma model LTL-484 (FIG. 21D). In LTL-484 tumors, R-ADC [225Ac]Macropa-PEG4-YS5-MMAE markedly slowed growth, whereas saline controls reached endpoint rapidly (FIG. 21D). Comparable growth inhibition was observed in C4-2B and DU145 cohorts (FIG. 21B and 21C). Body -weight monitoring in these three cell-line models revealed no weight-loss-related endpoints, underscoring good tolerability (FIG. 21D). Both saline and treated LTL-484 groups showed a brief, early decline in body weight, which stabilized thereafter (FIG. 21D).Toxicity analysis study with histology, CBC, and chemistriesStudy Design:

[0257] Nude mice from therapy study injected with [225Ac]Macropa-PEG4-YS5-MMAE at a dose of 0.125 pCi (1.8 mg / kg) and observed for 90 days post-injection were used for toxicity analysis. Healthy control mice were included for comparison.78KILPATRICK TOWNSEND 79434972 1

[0258] Histopathology: Major organs (heart, lungs, liver, spleen, kidneys, and bone marrow) were collected and fixed for histological examination. Hematoxylin and eosin (H&E) staining was performed to assess tissue morphology and detect potential abnormalities.

[0259] Complete Blood Count (CBC): Blood samples were collected and analyzed for white blood cell (WBC) count, neutrophil %, lymphocyte %, monocyte %, eosinophil %, basophil %, hemoglobin, and red blood cell (RBC) count to evaluate hematological toxicity.

[0260] Serum Chemistry Analysis: Serum samples were analyzed for alanine transaminase (ALT), creatinine, albumin, blood urea nitrogen (BUN), aspartate transaminase (AST), alkaline phosphatase (ALP), and total bilirubin to assess liver and kidney function.

[0261] FIG. 22A-C, complete blood count (CBC), blood cell parameters, including WBC count, neutrophil %, lymphocyte %, monocyte %, eosinophil %, basophil %, hemoglobin, and RBC count, remained within the normal physiological range in both treated and control groups. These findings demonstrate the absence of hematological toxicity following treatment with the radiolabeled ADC.

[0262] FIG. 22D, Serum Chemistry Analysis: Key indicators of liver and kidney function showed no significant differences between treated and control groups. ALT, creatinine, albumin, BUN, AST, ALP, and total bilirubin levels remained within the normal range, confirming preserved organ function and minimal systemic toxicity.

[0263] FIG. 22E, histopathological analysis revealed no significant abnormalities in the major organs of treated mice compared to healthy controls. The tissues from the heart, lungs, liver, spleen, and bone marrow of treated mice showed normal architecture, indicating minimal off-target toxicity of [225Ac]Macropa-PEG4-YS5-MMAE. Kidneys showed mild renal injury in three out of 5 mice.

[0264] Toxicity evaluation 90 days post-injection demonstrated the excellent safety profile of [225Ac]Macropa-PEG4-YS5-MMAE at a dose of 0.125 pCi (1.8 mg / kg) in nude mice. The absence of histopathological abnormalities in major organs, along with normal hematological and serum chemistry parameters, highlights the minimal off-target and systemic toxicity of this radiolabeled ADC. The results confirm that the dual-modality [225Ac]Macropa-PEG4-YS5-MMAE is not only effective but also well-tolerated, making it a promising therapeutic candidate for prostate cancer.

[0265] Complete blood counts revealed no sustained abnormalities in either healthy control or R-ADC-treated mice (FIG. 22A, FIG. 22B, FIG. 22C). A transient decline in WBC count was noted in the R-ADC group on Day 24, but values recovered by Day 94, indicating mild and reversible myelosuppression (FIG. 22A). Serum biochemistry analysis showed no 79KILPATRICK TOWNSEND 79434972 1significant changes in kidney or liver function markers, including creatinine, blood urea nitrogen (BUN), ALT, AST, ALP, and bilirubin (FIG. 22D), confirming the absence of systemic organ toxicity. FIG. 22E shows representative histological analysis of major organs from saline- and R-ADC-treated mice (4.6 kBq, 1.8 mg / kg). On Day 40, histopathologic examination revealed no abnormalities in the heart, lungs, liver, spleen, or bone marrow. On Day 94, all R-ADC-treated mice similarly showed normal histology in these organs.However, all mice exhibited mild renal tubular injury, with final renal injury scores of 2 (50% mice with cumulative scores of 3, and 50% mice with cumulative scores of 4), consistent with low-grade nephrotoxicity, as seen in FIG. 6. This finding is most likely due to non-target mediated nephrotoxicity related to redistribution of daughter213Bi. Consistent with the combination therapy studies, the R-ADC was well tolerated.Example 2Dual Modality Radionuclide-Conjugated Antibody-Drug Conjugates in vivo activity in Multiple Myeloma Cells

[0266] The binding affinity of [225Ac]Macropa-PEG4-YS5-MMAE was assessed using MM. IS multiple myeloma cells. MM. IS cells (2 million) were incubated with high specific activity (0.012 pCi / pg) of [225Ac]Macropa-PEG4-YS5-MMAE (6.44 ng) and low specific activity [225Ac]Macropa-PEG4-YS5-MMAE (0.03 pCi / pg) (6.4 ng) and with [225Ac]Macropa-PEG4-YS5 (6.4 ng) respectively for 2 hours at 37°C in CO2 incubator.

[0267] 10,000 cells were plated in a 6 well plate. Various concentration of [225Ac]Macropa-PEG4-MMAE-YS5 (ranging from 0.024 nCi / ml to 75 nCi / ml) in triplicate were incubated with the cells for 96 hours. After treatment, the contents of each well were transferred to 1.5 mL centrifuge tubes, centrifuged, and the supernatant was discarded. The cell pellets were washed twice with PBS and subsequently resuspended in 1.5 mL of agar gel in 6-well plates. Agar gel provides a supportive medium for colony formation. The cells were incubated for 14 days at 37°C to allow colonies to form. On day 14, the colonies were counted using a microscope.

[0268] 2000 cells were plated in a 96 well black opaque plate. Various concentration of [225Ac]Macropa-PEG4-MMAE-YS5 and [225Ac]Macropa-PEG4-YS5 (ranging from 0.02 pCi / ml to 75 nCi / ml) were incubated with the cells for 96 hours. After treatment, bioluminescence signals of the cells were recorded with multiplate reader (TEC AN software) after 10 minutes incubation by Cell Titre Gio® The % cell viability was fitted into a sigmoidal dose-response curve to determine IC50 value using GraphPad Prism.80KILPATRICK TOWNSEND 79434972 1

[0269] 0.5 million MM. IS cells were injected into NRG mice, and 15 days after the injection, the mice were imaged, and bioluminescence imaging (BLI) was performed to monitor tumor burden. On day 16 post cell inoculation, (considered day 0 of the treatment), the treatment study was initiated. Mice were randomized into two treatment groups (n=8) based on the average BLI signal, with a radiance range of 104to 106p / sec / cm2 / sr. The treatment arms included: 1) saline control (mice injected with 100 pL of saline + 0.5 mg non-specific binding IgG), 2) 0.125 pCi of [225Ac]Macropa-MMAE-YS5 and 1.8 mg / kg of Macropa-PEG4-YS5-MMAE, Fc blocking with 0.5 mg of cold IgG was performed in each cohort. Tumor growth in each mouse was monitored weekly using BLI. Body condition score, mobility, and body weight were monitored every other day. If body weight loss exceeded 20% or the mice exhibited deteriorating conditions, such as paralysis, hyperactivity, or head tilting, they were euthanized.

[0270] FIG. 30A: Cell Binding Assay: Incubation with MM. IS cells with high specific activity of [225Ac]Macropa-PEG4-YS5-MMAE showed 3% binding and with low specific activity of [225Ac]Macropa-PEG4-YS5-MMAE showed 1.8% binding which is almost equal to binding of [225Ac]Macropa-PEG4-YS5.

[0271] FIG. 30B: Colony formation assay with low specific activity of [225Ac]Macropa-PEG4-YS5-MMAE demonstrate concentration dependent formation of colonies and less cell proliferation as compared to control cells.

[0272] FIG. 30C: Cytotoxicity of [225Ac]Macropa-PEG4-MMAE-YS5 (High specific Activity and low specifc activity) was compared with [225Ac]Macropa-PEG4-YS5 for their therapeutic efficacy. The treatment efficacy at the same activities resulted in a decrease in cell viability. The high specific activity and low specific activity of [225Ac]Macropa-PEG4-YS5-MMAE showed potent killing action with IC50 1.67 nCi / ml and 3.34 nCi / ml respectively, compared to [225Ac]Macropa-PEG4-YS5 with IC50 10.85 nCi / ml. In addition, high and low specific activity of [225Ac]Macropa-PEG4-YS5-MMAE showed 10 fold lower IC50 as compared to [225Ac]Macropa-PEG4-YS5. These results confirm the affinity and therapeutic efficacy of [225Ac]Macropa-PEG4-YS5-MMAE, supporting its use as a targeted radiopharmaceutical for theranostic applications in CD46-expressing cancer.

[0273] The monotherapy was then compared with [225Ac]Macropa-PEG4-YS5 (2.3 kBq), and YS5-MMAE (1.8 mg / kg), to their combination in the disseminated MM. IS myeloma model (FIG. 31A, 32A-B). All vehicle treated mice succumbed by day 42, with median survival of 39 days. Bioluminescence imaging revealed that monotherapy with the alpha conjugate [225Ac]Macropa-PEG4-YS5 or the YS5-MMAE slowed tumor growth but eventually failed,81KILPATRICK TOWNSEND 79434972 1giving median survivals of 122.5 days and 59 days, respectively. In contrast, the combination delivered durable control: two mice relapsed late, but three remained tumor-free for more than 190 days, and median survival extended to 205 days (p <0.001 versus each monotherapy; FIG. 31A, FIG. 32A). Throughout the experiment, body weight varied by less than ten percent in all cohorts, indicating an absence of overt toxicity (FIG. 32B).

[0274] Encouraged by these combination therapy data on multiple myeloma tumor models, the dual-payload (R-ADC, [225Ac]Macropa-PEG4-YS5-MMAE) formulation was then tested in two disseminated myeloma models. In MM.lS-luc bearing mice, a single 4.6 kBq dose of [225Ac]Macropa-PEG4-YS5-MMAE kept tumor bioluminescence below the detection threshold in all the animals except one until day 35, whereas vehicle-treated mice showed progressive disease and reached endpoint by day 42 (FIG. 31B, 31C). Median survival for the vehicle cohort was 31.5 days; it was 54 days in the treated group (FIG. 31C). Body weight remained stable, confirming tolerability (FIG. 33). The RPMI-luc model yielded similar results (FIG. 31D-E). Vehicle animals progressed rapidly and with median survival of 37 days, while treated mice maintained low tumor signals through the same period and survived beyond it; with median survival of 73 days (p = 0.0006) (FIG. 31E). No weight loss occurred in either group (FIG. 34). These results demonstrate that combination therapy or R-ADC therapy demonstrates improved efficacy in multiple myeloma, in addition to prostate cancer.Example 3Combination Therapy in Disseminated Tumor Model

[0275] This example describes the combination therapy of [225Ac]Macropa-PEG4-YS5 combined with YS5-MMAE in a disseminated mice tumor model.

[0276] A disseminated tumor model of PCa (22R1-Luc) was prepared by inoculating mice with cells via intracardiac injections, followed by treatment and bioluminescence imaging (BLI). Longitudinal BLI (FIG. 8 A) confirmed that combining [225Ac]Macropa-PEG4-YS5 (4.6 kBq or 0.74 pg) with YS5-MMAE (at 1.8 mg / kg) achieved the most durable tumor control in the disseminated 22Rvl-luc model. All cohorts were injected at week 3 post inoculation; however, by week 5 the signals diverged markedly. Saline-treated and YS5-MMAE-only mice showed a rapid surge in photon flux, indicating unchecked tumor expansion, whereas [225Ac]Macropa-PEG4-YS5 monotherapy slowed growth but still allowed the signal to climb again by week 6 post-treatment. In contrast, the combination regimen kept the BLI signal near baseline until at least week 10, demonstrating sustained suppression of 82KILPATRICK TOWNSEND 79434972 1tumor burden (FIG. 8A). Median survival was 6 weeks for both the saline and YS5-MMAE groups, 16.5 weeks for [225Ac]Macropa-PEG4-YS5, and 20-weeks for combination cohort (FIG. 8B). Log-rank analysis showed that the combination significantly outperformed YS5-MMAE monotherapy and saline (p < 0.05 for all pairwise comparisons, p=0.039 for [225Ac]Macropa-PEG4-YS5 vs combination group). Throughout the observation period, body weight varied by less than ten percent in every cohort, indicating good tolerability of both the single-dose and combination regimens (FIG. 8C). It is worth mentioning that 3 / 10 mice in the [225Ac]Macropa-PEG4-YS5 group and 2 / 10 in the combination group died without exhibiting signs of systemic toxicity, such as body weight loss, reduced body condition scores or tumor burden.Example 4Drug and Functional Stability Assessment of [225Ac]Macropa-PEG4-YS5-MMAE

[0277] This example describes the evaluation of the drug and functional stability of the dualmodality conjugate (R-ADC) of [225Ac]Macropa-PEG4-YS5-M AE.

[0278] The stability of the dual-modality conjugate [225Ac]Macropa-PEG4-YS5-M AE was evaluated following storage in phosphate-buffered saline (PBS) at 37°C for up to 7 days to assess structural integrity, antigen-binding capability, and functional cytotoxic potency. Multiple complementary analytical methods were performed to characterize the conjugate during the storage period.

[0279] Drug-to- Antibody Ratio Stability: The drug-to-antibody ratio (DAR) of [225Ac]Macropa-PEG4-YS5-MMAE was quantified on Day 0, Day 4, and Day 7 using UV-based spectrophotometric analysis. The DAR values remained consistent, ranging from 2.2 to 2.8 throughout the study period (FIG. 16A). These results indicate that the MMAE payload remained stably conjugated to the antibody and that no significant payload loss or degradation occurred during storage.

[0280] Binding Specificity After Storage: A magnetic bead-based binding assay was performed on Day 7 to evaluate retention of antigen recognition following storage.[225Ac]Macropa-PEG4-YS5-MMAE demonstrated strong binding to CD46-coated beads, comparable to Day 0 activity. Binding was markedly reduced in the presence of excess unlabeled YS5 antibody (blocking control) and in samples lacking CD46 antigen, confirming that immunoreactivity and CD46-dependent specificity were preserved after storage (FIG.16B)83KILPATRICK TOWNSEND 79434972 1

[0281] Cytotoxic Activity Following Storage: The functional potency of the stored conjugate was evaluated using a CellTiter-Glo viability assay in 22Rvl prostate cancer cells. Cells were treated with [225Ac]Macropa-PEG4-YS5-MMAE freshly prepared (Day 0) or after 7 days of storage. Both preparations demonstrated comparable cytotoxic activity following 96-hour incubation, with ICso values of 1.3 nCi / mL (Day 0) and 0.6 nCi / mL (Day 7), indicating no loss of therapeutic efficacy (FIG. 16C).

[0282] Collectively, the DAR analysis, binding assay, and cytotoxicity evaluation confirm that [225Ac]Macropa-PEG4-YS5-MMAE maintains structural integrity, antigen specificity, and functional potency for at least 7 days under storage conditions. These results demonstrate that the R-ADC can be synthesized reproducibly with preserved radiochemical and biological properties, supporting its suitability for practical handling, batching, and clinical translation.

[0283] The examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.84KILPATRICK TOWNSEND 79434972 1

Claims

1. WHAT IS CLAIMED IS:

1. An antibody that specifically binds to CD46, wherein the antibody is linked to (i) a cytotoxic effector and (ii) a chelator, wherein a radionuclide is bound to the chelator via coordinate bonding.

2. The antibody of claim 1, wherein the antibody comprises an Fc domain.

3. The antibody of claim 1, wherein the antibody comprises heavy chain CDRs 1, 2 and 3 and light chain CDRs 1, 2, and 3 of any one of YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, orUA8kappa.

4. The antibody of claim 1, wherein the antibody comprises a heavy chain (HC) variable region that comprises three complementarity determining regions (CDRs): HC CDR1, HC CDR2 and HC CDR3 and a light chain (LC) variable region that comprises three CDRs: LC CDR1, LC CDR2, and LC CDR3, wherein said HC CDR1, HC CDR2, HC CDR3 comprise an amino acid sequence of SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82, respectively, and said LC CDR1, LC CDR2, and LC CDR3 comprise an amino acid sequence of SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO: 85, respectively.

5. The antibody of claim 4, wherein the HC comprises SEQ ID NO:86 and the LC comprises SEQ ID NO:87.

6. The antibody of claim 1, wherein the cytotoxic effector is linked to the antibody via a cleavable or non-cleavable linker via a cysteine sulfhydryl moiety in the antibody.

7. The antibody of claim 6, wherein the cleavable linker comprises valine-citrulline.

8. The antibody of claim 1, wherein the cytotoxic effector is a chemotherapeutic agent.

9. The antibody of claim 1, wherein the cytotoxic effector is a microtubule inhibitor, a DNA-damaging agent, or a polymerase inhibitor.

10. The antibody of claim 1, wherein the cytotoxic effector is selected from the group consisting of an auristatin, Dolastatin-10, synthetic derivatives of the natural product Dolastatin-10, and maytansine or a maytansine derivative.85KILPATRICK TOWNSEND 79434972 111. The antibody of claim 1, wherein the cytotoxic effector is selected from the group consisting of Monomethylauristatin F (MMAF), Auristatin E (AE), Monomethylauristatin E (MMAE), vcMMAE, and vcMMAF.

12. The antibody of claim 11, wherein monomethylauristatin E (MMAE) is conjugated to said antibody via a maleimidocaproyl-valine-citrulline-para-amino benzyloxy carbonyl (mc-vc-PAB) linker.

13. The antibody of claim 1, wherein the cytotoxic effector is selected from the group consisting of mertansine (DM1), ravtansine (DM4), calicheamicin, a duocarmycin analog, a pyrrol obenzodiazepine dimer, Exatecan derivative for ADC (DXd), 7-ethyl-10-hydroxy-camptothecin (SN-38), topotecan, or a derivative thereof.

14. The antibody of claim 13, wherein the duocarmycin analog is selected from the group consisting of Duocarmycin Bl (DUMB1), duocarmycin B2 (DUMB2), duocarmycin Cl (DUMC1), duocarmycin C2 (DUMC2), duocarmycin D 1 (DUMD1), duocarmycin D2 (DUMD2) and DSA.

15. The antibody of claim 1, wherein the chelator comprises Macropa, mercaptoacetyltriglycine (MAGs), diethylenetriaminepentaacetic acid (DTP A), Deferoxamine (DFO), DFO*, DFO-Km, DFO-HOPO, l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA) or triethylenetetramine (TETA).

16. The antibody of claim 1, wherein the chelator is linked to the antibody by a linker.

17. The antibody of claim 16, wherein the linker is linked to the antibody via an amine on a lysine side chain of the antibody.

18. The antibody of claim 16, wherein the linker comprises polyethylene glycol (PEG).

19. The antibody of claim 18, wherein the PEG comprises PEG2-10.

20. The antibody of claim 18, wherein the linker comprises PEG2, PEGr, PEGe, or PEGs.

21. The antibody of claim 1, wherein the radionuclide emits alpha particles.86KILPATRICK TOWNSEND 79434972 122. The antibody of claim 21, wherein the radionuclide is selected from the group consisting of Actinium-225, Thorium-227, Bismuth-213, Astatine-211, Terbium-149, Lead-212 and Bismuth-213.

23. The antibody of claim 1, wherein the radionuclide emits beta particles.

24. The antibody of claim 23, wherein the radionuclide is selected from the group consisting of Lutetium- 177, Terbium-161, Yttrium-90 and Lead-212.

25. The antibody of claim 1, wherein the radionuclide emits positrons.

26. The antibody of claim 25, wherein the radionuclide is selected from the group consisting of Zirconium-89, Copper-64, Gallium-68, Cerium-134 and Fluorine-18.

27. A pharmaceutical composition comprising a therapeutically-effective amount of the antibody of claim 1.

28. A composition comprising:(i) a first antibody that binds to CD46, wherein the first antibody is linked to a cytotoxic effector; and(ii) a second antibody that binds to CD46, wherein the second antibody is linked to a chelator, wherein a radionuclide is bound to the chelator via coordinate bonding.

29. The composition of claim 28, wherein the composition is a pharmaceutical composition comprising a therapeutically-effective amount of the first antibody and the second antibody.

30. The composition of claim 28, wherein the first antibody, or the second antibody, or both the first antibody and the second antibody, comprises heavy chain CDRs 1, 2 and 3 and light chain CDRs 1, 2, and 3 of any one of YS5, YS5F, YS5vlD, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, or UA8kappa.

31. The composition of claim 28, wherein the first antibody, or the second antibody, or both the first antibody and the second antibody, comprises a heavy chain (HC) variable region that comprises three complementarity determining regions (CDRs): HC CDR1, HC87KILPATRICK TOWNSEND 79434972 1CDR2 and HC CDR3 and a light chain (LC) variable region that comprises three CDRs: LC CDR1, LC CDR2, and LC CDR3, wherein said HC CDR1, HC CDR2, HC CDR3 comprise an amino acid sequence of SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82, respectively, and said LC CDR1, LC CDR2, and LC CDR3 comprise an amino acid sequence of SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO: 85, respectively.

32. The composition of claim 31, wherein the HC comprises SEQ ID NO:86 and the LC comprises SEQ ID NO:87.

33. The composition of claim 28, wherein the cytotoxic effector is linked to the first antibody via a cleavable or non-cleavable linker via a cysteine sulfhydryl moiety in the first antibody .

34. The composition of claim 33, wherein the cleavable linker comprises valine-citrulline.

35. The composition of claim 28, wherein the cytotoxic effector is a chemotherapeutic agent.

36. The composition of claim 35, wherein the cytotoxic effector is a microtubule inhibitor, a DNA-damaging agent, or a polymerase inhibitor.

37. The composition of claim 28, wherein the cytotoxic effector is selected from the group consisting of an auristatin, Dolastatin-10, synthetic derivatives of the natural product Dolastatin-10, and maytansine or a maytansine derivative.

38. The composition of claim 28, wherein the cytotoxic effector is selected from the group consisting of Monomethylauristatin F (MMAF), Auristatin E (AE), Monomethylauristatin E (MMAE), vcMMAE, and vcMMAF.

39. The composition of claim 38, wherein monomethylauristatin E (MMAE) is conjugated to the first antibody via a maleimidocaproyl-valine-citrulline-para-amino benzyloxycarbonyl (mc-vc-PAB) linker.

40. The composition of claim 28, wherein the cytotoxic effector is selected from the group consisting of mertansine (DM1), ravtansine (DM4), calicheamicin, a duocarmycin analog, a pyrrolobenzodiazepine dimer, Exatecan derivative for ADC (DXd), 7-ethyl-10-hydroxy-camptothecin (SN-38), topotecan, or a derivative thereof.88KILPATRICK TOWNSEND 79434972 141. The composition of claim 40, wherein the duocarmycin analog is selected from the group consisting of Duocarmycin B 1 (DUMB1), duocarmycin B2 (DUMB2), duocarmycin Cl (DUMC1), duocarmycin C2 (DUMC2), duocarmycin DI (DUMD1), duocarmycin D2 (DUMD2) and DSA.

42. The composition of claim 28, wherein the chelator comprises Macropa, mercaptoacetyltriglycine (MAGs), diethylenetriaminepentaacetic acid (DTP A), Deferoxamine (DFO), DFO*, DFO-Km, DFO-HOPO, l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA) or l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA) or tri ethyl enetetramine .

43. The composition of claim 28, wherein the chelator is linked to the second antibody by a linker.

44. The composition of claim 43, wherein the linker is linked to the second antibody via an amine on a lysine side chain of the second antibody.

45. The composition of claim 43, wherein the linker comprises polyethylene glycol (PEG).

46. The composition of claim 45, wherein the PEG comprises PEG2-10.

47. The composition of claim 45, wherein the linker comprises PEG2, PEG4, PEGe, or PEGs.

48. The composition of claim 28, wherein the radionuclide emits alpha particles.

49. The composition of claim 48, wherein the radionuclide is selected from the group consisting of Actinium-225, Thorium-227, Bismuth-213, Astatine-211, Terbium-149, Lead-212 and Bismuth-213.

50. The composition of claim 28, wherein the radionuclide emits beta particles.

51. The composition of claim 50, wherein the radionuclide is selected from the group consisting of Lutetium- 177, Terbium-161, Yttrium-90 and Lead-212.

52. The composition of claim 28, wherein the radionuclide emits positrons.89KILPATRICK TOWNSEND 79434972 153. The composition of claim 52, wherein the radionuclide is selected from the group consisting of Zirconium-89, Copper-64, Gallium-68, Cerium-134 and Fluorine-18.

54. A method of killing a CD46-expressing cancer cell, the method comprising contacting the antibody of claim 1 to the cancer cell, thereby killing the cancer cell.

55. The method of claim 54, wherein the cancer cell is in a human and the contacting comprises administering the antibody or pharmaceutically composition or the composition to the human.

56. The method of claim 54, wherein the cancer is selected from the group consisting of prostate cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer and a solid tumor cancer.

57. The method of claim 56, wherein the prostate cancer is a resistant form of prostate cancer.

58. The method of claim 56, wherein the prostate cancer is selected from the group consisting of Prostate-Specific Membrane Antigen (PSMA)-low, neuroendocrine, or androgen signaling inhibitor resistant form of prostate cancer.

59. The method of claim 54, wherein the cancer is multiple myeloma or a hematological cancer.

60. The method of claim 59, wherein the multiple myeloma is relapsing or refractory multiple myeloma.

61. A method for imaging a tumor in a subject, the method comprising, administering the antibody of claim 1 to a tumor; anddetecting emitted signal from the radionuclide.

62. The method of claim 61, wherein the radionuclide is selected from the group consisting of Zirconium-89, Cerium-134, Copper-64, Gallium-68, and Fluorine-18.

63. The method of claim 61, wherein the emitted signal is detected by a positron emission tomography (PET) scanner, single photon emission computed tomography (SPECT), or magnetic resonance imaging (MRI) system.90KILPATRICK TOWNSEND 79434972 164. The method of claim 61, wherein the detected signal is used to differentiate tumor tissue from non-tumor tissue, assess tumor progression, or evaluate response to therapy.

65. The method of claim 61, wherein the tumor is a cancer selected from the group consisting of prostate cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer and a solid tumor cancer.

66. The method of claim 65, wherein the prostate cancer is a resistant form of prostate cancer.

67. The method of claim 65, wherein the prostate cancer is selected from the group consisting of Prostate-Specific Membrane Antigen (PSMA)-low, neuroendocrine, or androgen signaling inhibitor resistant form of prostate cancer.91KILPATRICK TOWNSEND 79434972 1

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