Radioimmunotherapy for treatment of acute myeloid leukemia

Description

PATENT Attorney Docket No.084850-1498865-019510WO Client Ref. No. SF-2024-160-1-PCT RADIOIMMUNOTHERAPY FOR TREATMENT OF ACUTE MYELOID LEUKEMIA CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Application No. 63 / 640,158, filed April 29, 2024, which is incorporated by reference for all purposes. BACKGROUND

[0002] Acute myeloid leukemia (AML) is diagnosed in over 20,000 Americans per year, but has typically has poor outcomes. Although there have been newly approved therapies for treating AML, these therapies only lead to modest lifespan extension for a small subset of patients (Koenig, K. & Mims, A. Curr Opin Hematol 27, 108-114 (2020)).

[0003] While antibody-based immunotherapies have shown remarkable clinical efficacy in other blood cancers (such as B cell leukemias, B cell lymphomas, and multiple myeloma), antibody-based therapeutics in AML have thus far exhibited limited utility. The leading targets in treating AML (e.g., CD33, CD123, and CLL-1) are expressed on AML cells and on normal hematopoietic stem and progenitor cells (HSPCs), mature myeloid cells, and / or endothelial cells, which can lead to suboptimal efficacy.

[0004] Interest in radionuclide-based immunotherapies (RITs) has been reinvigorated through recent FDA approvals of radiopharmaceutical therapies in solid tumors, including prostate cancer and neuroendocrine tumors. In addition, RIT targeting CD33 in AML,225Ac- lintuzumab, has shown some efficacy when tested clinically. There is, however, a need for additional therapies for AML. BRIEF SUMMARY OF ASPECTS OF THE DISCLOSURE

[0005] The present disclosure provides radioimmunoconjugates comprising an antibody that specifically binds to an activated conformation of integrin beta-2 (activated integrin 2; aITGB2); a radionuclide; and a chelator, wherein the chelator chelates the radionuclide, and wherein the chelator is coupled to the antibody through a linker comprising poly(ethylene glycol), i.e., a (PEG)n–linker, wherein n is 4, 6, 8, 10, 12, 14, or 16. Such immunoconjugates KILPATRICK TOWNSEND 795667431are useful for treating cancer and for detecting cancer cells that express aITGB2. Also provided herein are methods comprising administering the radioimmunoconjugates for the treatment or detection of cancer. In some instances, the cancer is acute myeloid leukemia (AML). Thus, in some aspects, the disclosure features the following methods and compositions employed in such methods.

[0006] In one aspect, provided is a method of treating cancer in a subject, the method comprising administering to the subject an immunoconjugate according to Formula I as follows,In Formula I, X is a chelator moiety; Y is selected from the group consisting of -O- and -NR-; Z is a moiety selected from the group consisting of:A is an antibody that specifically binds to activated integrin beta-2 (aITGB2); subscript m is 3 or 5; subscript n is 4, 6, 8, 10, 12, 14, or 16; and R selected from the group consisting of H, OH, and a negative charge. In some embodiments, the cancer is a cancer that comprises cells expressing aITGB2. In some embodiments, the antibody comprises an aITGB2 binding domain comprising: (1) a heavy chain variable region (VH) comprising an HCDR1 sequence comprising ISYYYM, an HCDR2 sequence comprising SISSSSGYTY; and an HCDR3 sequence comprising GAM; and (2) a light chain variable region (VL) comprising an LCDR1 sequence comprising SVSSA, an LCDR2 sequence comprising SASSLYS; and an LCDR3 sequence comprising FSSGSWAPI. In some embodiments, the antibody comprises an aITGB2 binding domain comprising: (1) a heavy chain variable region (VH) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:1 and comprising an HCDR1 sequence comprising ISYYYM, an HCDR2 sequence comprising SISSSSGYTY; and an HCDR3 sequence comprising GAM; and (2) a light chain variable region (VL) comprising an amino aicd sequence having at least 95% identity to SEQ ID NO:2 comprising an LCDR1 sequence comprising SVSSA, an LCDR2 sequence comprising SASSLYS; and an LCDR3 sequence KILPATRICK TOWNSEND 795667431comprising FSSGSWAPI. In some embodiments, the antibody comprises a VH comprising amino acid sequence SEQ ID NO:1 and a VLcomprising amino acid sequence SEQ ID NO: 2. In some embodiments, the cancer is acute myeloid leukemia (AML).

[0007] In some embodiments, the immunoconjugate comprises a structure according to Formula Ia as follows:(Ia).

[0008] In some embodiments, the immunoconjugate comprises a structure according to Formula Ib as follows:

[0009] In some embodiments, the chelator moiety X includes, but is not limited to, one of the following structures:KILPATRICK TOWNSEND 795667431.

[0010] In some embodiments, the chelator moiety X has the following structure:Y is -O-; and subscript m is 3.

[0011] In some embodiments, the chelator moiety X has the following structure:; Y is -NR-; and subscript m is 3.

[0012] In some embodiments, the chelator moiety X has the following structure: KILPATRICK TOWNSEND 795667431; Y is -NR-; and subscript m is 3.

[0013] In some embodiments, the chelator moiety X has the following structure:; Y is -NR-; and subscript m is 5.

[0014] In some embodiments, subscript n is 4, 6, 8, or 12. In some embodiments, subscript n is 4 or 8.

[0015] In some embodiments, the immunoconjugate further comprises an alpha-emitting radionuclide, wherein the chelator moiety of the immunoconjugate chelates the alpha-emitting radionuclide. In some embodiments, the alpha-emitting radionuclide is selected from the group consisting of225Ac,134Ce,213Bi,224Ra,212Pb,227Th,223Ra,211At, and149Tb. In some embodiments, the alpha-emitting radionuclide is225Ac.

[0016] In some embodiments, the immunoconjugate comprises a structure according to Formula IIa as follows: KILPATRICK TOWNSEND 795667431(IIa), and M is the alpha-emitting radionuclide.

[0017] In some embodiments, the immunoconjugate comprises a structure according to Formula IIb as follows:and M is the alpha-emitting radionuclide, and subscript p is 0 or 1.

[0018] In some embodiments, the immunoconjugate comprises a structure according to Formula IIc as follows:and M is the alpha-emitting radionuclide, and subscript p is 0 or 1.

[0019] In some embodiments, the immunoconjugate comprises a structure according to Formula IId as follows: KILPATRICK TOWNSEND 795667431(IId), and M is the alpha-emitting radionuclide.

[0020] In some embodiments, wherein the alpha-emitting radionuclide is225Ac.

[0021] In another aspect, provided is a method of treating cancer in a subject, the method comprising administering to the subject an immunoconjugate according to Formula IIa as follows:(IIa). In Formula IIA, M is an alpha-emitting radionuclide225Ac;,A is an antibody that specifically binds to activated integrin beta-2 (aITGB2), wherein the antibody comprises a VH comprising amino acid sequence SEQ ID NO: 1 and a VL comprising amino acid sequence SEQ ID NO: 2; and subscript n is 4. In some embodiments, the cancer is a cancer that comprises cells expressing aITGB2. In some embodiments, the cancer is AML. In some embodiments, the immunoconjugate is administered with a pharmaceutically acceptable excipient.

[0022] In a further aspect, the disclosure features an antibody composition comprising [225Ac]Macropa-PEG4-7065 or comprising [89Zr]DFO*-7065, wherein 7065 is an antibody that specifically binds to activated integrin beta-2 (aITGB2), wherein the antibody comprises a VH comprising amino acid sequence SEQ ID NO: 1 and a VL comprising amino acid sequence SEQ ID NO: 2. In some embodiments, the composition comprising [225Ac]Macropa-PEG4-706 is a pharmaceutical composition for the treatment of cancer that 7 KILPATRICK TOWNSEND 795667431expresses aITGB2, e.g., AML. In some embodiments, the composition comprising i[89Zr]DFO*-7065 is formulated in a pharmaceutical compositions for using in radioimaging, e.g., for diagnostic and prognostic applications to identify a cancer, e.g., for aITGB2 expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG.1A-F Schematic representation of radioimmunoconjugates (A) Conjugation of aITGB2 targeting 7065 antibody with DFO*-NCS followed by radiolabeling with89Zr(C2O4)2 (B) Conjugation of IgG (non-specific binding) antibody with DFO*-NCS followed by radiolabeling with89Zr(C2O4)2 (1C) Conjugation of 7065 antibody with Macropa-PEG4-TFP followed by radiolabeling with225Ac(NO3)3 and134CeCl3 (D) Conjugation of IgG antibody with Macropa-PEG4-TFP followed by radiolabeling with225Ac(NO3)3 (E) Conjugation of anti-CD33 antibody (Lintuzumab) with DOTA-NCS followed by radiolabeling with225Ac(NO3)3.

[0024] FIG.2A-F [89Zr]DFO*-7065 detects aITGB2 expression in various AML cell lines A) Flow Cytometry analysis of aITGB2 cell surface expression in various AML cell lines (n = 3) B) Flow Cytometry analysis of aITGB2 cell surface expression in various Patient Derived Xenografts samples C) Flow Cytometry analysis of aITGB2 cell surface expression in Patients (diagnosed vs relapsed D) Cell binding assay to measure the percentage cell-associated activity of [89Zr]DFO*-7065 using different AML cell lines E) Kd measurement of [89Zr]DFO*-7065 on Nomo-1 cell line (n=3), determined by a saturation binding assay (Kd= 2.09 ± 2.6 nM), for HL-60 cell line (n=3), (Kd= 0.86 ± 0.23 nM) and for THP-1 Kdvalue is 1.19 ± 0.65 nM) F) Percentage membrane-bound and internalized binding of [89Zr]DFO*-7065 with Nomo-1 and HL-60 cell line; left to right, for each time period: Nomo-1, membrane-bound; Nomo-1, internalized; HL-60, membrane-bound, HL-60, internalized.

[0025] FIG.3A-Q [89Zr]DFO*-7065 detects aITGB2 positive AML disseminated lesions using PET imaging. (A) Schematic of in vivo and ex vivo imaging and biodistribution studies in disseminated AML models. BLI, MIP and PET / CT fusion images of (B) [89Zr]DFO*-7065 in Nomo-1-Luc model, (C) [89Zr]DFO*-7065 + 25fold excess of 7065 in Nomo-1-Luc model (D) [89Zr]DFO*-IgG in Nomo-1-Luc model (E) [89Zr]DFO*- 7065 in Nomo-1-ITGB2 KO-Luc model (F) Ex vivo BLI and PET / CT fusion images of KILPATRICK TOWNSEND 795667431[89Zr]DFO*-7065 in Nomo-1-Luc, BLI and PET / CT fusion images of [89Zr]DFO*-7065 +25fold excess of 7065 in Nomo-1 ITGB2-KO-Luc, BLI and PET / CT fusion images of [89Zr]DFO*-IgG in Nomo-1-Luc and BLI and PET / CT fusion images of [89Zr]DFO*-7065 in Nomo-1 ITGB2-KO-Luc model in Femur (G) Ex vivo biodistribution indicates significantly higher accumulation of % IA / gram of [89Zr]DFO*-7065 in bone marrow (primary site of the disease) in Nomo-1 as compared to other three negative controls at 4 days post injection. (H) Tumor / Blood ratio in all the groups at four days post injection (I) Tumor / Muscle ratio in all cohorts at 4 days post injection (K) BLI and PET / CT fusion images of [89Zr]DFO*-7065 in MV411-Luc model (K) THP-1-Luc model (L) HL-60-Luc model (M) Ex vivo BLI and PET / CT fusion images of [89Zr]DFO*-7065 in MV411-Luc, HL-60-Luc, THP-1-Luc model in femur and liver (N) Ex vivo biodistribution indicating %IA / gram accumulation of [89Zr]DFO*-7065 in bone marrow tumor (O) Ex vivo biodistribution indicating %IA / gram accumulation of [89Zr]DFO*-7065 in liver (P) Tumor / Blood ratio in all groups at 4 days post injection (Q) Tumor / Muscle ratio in all groups at 4 days post injection. For each of (N)-(Q), cell lines are MV411, THP-1, and HL-60, left to right.

[0026] FIG.4A-H [225Ac]Macropa-PEG4-7065 demonstrates high therapeutic efficacy as compare to controls and [134Ce / 225Ac]Macropa-PEG4-7065 demonstrates favorable tumoral and whole animal biodistribution. (A) Colony formation assay [225Ac]Macropa- PEG4-7065 in Nomo-1 and Nomo-1 ITGB2 KO cell line and HL-60 and MV411 cell line (B) Cell killing assay with [225Ac]Macropa-PEG4-7065 in Nomo-1 and Nomo-1 ITGB2 KO cell line and with [225Ac]DOTA-anti-CD33 in Nomo-1 cell line (C) BLI of representative mouse bearing Nomo-1-Luc disseminated diseases. MIP and PET / CT fusion images of [134Ce]Macropa-PEG4-7065 in Nomo-1-Luc bearing mouse at 24 h, 48 h, 96 h, 168 h post injection. Ex vivo PET / CT and BLI images of the organs injected with [134Ce]Macropa-PEG4- 7065 at 168 h post injection (D) Ex vivo biodistribution study comparing the IA / gram accumulation of [134Ce]Macropa-PEG4-7065 and [225Ac]Macropa-PEG4-7065 in all organs at 168 h post injection (E) Ex vivo biodistribution study in Nomo-1-Luc model indicating % IA / gram accumulation of [225Ac]Macropa-PEG4-7065 (n=4 in each group) (F) Tumor to blood ratio and (G) Tumor to muscle ratio at indicated time points of [225Ac]Macropa-PEG4- 7065 (H) Dosimetry study demonstrates amount of absorbed dose (Gy) in each organ. KILPATRICK TOWNSEND 795667431

[0027] FIG.5A-I [225Ac]Macropa-PEG4-7065 based radiopharmaceutical therapy for the effective treatment of AML (A) Schematic showing the workflow for the therapy study (B) Serial BLI imaging indicates reduce tumor burden in treatment group 9.25 KBq single dose as well as fractionated dose of 9.25 KBq of [225Ac]Macropa-PEG4-7065 (n=8) as compared to saline, one negative control [225Ac]Macropa-PEG4-IgG(n=8) and positive control [225Ac]DOTA-anti-CD33 (n=8). (C) Body weight plot indicating the measurements in all respective groups (D) Kaplan Meier curve demonstrates improvement in median time period survival in single 9.25 KBq as well as fractionated 9.25 KBq groups as compared to saline, [225Ac]Macropa-PEG4-IgG and [225Ac]DOTA-anti-CD33 cohort. FACS analysis indicating CD45+ CD33+ tumor population and aITGB2 expression for one of the representative mouse injected with (E) fractionated dose of 9.25 KBq [225Ac]Macropa-PEG4- 7065 (F) single dose of [225Ac]Macropa-PEG4-7065 (G) 9.25 KBq [225Ac]DOTA-anti-CD33 (H) 9.25 KBq [225Ac]Macropa-PEG4-IgG (I) saline + IgG.

[0028] FIG.6A-G [225Ac]Macropa-PEG4-7065 based radiopharmaceutical therapy for the effective treatment of Patient Derived Xenografts (A) Schematic showing the workflow for the therapy study. Flow analysis of peripheral blood at day 30 for one of the representative mouse in each group (B) saline indicates higher population of CD45+ tumor cells whereas treatment group (C), (D) 4.62 KBq of [225Ac]Macropa-PEG4-7065 and [225Ac]DOTA-anti-CD33 respectively has significantly less CD45+ tumor population. (E) Analysis of CD45+ population in all the groups at day 15, day 30, day 41, day 52 and day 60 after treatment (F) Body weight plot indicating the measurements in all respective groups (G) Kaplan Meier curve demonstrates statistically significant improvement in median time period survival each group, left to right: saline, [225Ac]-anti-CD33, [225Ac]-7065. DEFINITIONS

[0029] As used herein, the term “integrin beta 2,” “integrin2” or “ITGB2”, also known as CD18, LAD, LCAMB, LFA-1, MAC-1, MF17, MFI7, or integrin subunit beta 2, refer to a polypeptide that is encoded by a ITGB2 gene (chr21:44,885,949-44,931,989 (GRCH38 / hg38), cytogenetically localized to human chromosome 21q22.3 by HGNC, Entrez Gene, and Ensembl (genomic coordinates (GRCh38 / hg38 assembly December 2013:) and plays a role in cell adhesion, cell-surface-mediated sequencing, and immune responses. An illustrative human ITGB2 protein sequence encoded by a human ITGB2 gene, P05107-1, is available under Uniprot number P05107. ITGB2 can bind to a number of alpha chains and thus can from KILPATRICK TOWNSEND 795667431multiple heterodimers, but also exists in soluble, ligand binding forms. Deficiencies in ITGB2 expression can lead to adhesion defects in circulating white blood cells in humans, reducing the immune system's ability to fight off foreign invaders. Illustrative ITGB2 heterodimers include, e.g., integrin ITGAL / ITGB2, which is a receptor for ICAM1, ICAM2, ICAM3 and ICAM4, and is also a receptor for the secreted form of ubiquitin-like protein ISG15; integrins ITGAM / ITGB2 and ITGAX / ITGB2, which are receptors for the iC3b fragment of the third complement component and for fibrinogen; integrin ITGAX / ITGB2, which recognizes the sequence G-P-R in fibrinogen alpha-chain, Integrin ITGAM / ITGB2, which recognizes P1 and P2 peptides of fibrinogen gamma chain and is also a receptor for factor X; and integrin ITGAD / ITGB2, which is a receptor for ICAM3 and VCAM1.

[0030] The terms “anti-ITGB2 antibody,” “ITGB2 specific antibody,” “ITGB2 antibody,”and “anti-Itg ” are used synonymously herein to refer to an antibody that specifically bindsto Itg An illustrative human ITGB2 protein sequence encoded by a human ITGB2 gene,P05107-1, is available under Uniprot number P05107.

[0031] An anti-ITGB2 antibody of the present disclosure binds to an active form of ITGB2 (activated ITGB2; aITGB2). An active state of ITGB2 is an extended-open conformation (see, e.g., Nishida et al, Immunity 25:583-94, 2006; Li et al, EMBO J.36:629-45, 2017). The active conformation (extended-open) has a 4,000-fold increase in ligand affinity compared to the other two states (bent-closed, inactive; and extended-closed (intermediate) (Li et al., 2017, supra). Integrin activation takes place upon cell stimulation through various cell surface receptors. Cell stimulation triggers an inside-out signaling pathway that ultimately recruits cytoplasmic factors such as talin and kindlin to the NPxY motifs of the cytoplasmic tail of the integrin's beta-chain, which causes the cytoplasmic tails of the integrin subunits to separate and switches the integrin to the active (extended-open) conformation.

[0032] An “anti-ITGB2 binding domain” as used herein refers to an antigen binding domain comprising a VHand a VLregion of an anti-ITGB2 antibody as described herein, which antigen binding domain binds to active conformation ITGB2.

[0033] The term “antibody” or “immunoglobulin” are used interchangeably to refer to a polypeptide comprising a framework region encoded by an immunoglobulin gene, or fragments thereof, that specifically binds and recognizes an antigen, e.g., the activated form of ITGB2. Typically, the “variable region” contains the antigen-binding region of the antibody (or its KILPATRICK TOWNSEND 795667431functional equivalent) and is important in specificity and affinity of binding. The term “antibody” as used herein thus encompasses antigen binding fragments, e.g., an antigen binding domain, or other antigen binding fragment. Antigen binding fragments may be produced by modification of whole antibodies, or produced using recombinant DNA methodologies (e.g., single chain Fv (scFv) formats).

[0034] An illustrative immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.

[0035] As used herein, “V-region” refers to an antibody variable region domain comprising the segments of Framework 1, CDR1, Framework 2, CDR2, and Framework 3, including CDR3 and Framework 4, which segments are added to the V-segment as a consequence of rearrangement of V-region genes during B-cell differentiation.

[0036] 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 amino acid residues from a “complementarily determining region” or “CDR” (i.e., residues 24- 34 (CDRL1), 50-56 (CDRL2), and 89-97 (CDRL3) in the light-chain variable domain and 31- 35 (CDRH1), 50-65 (CDRH2), and 95-102 (CDRH3) 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 (L1), 50-52 (L2), and 91-96 (L3) in the light-chain variable domain and (H1), 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.

[0037] The amino acid sequences of the CDRs and framework regions can be determined using various well known definitions in the art, e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT), and AbM (see, e.g., Johnson et al., supra; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. KILPATRICK TOWNSEND 795667431Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, structural repertoire of the human VH segments J. Mol. Biol.227, 799-817; Al-Lazikani et al., J.Mol.Biol 1997, 273(4)). Definitions of antigen combining sites are also described in the following: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219–221 (2000); and Lefranc,M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1;29(1):207-9 (2001); MacCallum et al., Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al., Proc. Natl Acad. Sci. USA, 86, 9268–9272 (1989); Martin, et al., Methods Enzymol., 203, 121–153, (1991); Pedersen et al., Immunomethods, 1, 126, (1992); and Rees et al., In Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141–172 1996). Reference to CDRs as determined by Kabat numbering are based, for example, on Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)). Chothia CDRs are determined as defined by Chothia (see, e.g., Chothia and Lesk J. Mol. Biol.196:901-917 (1987)).

[0038] 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 35A and 35B) (CDRl), 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 (CDRl), 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.

[0039] An “isotype” is a class of antibodies defined by the heavy chain constant region. Antibodies described herein can be of any isotype of isotype class. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant 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 isotype classes, IgG, IgM, IgA, IgD and IgE, respectively. In some embodiments, the IgG is an IgG1, IgG2, IgG3 or IgG4. The subunit KILPATRICK TOWNSEND 795667431structures and three-dimensional configurations of different classes of immunoglobulins are well known. Different isotypes have different effector functions. For example, human IgG1 and IgG3 isotypes have ADCC (antibody dependent cell-mediated cytotoxicity) activity. The light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned toone of two clearly distinct types, called kappa ( ) and lambda ( ), based on the amino acidsequences of their constant domains.

[0040] Antibodies can exist as intact immunoglobulins or as any of a number of well- characterized fragments that include specific antigen-binding activity. Such fragments can be produced by digestion with various peptidases. Pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)’2,a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)’2may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)’2dimer into an Fab’ monomer. The Fab’ monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology.

[0041] Antibodies or antigen-binding molecules of the present disclosure further include one or more immunoglobulin chains that are chemically conjugated to, or expressed as, fusion proteins with other proteins. The term “antibody” thus additionally encompasses bispecific and multi-specific antibodies as well as any other monovalent, bivalent, or multivalent antibody format.

[0042] The various antibodies or antigen-binding fragments described herein can be produced by enzymatic or chemical modification of the intact antibodies, or synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv), or identified using yeast or phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554, 1990; Boder, et al (2000) Proc. Natl. Acad. Sci. U. S. A.97:10701).

[0043] A “humanized” antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts. In one embodiment, some, most or all of the amino acids outside the KILPATRICK TOWNSEND 795667431CDR domains are replaced with amino acids corresponding to the human immunoglobulin germline, while amino acids within one or more CDR regions are unchanged. In some embodiments, one or more CDR residues may be altered, e.g., to provide a sequence closer to germline or to replace a residue that may impede production.

[0044] 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.

[0045] The term “specifically bind” refers to a molecule (e.g., antibody or antibody fragment) that binds to a target with at least 2-fold greater affinity than non-target compounds, e.g., at least 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, or 100-fold greater affinity. For example, an antibody that specifically binds ITGB2, typically binds to ITGB2 with at least a 2-fold greater affinity than a non-ITGB2 target. [ For example, an antibody that specifically binds activated ITGB2 (aITGB2), typically binds to aITGB2 with at least a 2-fold greater affinity than an inactive form of ITGB2. In some embodiments, an antibody binds to aITGB2 with a KDthat is at least 100-fold greater than its affinity for inactive ITGB2. The ability of an antibody or antibody fragment 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, an antibody that binds to the antigen has a dissociation constant (Kd) of 1 M, 100 nM, 10nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM (e.g., 10 7 M or less, e.g., from 10 7 M to10 13 M, e.g. from 10 9 M to 10 13 M).

[0046] “Epitope” or “antigenic determinant” refers to a site on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids or from noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An KILPATRICK TOWNSEND 795667431epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).

[0047] 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.

[0048] 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 VHand VLregions 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.

[0049] 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 “trivalent” 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.

[0050] 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. KILPATRICK TOWNSEND 795667431

[0051] The term “linker” as used herein means a chemical moiety comprising or derived from a group of atoms that is covalently attached to an antibody and that is also covalently attached to a chelator. The linker used in the immunoconjugates described herein comprises poly(ethylene glycol) (PEG). PEGs of varying chain lengths can be used in the linker that covalent attaches the antibody to the chelator. In some embodiments, the poly(ethylene glycol) portion of the linker is –(PEG)n–, wherein n is 4, 6, 8, or 12. An exemplary linker comprising poly-ethylene glycol is a (PEG)4,6,8,12linker with malemide and N-hydroxysuccinamide (NHS) functional groups (Mal-PEGn-NHS, wherein n is 4, 6, 8, 12).

[0052] The words “protein,” “peptide,” and “polypeptide” are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non- naturally occurring amino acid polymer.

[0053] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, -carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.

[0054] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. KILPATRICK TOWNSEND 795667431

[0055] “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical or associated, e.g., naturally contiguous, sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to another of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes silent variations of the nucleic acid. One of skill will recognize that in certain contexts each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, silent variations of a nucleic acid which encodes a polypeptide is implicit in a described sequence with respect to the expression product, but not with respect to actual probe sequences.

[0056] A “conservative” substitution as used herein refers to a substitution of an amino acid such that charge, hydrophobicity, and / or size of the side group chain is maintained. Illustrative sets of amino acids that may be substituted for one another include (i) positively-charged amino acids Lys, Arg and His; (ii) negatively charged amino acids Glu and Asp; (iii) aromatic amino acids Phe, Tyr and Trp; (iv) nitrogen ring amino acids His and Trp; (v) large aliphatic nonpolar amino acids Val, Leu and Ile; (vi) slightly polar amino acids Met and Cys; (vii) small-side chain amino acids Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gln and Pro; (viii) aliphatic amino acids Val, Leu, Ile, Met and Cys; and (ix) small hydroxyl amino acids Ser and Thr. Reference to the charge of an amino acid in this paragraph refers to the charge at physiological pH.

[0057] The terms “nucleic acid” and “polynucleotide” are used interchangeably and as used herein refer to both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. In particular embodiments, a nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide, and combinations thereof. The terms also include, but is not limited to, single- and double-stranded forms of DNA. In addition, a polynucleotide, e.g., a cDNA or mRNA, may include either or KILPATRICK TOWNSEND 795667431both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages. Nucleic acid molecules, e.g. oligonucleotide probes or priomers, may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analogue, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). The above term is also intended to include any topological conformation, including single-stranded, double- stranded, partially duplexed, triplex, hairpinned, circular and padlocked conformations. A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. The term also includes codon-optimized nucleic acids that encode the same polypeptide sequence.

[0058] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. A “vector” as used here refers to a recombinant construct in which a nucleic acid sequence of interest is inserted into the vector. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0059] The terms “identical” or “percent identity,” in the context of two or more nucleic acids, or two or more polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides, or amino acids, that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters, or by manual alignment and visual inspection. See e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST. Such sequences are then said to be “substantially identical.” This KILPATRICK TOWNSEND 795667431definition also refers to, or may be applied to, the compliment of a nucleotide test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the algorithms can account for gaps and the like. Typically, identity exists over a region comprising an antibody epitope, or a sequence that is at least about 25 amino acids or nucleotides in length, or over a region that is 50-100 amino acids or nucleotides in length, or over the entire length of the reference sequence.

[0060] The terms “corresponding to,” “determined with reference to,” or “numbered with reference to” when used in the context of the identification of a given amino acid residue in a polypeptide sequence, refers to the position of the residue of a specified reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence. Thus, for example, an amino acid residue in a heavy chain variable domain polypeptide “corresponds to” an amino acid in the heavy chain variable domain polypeptide of SEQ ID NO: 1 when the residue aligns with the amino acid in SEQ ID NO: 1 when optimally aligned to SEQ ID NO: 1. The polypeptide that is aligned to the reference sequence need not be the same length as the reference sequence.

[0061] The term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0062] The term “heterologous” when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein). 20 KILPATRICK TOWNSEND 795667431

[0063] The term “isolated,” when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state. It can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest. The term "purified" denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

[0064] The terms “therapy,” “treatment,” and “amelioration” refer to any reduction in the severity of symptoms. For example, in the case of treating cancer, e.g., AML, treatment can refer to reducing the number of cancer cells or growth rate or cell death of non-cancer cells, etc. As used herein, the terms “treat” and “prevent” are not intended to be absolute terms. Treatment and prevention can refer to any delay in onset, amelioration of symptoms, improvement in patient survival, increase in survival time or rate, etc. Treatment and prevention can be complete (no detectable symptoms remaining) or partial, such that symptoms are less frequent of severe than in a patient without the treatment described herein. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. In some aspects, the severity of disease is reduced by at least 10%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some aspects the severity of disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, no longer detectable using standard diagnostic techniques.

[0065] The terms “effective amount,” “effective dose,” “therapeutically effective amount,” etc. refer to that amount of the therapeutic agent sufficient to ameliorate a disorder, as described above. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of therapeutic effect at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2- fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. KILPATRICK TOWNSEND 795667431

[0066] As used herein, the term “pharmaceutically acceptable” is used synonymously with physiologically acceptable and pharmacologically acceptable. A pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration.

[0067] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. For the present invention, the dose can refer to the concentration of the antibody or associated components, e.g., the amount of therapeutic agent or dosage of radiolabel. The dose will vary depending on a number of factors, including frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; the route of administration; and the imaging modality of the detectable moiety (if present). One of skill in the art will recognize that the dose can be modified depending on the above factors or based on therapeutic progress. The term “dosage form” refers to the particular format of the pharmaceutical, and depends on the route of administration. For example, a dosage form can be in a liquid, e.g., a saline solution for injection.

[0068] “Subject,” “patient,” “individual,” and like terms are used interchangeably and refer to, except where indicated, mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision. A patient can be an individual that is seeking treatment, monitoring, adjustment or modification of an existing therapeutic regimen, etc.

[0069] “Cancer”, “tumor,” “transformed,” and like terms include precancerous, neoplastic, transformed, and cancerous cells, and can refer to a solid tumor, or a non-solid cancer. Cancer includes both benign and malignant neoplasms (abnormal growth).

[0070] The term “co-administer” refers to the simultaneous presence of two active agents in the blood of an individual. Active agents that are co-administered can be concurrently or sequentially delivered.

[0071] The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, KILPATRICK TOWNSEND 795667431for example, reference to “a cell” includes a plurality of such cells and reference to “the agent” includes reference to one or more agents known to those skilled in the art, and so forth.

[0072] As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount, but also allows a reasonable amount of deviation of the modified term such that the end result is not significantly changed. The term about should be construed as including a deviation of at least ±5% (e.g., ± 20%, ± 10%, or ± 5%) of the modified term if this deviation would not negate the meaning of the word it modifies. Generally, the term “about” includes an amount that would be expected to be within experimental error. The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. For example, for KDand IC50values ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value.

[0073] 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. DETAILED DESCRIPTION A. Introduction

[0074] Provided herein are compositions and methods related to radioimmunoconjugates for the treatment of cancer. Cancer-specific cell-surface antigens are ideal targets for monoclonal antibody (mAb)-based immunotherapy. The radioimmunoconjugates disclosed herein include an mAb that binds to an activated conformation of integrin beta-2 (integrin 2; ITGB2), which has been identified as a tumor-selective immunotherapy target in acute myeloid leukemia (AML). See Mandal, Kamal et al. Nature Cancer vol.4,11 (2023).

[0075] As noted above, ITGB2 is an integrin chain protein that can form a heterodimer by binding to an integrin chain protein, such as L (CD11a, ITGAL), M (CD11b, ITGAM), X (CD11c, ITGAX), or D (CD11d, ITGAD). ITGB2 dimerizes with an chain protein 23 KILPATRICK TOWNSEND 795667431intracellularly and the dimer is subsequently embedded in the cell membrane. Integrins exhibit structural diversity, flexibility, and dynamism, and are capable of conformational changes (as opposed to surface expression or clustering) that are central to the regulation of receptor function. Integrin dimers shift between a low-affinity bent-closed (inactive) conformation and a high-affinity extended-open (active) conformation; this shift is defined as integrin activation. Activation of high-affinity binding and of intracellular signal transduction can occur via extracellular signals (also referred to as “outside-in signaling”) or via intracellular signals (also referred to as “inside-out signaling”). Integrins are discussed, e.g., in Hynes, R O. Cell vol. 69,1 (1992): 11-25; and Askari, Janet A et al. Journal of Cell Science vol. 122,Pt 2 (2009).

[0076] ITGB2 has been identified on several immune cell types, including monocytes, neutrophils, natural killer cells and T cells, and is known to largely remain in the closed, inactive conformation until cellular activation by binding to appropriate cytokines, adhesion molecules, or other proteins. Constitutive signaling through ITGB2, in some cases, has been thought to maintain proliferation in AML blasts. It has been suggested that aberrant AML biology may lead to constitutive activation of ITGB2, thus creating a possible tumor-specific conformation that, when targeted, would largely spare normal resting hematopoietic cells. The role of ITGB2 in diseases, including cancer, autoimmune disease and inflammatory diseases, is discussed in, e.g., Bednarczyk, Monika et al. International Journal of Molecular Sciences vol. 21,41402. 19 Feb. 2020; Valdembri, Donatella, and Guido Serini. Faculty Reviews vol. 1045.7 May.2021; Fagerholm, Susanna C et al. Frontiers in Immunology vol.10254.19 Feb. 2019; Schittenhelm, Leonie et al. Frontiers in Immunology vol. 8 1866. 20 Dec. 2017; and Oellerich, Thomas et al. Blood vol.121,19 (2013): 3889-99, S1-66.

[0077] Provided herein are radioimmunoconjugates that include an antibody that binds the activated form or active conformation of ITGB2 (aITGB2), a radionuclide, a chelator that chelates the radionuclide and that is coupled to the antibody through a poly(ethylene glycol) (PEGn) linker. The radioimmunoconjugates described herein are useful for treating cancer, especially, aITGB2 expressing cancer (e.g., acute myeloid leukemia, AML), and for detecting tumor cells. The radioimmunoconjugates described herein, which include short PEGylated macrocyclic chelator complexes, advantageously alter the biodistribution and therapeutic efficacy of radionuclide imaging and therapy. Insertion of short poly(ethylene glycol) (PEG) linkers (PEG4-12) into the disclosed radioimmunoconjugates can promote higher tumor uptake KILPATRICK TOWNSEND 795667431and, in turn, lower the burden of unnecessary radiation on other major organs, such as the liver, spleen, etc. Thus, the radioimmunoconjugates of the present disclosure improve treatment efficacy by lowering the unnecessary radiation burden to the patient. Also provided herein are radioimmunotherapy methods comprising administering the disclosed radioimmunoconjugates to treat cancers, such as AML. B. Antibodies that Bind Activated Integrin2(aITGB2)

[0078] Provided herein are therapeutic conjugates, e.g., radioimmunotherapy reagents, that comprise an anti-ITGB2 antibody that binds the active conformation of ITGB2 (aITGB2). In some embodiments, the anti-ITGB2 antibody binds to aITGB2 with a KD of less than about 10 nM.

[0079] In some embodiments, the anti-ITGB2 antibody is internalized by cancer cells. In some embodiments, the anti-ITGB2 antibody is internalized by cancer cell lines or by cancer cells in situ, e.g., a cancer cell in a tissue microenvironment in vivo. In some embodiments, the anti-ITGB2 antibody is internalized by leukemia cells, e.g., acute myeloid leukemia (AML) cells. As indicated above, such antibodies are useful for targeting cancers that express aITGB2 and are used to deliver therapeutic agents.

[0080] In some embodiments, an anti-ITGB2 binding domain of the present disclosure has at least one, at least two, or three CDRs of a variable domain sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, an anti-ITGB2 binding domain of the present disclosure comprises an HCDR3 of SEQ ID NO: 1 and an LCDR3 of SEQ ID NO: 2. In some embodiments, an anti-ITGB2 binding domain comprises an HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 1 and LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 2.

[0081] In some embodiments, an anti-ITGB2 binding domain comprises an HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 1 in which one of the CDRs comprises a substitution relative to the corresponding CDR set forth in SEQ ID NO: 1. In some embodiments, an anti- ITGB2 binding domain comprises an HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 1 in which two of the CDRs comprise a substitution relative to the corresponding CDRs set forth in SEQ ID NO: 1; and LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 2. In some embodiments, an anti-ITGB2 binding domain comprises an HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 1 in which all three of the CDRs comprise a substitution relative to the corresponding CDR sequences set forth in SEQ ID NO: 1. 25 KILPATRICK TOWNSEND 795667431

[0082] In some embodiments, an anti-ITGB2 binding domain comprises an LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 2 in which one of the CDRs comprises a substitution relative to the corresponding CDR set forth in SEQ ID NO: 2. In some embodiments, an anti- ITGB2 binding domain comprises an LCDR1, LCDR2, and LCDR3 of CDR3 in which two of the CDRs comprise a substitution relative to the corresponding CDRs set forth in SEQ ID NO: 2; and LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 2. In some embodiments, an anti- ITGB2 binding domain comprises an LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 2 in which all three of the CDRs comprise a substitution relative to the corresponding CDR sequences set forth in SEQ ID NO: 2.

[0083] In some embodiments, an anti-ITGB2 binding domain of the present disclosure comprises a variable region having at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of a variable region sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and comprises an HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 1 and an LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 2. In some embodiments, the variable domain comprises substitutions, insertions, or deletions in the framework of a variable region as shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, an anti-ITGB2 binding domain of the present disclosure comprises a heavy chain variable region comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 1 and having at least 90% identity, or at least 95% identity, to SEQ ID NO: 1; and a light chain variable region comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 2 and having at 90% identity, or at least 95% identity, to SEQ ID NO: 2. SEQ ID NO: 1, Antibody D3-7065 heavy chain variable region; CDRs are shown in bold letters EVQLVESGGGLVQPGGSLRLSCAASGFTISYYYMHWVRQAPGKGLEWVASI SSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGAMD YWGQGTLVTVSS SEQ ID NO: 2, Antibody D3-7065 light chain variable region; CDRs are underlined DDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSAS SLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQFSSGSWAPITFGQGTK VEIK KILPATRICK TOWNSEND 795667431SEQ ID NO: 3, HCDR1 – ISYYYM SEQ ID NO: 4, HCDR2 – SISSSSGYTY SEQ ID NO: 5, HCDR3 – GAM SEQ ID NO: 6, LCDR1 – SVSSA SEQ ID NO: 7, LCDR2 – SASSLYS SEQ ID NO: 8, LCDR3 – FSSGSWAPI

[0084] In some embodiments, the antibody comprises VH and VL regions, e.g., in an scFv format, that are joined by a polypeptide flexible linker. In some embodiments, the VH region, e.g., of the scFv, is N-terminal to the VL region. In some embodiments, the VL region, e.g., of the scFv, is N-terminal to the VH region. In some embodiments, the linker comprises 1-4 tandem repeats of a Gly4Ser. In some embodiments, the VH and VL regions are joined by a (Gly4Ser)3 (SEQ ID NO: 9) linker, however it will be recognized that other antibody forms comprising the CDRs (or the VH and / or VL domains) are possible.

[0085] 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 (Gly4Ser)3) (SEQ ID NO: 9), GGGGS GGGGS (SEQ ID NO: 10), GGGGS (SEQ ID NO: 11), GS GGGGS GGGGS GGS GGGGS (SEQ ID NO: 12), SGGGGS (SEQ ID NO: 13), GGGS (SEQ ID NO: 14), VPGV (SEQ ID NO: 15), VPGVG (SEQ ID NO: 16), GVPGVG (SEQ ID NO: 17), GVG VP GVG (SEQ ID NO: 18), VP GVG VP GVG (SEQ ID NO: 19), GGSSRSS (SEQ ID NO: 20), and GGSSRSSSSGGGGSGGGG (SEQ ID NO: 21), and the like.

[0086] In some embodiments, the anti-ITGB2 antibody disclosed herein comprises an immunoglobulin constant region (e.g., an Fc region). Thus, for example, Fc regions can be selected from IgG1, IgG2, IgG3 or IgG4 human heavy chain constant regions; more in some embodiments, the heavy chain constant region of human IgG1 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. 27 KILPATRICK TOWNSEND 795667431

[0087] Other antibody forms include, without limitation, 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, IgG CH2, a minibody, and the like), a single chain antibody (e.g., scFv),a diabody, a unibody, an affibody, and the like.

[0088] Using the sequence information provided herein antibodies comprising one or more of the CDRs disclosed herein, or antibodies comprising the VH and / or VL domain(s) disclosed herein 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.

[0089] In addition, other “related” aITGB2-specific antibodies can be identified by screening for antibodies that bind to the same epitope (i.e., that compete with the antibodies disclosed herein that bind to aITGB2 and / or to a cell expressing or overexpressing aITGB2, e.g., in a leukemia cell, e.g., an AML cell) and / or by modification of the aITGB2-specific 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 aITGB2, e.g., leukemia cells, e.g., an AML cells.

[0090] In some embodiments, that antibody is a recombinant antibody (or antigen binding fragment thereof) that specifically binds aITGB2. In some embodiments, antibody or antigen binding fragment or variant thereof is a monoclonal antibody. In some embodiments, 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 function 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 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 an immunoglobulin variable light chain domain (VL). In some embodiments, the recombinant antibody (or antigen binding fragment thereof) comprises a VH and a VL.

[0091] 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 KILPATRICK TOWNSEND 795667431full 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, an antibody of the present disclosure comprises an HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1 and has at least have at least 90%, 91%, 92%, 93%, or 94% identity to SEQ ID NO:1. In some embodiments, an antibody of the present disclosure comprises an HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1 and has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1. In some embodiments, an antibody of the present disclosure comprises an LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 2 and has at least have at least 90%, 91%, 92%, 93%, or 94% identity to SEQ ID NO:2. In some embodiments, an antibody of the present disclosure comprises an LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 2 and has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2. In some embodiments, an antibody of the present disclosure comprises an HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1 and has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1; and comprises an LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 2 and has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2

[0092] 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. KILPATRICK TOWNSEND 795667431C. Chelators

[0093] The radioimmunoconjugates of the present invention include a chelator that chelates the radionuclide and that has a moiety that is or can be coupled to an antibody. Chelators for radionuclides are known to those of skill in the art. The chelator is typically a bifunctional chelator. As used herein, the term “bifunctional chelator” refers to a chelator that has a metal binding function as well as a chemically reactive functional group that provides the requisite chemistry for coupling to the antibody through a PEG linker.

[0094] In some embodiments, the chelator can be Macropa.NH2, which was developed by Thiele et al., (Thiele NA et al. (2017) Angew Chem Int Ed Engl, 56(46),14712-14717), the teachings of which are incorporated herein by reference. The chelator can also be DOTA (1,4,7,10-Tetraazacyclododecane-l,4,7,10-tetraacetic acid; tetraxetan), and derivatives thereof such as p-SCN-Bn-DOTA and MeoDOTA-NCS or DOTP (1,4,7,10-Tetraazacyclododecane- l,4,7,10-tetra(methylene phosphonic) acid). In some embodiments, the chelator can be DFO or Desferoxamine (N'-[5-(acetyl-hydroxy-amino)pentyl]-N-[5-[3-(5-aminopentyl-hydroxy- carbamoyl)propanoylamino]-pentyl]-N-hydroxy-butane diamide). In some embodiments, thechelator can also be NOTA (2,2 ,2”-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid).

[0095] In other embodiments, the chelator can include, but is not limited to, the following: isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (NCS-DTPA) (see, e.g., PCT Publication No. WO94 / 11026), isothiocyanatobenzyl-1,4, 7, 1O-Tetraazacyclododecane- 1,4,7,10-tetra(methylenephosphonic acid) (p-NCS-DOTP) and Macropa-NCS (6-((16-((6- carboxypyridin-2-yl)methyl)-1,4, 10, 13-tetraoxa-7, 16-diazacyclooctadecan-7-yl)methyl)-4- isothiocyanatopicolinicacid).

[0096] Other examples of chelators that can be used include, but are not limited to, the following: 1,4, 7,10-Tetraazacyclododecane-1,4, 7-tris(aceticacid)-10-(2-thioethyl)acetamide (D03A), [(R)-2-Amino-3-(4-isothiocyanatophenyl)propyl ]-trans-(S, S)-cyclohexane-1,2- diamine-pentaacetic acid (CHX-DTPA), 2-S-( 4-lsothiocyanatobenzyl)-1,4, 7- triazacyclononane-1,4, 7-triacetic acid (SCN-NOTA), 1,4, 7-Triazacyclononane-1,4-bis-acetic acid-7-maleimidoethylacetamide (maleimide-NOTA), 4,11-bis(carboxymethyl)-1,4,8, 11- tetraazabicyclo[ 6.6.2]hexadecane)(CB-TE2A) and Triethylenetetramine (TETA) derivatives.

[0097] The chelator can be directly or indirectly coupled to the antibody. For example, the chelator can be coupled to the antibody by any chemical reaction that will bind the chelator KILPATRICK TOWNSEND 795667431and the antibody, so long as these retain their respective activities / characteristics for the intended use thereof. This coupling can include chemical mechanisms including for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. In some embodiments, the chelator is attached to the antibody through a PEG linker.

[0098] In some embodiments, each chelator carries one radionuclide. Optionally, each antibody is coupled to 1-3 chelator for an antibody:radionuclide ratio of 1:1 to 1:3. The number of chelators per antibody may be controlled for example by adjusting the pH of the reaction, the reaction time and the of fold excess of the chelator to antibody. D. Radionuclides

[0099] The complexes of the present invention include a radionuclide. The radionuclide is optionally an alpha emitter (a radionuclide that emits alpha particles), a beta emitter (a radionuclide that emits beta particles), or a gamma emitter (a radionuclide that emits gamma particles). Examples of radionuclides include, but are not limited to,225Ac,67Cu,177Lu,213Bi,90Y,188Re,47Sc,227Th,212Ph, lllIn,124I,131I,134Ce,213Bi,89Zr,211At,212B, and186Re. Other suitable radionuclide suitable for use in the radioimmunoconjugates disclosed herein will be known to those skilled in the art.

[0100] In some embodiments, the radionuclide is an alpha emitter (a radionuclide that emits alpha particles). The alpha-emitting radionuclide can include, but is not limited to, the following:225Ac,134Ce,213Bi,224Ra,212Pb,227Th,223Ra,211At, and149Tb. In some embodiments, the radionuclide is Actinium-225 (225Ac), an alpha particle emitter. Use of225Ac in the compositions of the present disclosure is particularly advantageous because it has a long half- life of 10 days due to its unique properties such as “nanogenerator” status and due to its uniqueability to produce a total of 4 and 2 particles in its decay chain.E. Methods of Treating Integrin2Expressing Cancers

[0101] In another aspect, provided herein are methods of treating certain cancers by administering to a subject a radioimmunoconjugate described herein. As is known to those skilled in the art, aberrant activation of ITGB2 is associated with human cancer cells such as leukemia cells, e.g., acute myeloid leukemia (AML) cells. The radioimmunoconjugates described herein can be used to treat aITGB2-expressing cancers. KILPATRICK TOWNSEND 795667431

[0102] As used herein, “treating a cancer” includes, but is not limited to, reversing, alleviating or inhibiting the progression of the cancer or symptoms or conditions associated with the cancer. “Treating the cancer” also includes extending survival in a subject. Survival is optionally extended by at least 1, 2, 3, 6 or 12 months, or at least 2, 3, 4, 5 or 10 years over the survival that would be expected without treatment with a radioimmunoconjugate as described herein. “Treating the cancer” also includes reducing tumor mass and / or reducing tumor. Optionally, tumor mass and / or tumor burden is reduced by at least 5, 10, 25, 50, 75 or 100% following treatment with a radioimmunoconjugate as described herein. “Treating the cancer” also includes reducing the aggressiveness, grade and / or invasiveness of a tumor.

[0103] In one embodiment, the cancer is an aITGB2-expressing cancer. In some embodiments, the cancer is leukemia. In some embodiments, the leukemia is AML.

[0104] As used herein, the terms “subject,” patient,” and “animal” include all members of the animal kingdom. In one embodiment, the subject is a mammal. In a further embodiment, the subject is a human being. In one embodiment, the subject is a patient having a disease, such as a cancer, e.g., an aITGB2-expressing cancer, such as AML.

[0105] In some embodiments, the radioimmunoconjugates disclosed herein are administered for a period necessary to prevent occurrence or recurrence of disease, to alleviate symptoms, to diminish any direct or indirect pathological consequences of the disease, to decrease the rate of disease progression, to ameliorate or palliate the disease state, and / or to bring about remission or to improve prognosis. In some embodiments, the period of time is (e.g., once or more a day for) 1-90 days, e.g., 1-60, 15-45, 5-15 days, e.g., 5-10 days, e.g., 3-10 days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 days.

[0106] Pharmaceutical compositions of the radioimmunoconjugates as described herein 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, KILPATRICK TOWNSEND 7956674312005, 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 herein by reference. Pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or efficacious dose of the immunoconjugates (antibody and radionuclide) descried herein is employed in the pharmaceutical compositions. The radioimmunoconjugates 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.

[0107] 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.

[0108] A therapeutically effective amount of the antibodies and radionuclide 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 KILPATRICK TOWNSEND 795667431can 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, about 400 ng to about 4,500 mg, about 500 ng to about 4,000 mg, about 1 μg to about 3,500 mg, about 5 μg to about 3,000 mg, about 10 μg to about 2,600 mg, about 20 μg to about 2,575 mg, about 30 μg to about 2,550 mg, about 40 μg to about 2,500 mg, about 50 μg to about 2,475 mg, about 100 μg to about 2,450 mg, about 200 μg to about 2,425 mg, about 300 μg to about 2,000, about 400 μg to about 1,175 mg, about 500 μg 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-aITB2 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 detrimental effects (i.e., side effects) of an antibody or antigen binding portion thereof or of the radionuclide are minimized and / or outweighed by the beneficial effects.

[0109] 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. KILPATRICK TOWNSEND 795667431

[0110] In some embodiments, the anti-ITGB2 antibody or immune effector cells comprising the antibody are administered with an antineoplastic agent or a chemotherapeutic agent. Examples of antineoplastic agents include venetoclax (Venclexta®), azacitidine (Vidaza, Onureg®), cytarabine, daunorubicin (Cerubidine®), idarubicin, mitoxantrone, arsenic trioxide, fludarabine, cyclophosphamide, cladribine, decitabine (Dacogen®), cytarabine / daunorubicine liposomal (Vyxeos®), gemtuzumab (Mylotarg®), mydostaurin (Rydapt®), gilteritinib (Xospata®), quizartinib (Vanflyta®), glasdegib (Daurismo™). Examples of cancer chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil; folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine; urethan; 35 KILPATRICK TOWNSEND 795667431vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside; cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; docetaxel, platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as bexarotene, alitretinoin; denileukin diftitox; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. F. Methods of Synthesizing Radioimmunoconjugates

[0111] In another aspect, provided herein are methods for making the radioimmunoconjugates as disclosed herein. The methods generally involve coupling an antibody, such as an antibody that binds aITGB2, to a chelator through a PEGylated linker to produce an antibody-chelator conjugate (ACC), and then radiolabeling the ACC with a radionuclide.

[0112] As will be appreciated by those skilled in the art, the chelator can be coupled to the antibody (or a fragment or portion thereof) either through a lysine residue or a cysteine residue on the antibody and will depend, in part, on the functional group(s) present on end of the linker that will be attached to the antibody. Exemplary Chelator, Linker, Functional Groups, and Conjugation Reactions on YS5 are set forth in Table 1, infra.KILPATRICK TOWNSEND 795667431

[0113] When the chelator is to be coupled or attached to a lysine residue on the antibody, the following exemplary reaction scheme can be used:. When the chelator is to be coupled or attached to a cysteine residue on the antibody, the following exemplary reaction scheme can be used:. When the chelator is DOTA or NOTA, the chelator can first be functionalized using the following exemplary reaction scheme: KILPATRICK TOWNSEND 7956674310.1 M NaCO3 / NaHCO3, pH= 9.0, 37oC, 1 h Chelator-NHS esterChelator=DOTA / NOTA .

[0114] A detailed synthetic protocol for preparing the radioimmunoconjugates is described in the published PCT application WO 2023 / 225408, which is incorporated by reference in its entirety.

[0115] The radioimmunoconjugate compounds may be prepared using the synthetic protocols disclosed herein and routine modifications thereof, which will be apparent given the disclosure herein and methods well known in the art. One of skill in the art will appreciate that other synthetic routes may be employed for preparation of the radioimmunoconjugate products and intermediates thereof. Conventional and well-known synthetic methods may be used in addition to the teachings herein. The synthesis of typical compounds and conjugates described herein may be accomplished as described in the following examples. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

[0116] Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. Suitable protecting groups for various functional groups as well as suitable conditions for protecting and deprotecting particular functional groups are well known in the art. For example, numerous protecting groups are described in Wuts, P. G. M., Greene, T. W., & Greene, T. W. (2006). Greene’s protective groups in organic synthesis. Hoboken, N.J., Wiley-Interscience, and references cited therein.

[0117] Materials for the synthetic protocols disclosed herein are generally known compounds or can be prepared by known procedures or obvious modifications thereof. For example, the materials used in the preparation of the radioimmunoconjugates described herein are available from commercial suppliers such as Biopharma PEG (Biochempeg) Scientific Inc. KILPATRICK TOWNSEND 795667431(Watertown, MA, USA), Quanta Biodesign Ltd. (Plain City, Ohio, USA), Aldrich Chemical Co. (Milwaukee, Wisconsin, USA), Merck Millipore Ltd. or Millipore Sigma (Burlington, MA, USA), National Isotope Development Center, Los Alamos National Laboratory (New Mexico, USA), Sino Biological US Inc. (Chesterbrook, PA, USA), BioLegend (San Diego, CA, USA), Sigma-Aldrich (St. Louis, Missouri, USA), or Thermo Fisher Scientific. Others may be prepared by procedures or obvious modifications thereof, described in standard reference texts such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-15 (John Wiley, and Sons, 1991), Rodd’s Chemistry of Carbon Compounds, Volumes 1-5, and Supplementals (Elsevier Science Publishers, 1989) organic Reactions, Volumes 1-40 (John Wiley, and Sons, 1991), March’s Advanced Organic Chemistry, (John Wiley, and Sons, 5th Edition, 2001), and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Illustrative Embodiments of the Disclosure

[0118] Embodiments of the disclosure include, but are not limited to, embodiments listed below: Embodiment 1. A method of treating cancer in a subject, the method comprising administering to the subject an immunoconjugate according to Formula I,wherein, X is a chelator moiety; Y is selected from the group consisting of -O- and -NR-; Z is a moiety selected from the group consisting of:wherein A is an antibody that specifically binds to activated integrin beta-2 (aITGB2); and further wherein the antibody comprises an aITGB2 binding domain comprising: KILPATRICK TOWNSEND 795667431a heavy chain variable region (VH) comprising an HCDR1 sequence comprising ISYYYM, an HCDR2 sequence comprising SISSSSGYTY; and an HCDR3 sequence comprising GAM; and a light chain variable region (VL) comprising an LCDR1 sequence comprising SVSSA, an LCDR2 sequence comprising SASSLYS; and an LCDR3 sequence comprising FSSGSWAPI. subscript m is 3 or 5; subscript n is 4, 6, 8, 10, 12, 14, or 16; and R selected from the group consisting of H, OH, and a negative charge; and wherein the cancer is a cancer that comprises cells expressing aITGB2. Embodiment 2: The method of Embodiment 1, wherein the cancer is acute myeloid leukemia (AML). Embodiment 3: The method of Embodiment 1 or 2, wherein the antibody comprises a VH comprising an amino acid sequence that comprises at least 95% identity to SEQ ID NO: 1 and a VL comprising an amino acid sequence that comprises at least 95% identity to SEQ ID NO: 2. Embodiment 4: The method of Embodiment 3, wherein the antibody comprises a VH comprising amino acid sequence SEQ ID NO: 1 and / or a VL comprising amino acid sequence SEQ ID NO: 2. Embodiment 5: The method of any one of Embodiments 1-4, wherein the immunoconjugate comprises a structure according to Formula Ia:Embodiment 6: The method of any one of Embodiments 1-4, wherein the immunoconjugate comprises a structure according to Formula Ib:Embodiment 7: The method of any one of Embodiments 1-6, wherein the chelator moiety X is selected from the group consisting of: KILPATRICK TOWNSEND 795667431Embodiment 8: The method of any one of Embodiments 1-7, wherein: the chelator moiety X is:; Y is -O-; and subscript m is 3. Embodiment 9: The method of any one of Embodiments 1-7, wherein: KILPATRICK TOWNSEND 795667431the chelator moiety X is:; Y is -NR-; and subscript m is 3. Embodiment 10: The method of any one of Embodiments 1-7, wherein: the chelator moiety X is:; Y is -NR-; and subscript m is 3. Embodiment 11: The method of any one of Embodiments 1-7, wherein: the chelator moiety X is:Y is -NR-; and subscript m is 5. KILPATRICK TOWNSEND 795667431Embodiment 12: The method of any one of Embodiments 1-11, wherein subscript n is 4, 6, 8, or 12. Embodiment 13: The method of any one of Embodiments 1-12, wherein subscript n is 4 or 8. Embodiment 14: The method of any one of Embodiments 1-13, wherein the immunoconjugate further comprises an alpha-emitting radionuclide, wherein the chelator moiety of the immunoconjugate chelates the alpha-emitting radionuclide. Embodiment 15: The method of any one of Embodiments 1-14, wherein the alpha-emitting radionuclide is selected from the group consisting of225Ac,134Ce,213Bi,224Ra,212Pb,227Th,223Ra,211At, and149T. Embodiment 16: The method of Embodiment 15, wherein the alpha-emitting radionuclide is225Ac. Embodiment 17: The method of Embodiment 14, wherein the immunoconjugate comprises a structure according to Formula IIa:and M is the alpha-emitting radionuclide. Embodiment 18: The method of Embodiment 14, wherein the immunoconjugate comprises a structure according to Formula IIb:KILPATRICK TOWNSEND 795667431and M is the alpha-emitting radionuclide, and subscript p is 0 or 1. Embodiment 19: The method of Embodiment 14, wherein the immunoconjugate comprises aand M is the alpha-emitting radionuclide, and subscript p is 0 or 1. Embodiment 20: The method of Embodiment 14, wherein the immunoconjugate comprises aand M is the alpha-emitting radionuclide. Embodiment 21: The method of any one of Embodiments 17-20, wherein the alpha-emitting radionuclide is225Ac. Embodiment 22: A method of treating cancer in a subject, the method comprising administering to the subject an immunoconjugate according to Formula IIa,KILPATRICK TOWNSEND 795667431wherein M is an alpha-emitting radionuclide225Ac; A is an antibody that specifically binds to activated integrin beta-2 (aITGB2), wherein the antibody comprises a VHcomprising amino acid sequence SEQ ID NO: 1 and a VLcomprising amino acid sequence SEQ ID NO: 2 ; and subscript n is 4; and wherein the cancer is a cancer that comprises cells expressing aITGB2. Embodiment 23: The method of Embodiment 22, wherein the cancer is AML. Embodiment 24: The method of any one of Embodiments 1-23, wherein the immunoconjugate is administered with a pharmaceutically acceptable excipient. Embodiment 25: An immunoconjugate according to Formula I,wherein, X is a chelator moiety; Y is selected from the group consisting of -O- and -NR-; Z is a moiety selected from the group consisting of:wherein A is an antibody that specifically binds to activated integrin beta-2 (aITGB2); and further wherein the antibody comprises an aITGB2 binding domain comprising: a heavy chain variable region (VH) comprising an HCDR1 sequence comprising ISYYYM, an HCDR2 sequence comprising SISSSSGYTY; and an HCDR3 sequence comprising GAM; and a light chain variable region (VL) comprising an LCDR1 sequence comprising SVSSA, an LCDR2 sequence comprising SASSLYS; and an LCDR3 sequence comprising FSSGSWAPI. KILPATRICK TOWNSEND 795667431subscript m is 3 or 5; subscript n is 4, 6, 8, 10, 12, 14, or 16; and R selected from the group consisting of H, OH, and a negative charge. Embodiment 26: The immunoconjugate of Embodiment 25, wherein the antibody comprises a VH comprising at least 95% identity to SEQ ID NO: 1 and a VL comprises an amino acid sequence comprising at least 95% identity to SEQ ID NO: 2. Embodiment 27: The immunoconjugate of Embodiment 26, wherein the antibody comprises a VH comprising amino acid sequence SEQ ID NO: 1 and / or a VL comprising amino acid sequence SEQ ID NO: 2. Embodiment 28: The immunoconjugate of Embodiment 25, 26, or 27, comprising a structure according to Formula Ia:Embodiment 29: The immunoconjugate of Embodiment 25, 26, or 27, comprising a structure according to Formula Ib:Embodiment 30: The immunoconjugate of any one of Embodiments 25-29, wherein the chelator moiety X is selected from the group consisting of:KILPATRICK TOWNSEND 795667431. Embodiment 31: The immunoconjugate of any one of Embodiments 25-30, wherein: the chelator moiety X is:; Y is -O-; and subscript m is 3. Embodiment 32: The immunoconjugate of any one of Embodiments 25-30, wherein: the chelator moiety X is: KILPATRICK TOWNSEND 795667431; Y is -NR-; and subscript m is 3. Embodiment 33: The immunoconjugate of any one of Embodiments 25-30, wherein: the chelator moiety X is:Y is -NR-; and subscript m is 3. Embodiment 34: The immunoconjugate of any one of Embodiments 25-30, wherein: the chelator moiety X is:Y is -NR-; and subscript m is 5. KILPATRICK TOWNSEND 795667431Embodiment 35: The immunoconjugate of any one of Embodiments 25-34, wherein subscript n is 4, 6, 8, or 12. Embodiment 36: The immunoconjugate of Embodiment of claim 35, wherein subscript n is 4 or 8. Embodiment 37: The immunoconjugate of any one of Embodiments 25-36, wherein the immunoconjugate further comprises an alpha-emitting radionuclide, wherein the chelator moiety of the immunoconjugate chelates the alpha-emitting radionuclide. Embodiment 38: The immunoconjugate of Embodiment 37, wherein the alpha-emitting radionuclide is selected from the group consisting of225Ac,134Ce,213Bi,224Ra,212Pb,227Th,Embodiment 39: The immunoconjugate of Embodiment 38, wherein the alpha-emitting radionuclide is225Ac. Embodiment 40: The immunoconjugate of Embodiment 37, wherein the immunoconjugate comprises a structure according to Formula IIa:(IIa), and M is the alpha-emitting radionuclide. Embodiment 41: The immunoconjugate of Embodiment 37, wherein the immunoconjugate comprises a structure according to Formula IIb: KILPATRICK TOWNSEND 795667431and M is the alpha-emitting radionuclide, and subscript p is 0 or 1. Embodiment 42: The immunoconjugate of Embodiment 37, wherein the immunoconjugate comprises a structure according to Formula IIc:and M is the alpha-emitting radionuclide, and subscript p is 0 or 1. Embodiment 43: The immunoconjugate of Embodiment 37, wherein the immunoconjugate comprises a structure according to Formula IId:and M is the alpha-emitting radionuclide. Embodiment 44: The immunoconjugate of any one of Embodiments 40-43, wherein the alpha- emitting radionuclide is225Ac. KILPATRICK TOWNSEND 795667431Embodiment 45: A pharmaceutical composition comprising the immunoconjugate of any one of Embodiments 25-44 and a pharmaceutically acceptable excipient. Embodiment 46: An immunoconjugate according to Formula IIa,(IIa), wherein M is an alpha-emitting radionuclide225Ac; A is an antibody that specifically binds to activated integrin beta-2 (aITGB2), wherein the antibody comprises a VHcomprising amino acid sequence SEQ ID NO: 1 and a VLcomprising amino acid sequence SEQ ID NO: 2 ; and subscript n is 4; and wherein the cancer is a cancer that comprises cells expressing aITGB2. Embodiment 47: A pharmaceutical composition comprising the immunoconjugate of Embodiment 46 and a pharmaceutically acceptable excipient. EXAMPLES

[0119] The following examples are intended for illustrative purposes only and do not limit in any way the scope of the present invention. Material and Methods for Technical Evaluation

[0120] Therapeutic antibodies. 7065 scFv sequence (see Mandal, Kamal et al. Nature Cancer vol.4,11 (2023)) was closed into a human IgG1 framework. This clone was expressed in Expi293 mammalian cells and purified via Protein A resin, followed by endotoxin removal. The purified antibody’s molecular weight and heavy chain and light chain pattern were confirmed via Coomassie gel and size exclusion chromatography. The purified antibody also KILPATRICK TOWNSEND 795667431demonstrated binding to recombinant ITGB2 via BioLayer Interferometry with a KD of 1.56 ± 0.10 nM. See Mandal, Kamal et al. Nature Cancer vol.4,11 (2023).

[0121] The anti-CD33 biosimilar, Lintuzumab, was purchased from Creative Biolabs and certified for purity and molecular weight.

[0122] Radiopharmaceuticals. Synthesis of [225Ac]Macropa-PEG4-7065 was performed as described in Bobba, K. N. et al. J Nucl Med 64, 1076-1082 (2023) and International Patent Application Publication No. WO 2023 / 225408. Actinium-225 (225Ac) was obtained from U.S. Department of Energy from the229Th generator decay pathway. See van Cleve, S. et al. J Med Imag Rad Sci 50 (Supplement), S11-S12 (2019). [89Zr]DFO*-7065 was used for PET imaging.

[0123] The DFO* chelator was used instead of the more commonly used DFO chelator because of DFO* had demonstrated reduced de-chelation and non-specific bone uptake. See Chomet, M. et al. Eur J Nucl Med Mol Imaging 48, 694-707 (2021). PET imaging in the preclinical studies was used to determine the biodistribution of radiotherapeutic agents.

[0124] In contrast, owing to low administered activity and emission rates of imageable photons, of [225Ac]Macropa-PEG4-7065 biodistribution was determined with classic “cut and count” methodology, which is impractical and expensive across a larger panel of models. Radiopharmaceuticals were prepared as detailed below (see also, FIG.1A-F) and characterized by iTLC and size exclusion chromatography.

[0125] The [225Ac]Macropa-PEG4-Lintuzumab biosimilar (also referred to herein as “[225Ac]anti-CD33”) was generated as previously described in Garg, R. et al. Cancer Med 10, 1128-1140 (2021). This agent mimics the [225Ac]-lintuzumab drug now in clinical trials, e.g., ClinitalTrials.gov ID NCT03705858. For a radiotherapeutic negative control, commercially available non-targeting IgG was used to generate [225Ac]Macropa-PEG4-IgG.

[0126] Conjugation of antibodies with chelators, radiolabeling, and stability analyses are detailed in the following sections. Antibody Conjugation with Chelators

[0127] For conjugation of the 7065 antibody with DFO*-NCS, 173.9 L (1 mg) of 7065 IgG antibody, pre-dissolved in PBS, was exchanged with 0.1 M Na2CO3 / NaHCO3buffer at pH 9.5 through three cycles of centrifugation at 10,000 rpm for eight minutes, achieving a final concentration of 7.23 mg / ml. This exchange was performed using a YM30K MW centrifugal KILPATRICK TOWNSEND 795667431filter (Millipore, MA, USA). DFO*-NCS (ABX, Product Number 7272) was pre-dissolved in anhydrous DMSO (1 mg / 40 L). The 7065 antibody was incubated with 5 equivalents of DFO*-NCS (1.27 L) at 37°C for 45 minutes. For purification, the mixture was passed through a PD10 gel column (GE Healthcare) pre-treated with 0.25 M NaOAc (pH 6), and elution through the column was performed using 0.25 M NaOAc. A non-targeting IgG antibody (negative control) was purchased from AbCam (catalog number AB91102), and conjugation of IgG with DFO*-NCS was carried out in the same manner as for the 7065 antibody.

[0128] For conjugation with Macropa-PEG4-TFP, the 7065 antibody underwent bufferexchange through three cycles with 0.1 M Na CO / NaHCO buffer at pH 9.5 by centrifugationat 10,000 rpm for eight minutes, achieving a final concentration around 15 mg / ml. The bifunctional chelator Macropa-PEG4-TFP was prepared as previously described21. It was dissolved in DMSO (1 mg / 30 L). The 7065 antibody was incubated with 7.5 equivalents of Macropa-PEG4-TFP (1.45 L) at 37°C for 2 hours. Following the conjugation process, the reaction mixture was passed through a PD10 column, and elution was performed with 0.25 M NaOAc buffer. The conjugation of IgG with Macropa-PEG4-TFP was carried out similarly to the conjugation of the 7065 antibody. The resulting conjugate, Macropa-PEG4-7065 and Macropa-PEG4-IgG, were stored at -20°C and used without further purification for analysis and radiolabeling. Lintuzumab (anti-CD33) antibody was purchased from Creative Biolabs (catalog number TAB-756), and the conjugation of the anti-CD33 antibody with DOTA-NCS was performed according to a previously published procedure1. Radiolabeling with89Zr(C2O4)2,134CeCl3and225Ac(NO3)3

[0129] For labeling with Zirconium-89, a one step process was used.89Zr-oxalate was purchased from 3D Imaging (Little Rock, AK). 3-5 μL of89Zr-oxalate (37 MBq) was mixed with equal amount of 1 M Na2CO3(3-5 μL) and 200 μL of 1M NH4OAc, followed by the addition of 130 μg of DFO*-7065 (7.2 mg / ml, pre-dissolved in 0.25 M NaOAc) and the mixturewas incubated at 37 C for 1 hour. The mixture was subjected to instant thin-layerchromatography (iTLC) for radiochemical yield (eluted with 10 mM EDTA, pH 5.5) to confirm reaction completion, and purified by eluting through PD10 column with 0.9% saline as mobile phase. A similar procedure was followed for89Zr radiolabeling of DFO*-IgG.

[0130] 134Ce(NO3)3in 0.1 M HCl was purchased from Department of Energy Isotope Production Program22. For radiolabeling,134CeCl3(100 μL, 111 MBq) was mixed with 200 μl KILPATRICK TOWNSEND 795667431of 2M NH4OAc (pH 8), 200 μg of Macropa-PEG4-7065 (15.87 mg / ml) was added, and thereaction mixture was incubated at 37 C for 1 hour. The reaction yield was analyzed by iTLCSG (using 10 mM EDTA pH 5.5 as a mobile phase), allowing 60 minutes to pass to achieve secular equilibrium prior to plate imaging. The reaction mixture was purified by eluting through PD10 with 0.9% saline as mobile phase.

[0131] 225Ac(NO3)3 was purchased from the Department of Energy Isotope Production Program and produced at Oak Ridge National Laboratory via the229Th generator route23. A single step process was followed for radiolabeling with Ac-225. The Ac-225 received from ORNL (typically approximately 27.75 MBq- 29.6 MBq) was dissolved in 100 μL of 0.2 M HCl. An aliquot of 4 μL (approx.1.11 MBq of Ac-225) was added to a vial.50 μL of NH4OAc (2M, pH 5.8) and 20 μL of L-Ascorbic acid (150 mg / ml) and 120 μg of Macropa-PEG4-7065(7.6 μL) was added to the Ac-225 containing vial. The reaction was incubated at 37 C for 30minutes. The radiochemical yield was monitored by ITLC-SG, eluted with 10 mM EDTA (pH 5.5). For purification and buffer exchange to 0.9% saline, the reaction mixture was passed through YM30K centrifugation filter with three washes and final radioimmunoconjugate purity was analyzed by ITLC-SG. For labelling of Macropa-PEG4-IgG, similar procedure was followed as for radiolabeling of Macropa-PEG4-7065. For radiolabeling of DOTA-anti-CD33 with225Ac, an aliquot of 4 μL (approx. 1.11 MBq) of Ac-225 was added to a vial. 50 μL of NH4OAc (2M, pH 5.8) and 20 μL of L-Ascorbic acid (150 mg / ml) and followed by the addition of 180 μg of DOTA-anti-CD33 (19.07 μL) to the Ac-225 containing vial. The reaction wasincubated at 37 C for 1 hour. The radiochemical yield was determined by ITLC-SG, elutedwith 10 mM EDTA (pH 5.5). For purification and buffer exchange to 0.9% saline, the reaction mixture was passed through YM30K centrifugation filter by three washes and final radioimmunoconjugate purity was analyzed by ITLC-SG. Stability Studies

[0132] For stability studies, [89Zr]DFO*-7065 was purified using a PD10 pre-treated with 0.1 % ascorbic acid in saline to prevent radiolysis and reaction mixture was passed through this pre-treated PD10 column using the 0.1 % ascorbic acid as an eluent. [89Zr]DFO*-7065 (37 MBq radiochemical purity > 98%, 90-100 μL) was mixed with 500 μL of either saline or human serum at 37°C. At the indicated time points, 10 μL aliquots were analyzed by ITLC-SG using 10 mM EDTA (pH=5.5) as a mobile phase. KILPATRICK TOWNSEND 795667431

[0133] [225Ac]Macropa-PEG4-7065 (10-15 μCi, Radiochemical purity 95 % after purification through centrifugal filtration) was diluted with 500 μL of either saline or humanserum at 37 C. At the indicated time points, 5-10 μL aliquots were analyzed by ITLC-SG using10 mM EDTA (pH 5.5) as an eluent. All radio-ITLC’s were scanned after 24 h, to achieve secular equilibrium.

[0134] Size Exclusion Chromatography (SEC). The radiopharmaceuticals [89Zr]DFO*-7065 and [225Ac]Macropa-PEG4-7065 were analyzed using size exclusion chromatography. The LabLogic Logi-CHROM HPLC instrument was used, and the column used was a BioSep™ SEC-s3000290 Å with dimensions of 300 × 7.8 mm.

[0135] For optimization of size exclusion chromatography for [225Ac]Macropa-PEG4-7065, 10 μL of [225Ac]Macropa-PEG4-7065 was mixed 40 μl of a saline and injected in Lablogic HPLC instrument and passed through 3000 SEC column.0.1% IX PBS was used as the eluent (mobile phase) and 20 minutes of chromatogram has been recorded with a flow rate of 1ml / minute. 1 ml of fractions has been collected per tube and total 20 fraction were collected and counted on Hidex before and after secular equilibrium and the graph between time and Count per minute was plotted.

[0136] The following methods were employed to evaluate conjugates in vitro and in vivo. In vitro analyses

[0137] Cell lines. Nomo-1, HL-60, THP-1, MV411 cell lines were purchased from ATCC. All cells were maintained in RPMI1640 medium supplemented with 10% FBS, 100 U penicillin, and 100 μg / ml streptomycin in a humidified incubator at 37°C and 5% CO2. Cell lines used in our studies were negative from mycoplasma contamination when routinely tested with the bioluminescence based MycoAlert Mycoplasma Detection Kit.

[0138] Cell lines in which ItgB2 is knocked out were generated using in vitro nucleofectionof Cas9 ribonuclease protein complex. Unless otherwise specified, 2 l of each ItgB2 sgRNA(100 μM; Synthego Corporation) and recombinant Cas9 protein (40 μM; QB3 MacroLab,University of California, Berkeley) was incubated at 37 °C for 15 min to generate ribonucleasecomplex, which was then nucleofected using a 4D-Nucleofector (Lonza) with the built-in program DS-137 for cell lines (using Lonza V4XC-2032) unless otherwise specified. KILPATRICK TOWNSEND 795667431Knockout cell lines were allowed to recover before being purified through Fluorescent Activated Cell Sorting (FACS). sgRNA sequences were obtained from Brunello DNA Library. Generation of Luc+ cells

[0139] Generation of Luc+ cells. Nomo-1, HL-60, THP-1, MV411, Nomo-1 ITGB2 KO cells were modified to express mCherry and luciferase according to a previous established procedure24and transduced with a lentiviral expressing mCherry fluorescent and luciferase tag.

[0140] Cell Binding Assay. Five million cells (Nomo-1, Nomo-1 ITGB2 KO, HL-60, THP- 1, MV411) were dissolved in 100 μL of PBS (in triplicate).1 nM (0.076 ng; 0.50 μl, 6.92 MBq) solution of [89Zr]DFO*-7065 (original solution concentration is 433.3 nM) was prepared in 100 μl PBS and 1% nonfat milk (20 μl, 0.1 mg / ml) was added to each vial containing cells.The cells were incubated with [89Zr]DFO*-7065 solution for 1 hour at 37 C. Cells werecentrifuged after one hour and supernatant was removed. The pallet was washed with PBS twice and radioactivity bound to cell pellet was counted using a Hidex Gamma Counter using a 480 to 558 KeV counting window, comparing to standards of known radioactivity (6.92 MBq). Cell-associated activity percentage was calculated by cell pallet activity / 6.92 MBq [89Zr]DFO*-7065 activity.

[0141] In order to test for Fc-mediated binding, a separate experiment was conducted utilizing an excess of non-targeting IgG. 1 nM (0.099 ng; 0.66 μl, 6.92 MBq) solution of [89Zr]DFO*-7065 (original solution concentration is 333.3 nM) was prepared in 100 μl PBS and 1% nonfat milk (20 μl, 0.1 mg / ml) was added to each vial containing cells. Cells were centrifuged after one hour and supernatant was removed. The pallet was washed with PBS twice and radioactivity bound to cell pallet was counted using a Hidex Gamma Counter. The radioactivity associate with cell pallet and 0.5 nM (9.25 KBq solution) was counted using a Hidex Gamma Counter using a 480 to 558 KeV counting window. Cell-associated activity percentage was calculated by cell pallet activity / 9.25 KBq [89Zr]DFO*-7065 activity.

[0142] Saturation Binding Assay. The kd value of [89Zr]DFO*-7065 with aITGB2 expressing cell lines (Nomo-1, Nomo-1 ITGB2 KO, HL-60, THP-1, MV411) was determined through saturation binding assay. Aliquots of 1 million of each cell type were dissolved in 100 μL PBS, with 15 vials total for each cell line. 20 μL of 0.1% milk in PBS was added to blockthe non-specific binding. After 1 hour in incubation at 37 C, various concentrations of[89Zr]DFO*-7065 (0.001 nM- 10 nM, 100 uL / vial, triplicate) in PBS was added to each vial 56 KILPATRICK TOWNSEND 795667431and incubated at 37 C for 1 hour. Cells were centrifuged after one hour and supernatant wasremoved. The pellet was washed with 800 μL PBS twice and radioactivity bound to cell pallet was counted using a Hidex Gamma Counter. Kd values and Bmax was calculated by non-linear regression, utilizing one-site specific binding using GraphPad Prism Software. The Kd value of [225Ac]Macropa-PEG4-7065, and for negative control [225Ac]Macropa-PEG4-IgG as well as for positive control [225Ac]DOTA-anti-CD33 with the Nomo-1 cell line was determined by a similar procedure.

[0143] Membrane Binding and Internalization assay. Three million Nomo-1 or HL-60 cells were dissolved in 100 μL of PBS (in triplicate). 1 nM (0.099 ng; 0.66 μl) solution of [89Zr]DFO*-7065 (original solution concentration is 433.3 nM) was prepared in 100 μl PBS and 1% nonfat milk (20 μl, 0.1 mg / ml) and added to each vial containing cells. The total volume of the solution was 220 μl. The cells were incubated with [89Zr]DFO*-7065 solution for 1 h, 4h and 24 h at 37 C. After incubation, the cells were centrifuged and washed with PBS twice.Cells were treated with a solution of 50 mM glycine / 100 mM NaCl for 5 minutes at 4 C andsupernatant corresponding to the membrane bound fraction was removed, and separated from the pellet which contained the internalized fraction. The membrane bound and internalized fractions were calculated by activity associated with supernatant / 6.92 MBq solution of 1 nM [89Zr]DFO*-7065.

[0144] Immunoreactivity Assay: Lindmo Assay. In centrifuge tubes, six aliquots of 5 106Nomo-1 cells in 500 μL PBS were prepared (n =3 for both experiment and blocking group). Additional similar aliquots were prepared containing 2.5 106cells, 1.25 106cells, 0.0625 106cells, 0.3125 106and 0.1525 106cells.50 μg of cold 7065 IgG was added to the aliquots in the blocking group to saturate the antigen on the cells. All the samples were incubated on the ice for 30 min and manually tubes were agitated after every 10 mins to prevent the formation of cell pallet.

[0145] In each tube 11.9 μl of a solution containing 40 ng / ml (266.6 pM) of [89Zr]DFO*- 7065 in PBS was added and incubated on ice 1 h. After 1 hour, all the treated and blocked samples were centrifuged at 650 rpm for 3 min and supernatant was collected in different tubes. Cells were washed twice with PBS and cell pellet and supernatant activity was counted using hidex gamma counter. The ratio of cell associated activity to the total radioactivity of the each KILPATRICK TOWNSEND 795667431sample was plotted as a function of cells increasing concentration and inverse of Y-slope (1 / Bmax) of the resulting curve denotes the immunoreactive fraction of the [89Zr]DFO*-7065.

[0146] Cell killing Assay. For each indicated cell line, 2000 cells were plated in a 96 well black opaque plate. Various concentration of [225Ac]Macropa-PEG4-7065, [225Ac]DOTA-anti- CD33 (ranging from 0.001 pCi / ml to 10 nCi / ml) in 10% RPMI media were incubated with the cells for 96 hours. After treatment, bioluminescence signals of the cells were recorded with multiplate reader (TECAN software) after 10 minutes incubation by Cell Titre Glo® according to the manufacturer’s instructions. The % cell viability was fitted into a sigmoidal dose- response curve to determine IC50value using GraphPad Prism.

[0147] Colony formation assay. Two hundred Nomo-1, Nomo-1 ITGB2 KO, HL-60, or MV411 cells were seeded in 1 mL of media per well in a 6-well plate. The cells were treated with varying concentrations ranging from 0.1 nCi / ml to 50 nCi / ml in 10% RPMI media of [225Ac]Macropa-PEG4-7065 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 MethoCult media (H4230 without cytokines) in 6-well plates. MethoCult (#04230) was purchased from Stem Cell Technologies and was thawed overnight at 4°C or for 2–3 hours at room temperature. AML cells, being non-adherent, do not attach to surfaces; MethoCult 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. In vivo analyses

[0148] Animal models. All animal studies were performed according to Institutional Animal Care and Use Committed-approved protocols at the University of California (AN194778). For imaging purpose, a mixture of male and female NOD SCID gamma (commonly named as NSG, stock number 005557) aged 7 to 9 weeks old were purchased from Jackson laboratory. For therapy in AML disseminated model, a mixture of male and female NOD Rag gamma aged 7 to 9 weeks (commonly known as NRG, stock number from Jackson Lab 007799) were obtained from a breeding colony maintained at UCSF. The NRG mouse strain was characterized by the vendor Jackson Labs to be less sensitive to radiation-induced toxicity compared to the NSG mouse strain. Cells were introduced into mice by intravenous administration via the tail vein. KILPATRICK TOWNSEND 795667431

[0149] In vivo disseminated model formation. To generate the disseminated AML models, 4 million Nomo-1, Nomo-1 ITGB2 KO, HL-60, MV411, THP-1, or HL-60 cells in PBS were injected by tail vein into a cohort of mix of male and female NSG mice aged between 7 to 9 weeks. Bioluminescence Imaging was used to monitor the tumor engraftment. Tumor growth was heterogeneous and differed between cell lines, but mostly accumulated in the bone marrow and liver. When mice showed bioluminescence signal within a reference range of 105to 107photons / sec / cm2 / steradian, mice were injected with [89Zr]DFO*-7065 followed by PET imaging and biodistribution study.

[0150] Bioluminescence Imaging. In vivo BLI was performed to monitor the tumor burden / lesions in mice inoculated with luciferase-tagged AML cells. D-Luciferin was purchased from LUCK-1G-Gold Biotechnology. One gram of D-Luciferin was dissolved in 33 ml of PBS. 100 μl solution (approx. 150 mg / kg) of Luciferin in PBS was injected intraperitoneally into mice, and mice were allowed to move freely for 8-10 minutes. After 8 minutes, mice were anesthetized and imaged using IVIS 50 (PerkinElmer). Images were acquired after 60 seconds of exposure time and the intensity of Luciferase-tagged AML cells was quantified as radiance which has a unit photons / sec / cm2 / steradian using spherical region of interest using Living Image 4.0 software.

[0151] For ex vivo BLI, mice were injected 100 μl of D-Luciferin. After 8 minutes mice were sacrificed, organs were dissected and placed in a petri dish. All the organs are imaged with 60 seconds of exposure time and intensity of signal has been recorded in photons / sec / cm2 / steradian. In vivo PET Imaging

[0152] General PET Imaging method. Approximately 5 to 6 weeks after tumor implantation, when mice showed the BLI signal within a reference range 105to 107photons / sec / cm2 / steradian, each mouse was injected with 3.7-5.55 MBq, 10 μg (n=4) of the indicated radiopharmaceutical ([89Zr]DFO*-7065, [89Zr]DFO*-IgG, [89Zr]DFO*-7065 + 25 fold excess of 7065, [134Ce]Macropa-PEG4-7065). In all cases, 0.5 mg non-specific binding cold IgG was co-administered to block the Fc receptor in NSG mice25. The animals were imaged at day 1, day 2, and day 4 post injection of all the radiotracer. μPET / CT imaging data were acquired using a small animal PET / CT scanner (nanoScan PET123S / CT1512, Mediso Medical Imaging Solutions) using a multi-animal bed that enables scanning four mice KILPATRICK TOWNSEND 795667431simultaneously. The mice were anesthetized with ~2% isoflurane. A custom-made intravenous catheter was used for tail vein administration of radiotracer. PET data with 15 cm axial field of view (FOV) were acquired for 20 minutes in list mode, followed by helical CT for anatomical localization and correction for attenuation and scatter. The helical pitch for CT was 1.0 for 2.57 rotations to cover the matched axial FOV of PET. X-ray tube settings were 50 kVp and 0.98 mA with exposure time of 170 ms at each angle for 360 projections per rotation. CT data were reconstructed using the vendor-provided cone-beam filtered back projection (FBP) with a cosine filter. The reconstructed CT volume was in the matrix of 486´486´603 with the isotropic voxel size of 0.25 mm. PET data were reconstructed using the vendor-provided 3D iterative algorithm with 4 iterations and 6 subsets. All corrections (randoms, attenuation, and scatter) were applied for PET reconstruction, and the vendor-developed body-air segmentation method using coregistered CT was used for attenuation correction. The reconstructed PET volume was in the matrix of 225´225´366 with the isotropic voxel size of 0.4 mm.

[0153] For [18F]-FDG imaging in AML models Nomo-1 and THP-1 when BLI signal reached in the reference range, mice were injected with 7.4 MBq- 8.14 MBq of [18F]-FDG (n=4) / group and similar imaging procedure has been followed as previously described followed by a biodistribution study26.

[0154] Ex vivo Biodistribution and Ex vivo PET / CT imaging. Following PET / CT imaging of mice injected with [89Zr]DFO*-7065 in Nomo-1, MV411, THP-1, HL-60 model and as well as in negative model, mice were injected with 100 μl of D-Luciferin and BLI images were acquired with 60 seconds of exposure time. Mice were sacrificed after BLI and blood was collected by cardiac puncture. Major organs (femur, liver, heart, kidney, small intestine, large intestine, spleen, pancreas, lungs, stomach and brain) were harvested and collected in a Petri dish. Organs were imaged with BLI followed by PET / CT imaging using the imaging acquisition parameters after above. After PET / CT imaging, bone marrow was extracted usinga previously published procedure26. All the organs were collected in tubes and counted in HidexGamma Counter and

[0155] Dosimetry calculation. Dosimetry calculations were performed by determining time- integrated activity coefficients and applying curve-fitting techniques within the EXM module of OLINDA / EXM Version 1.127. For this analysis, the digital mouse phantom provided in OLINDA Version 2.0 was used. Biodistribution studies were performed at day 1, day 2, day 4 KILPATRICK TOWNSEND 795667431and day 7 using the same procedure as above to calculate % IA / gram of [225Ac]Macropa-PEG4- 7065 in NRG mice bearing Nomo-1 disseminated xenograft (n=4 per group). Data from biodistribution study were systematically organized to calculate the time and percentage of injected activity for each organ and tumor. These values served as inputs to obtain time- integrated activity coefficients. Finally, the equivalent dose (Sv) was calculated by multiplying the absorbed dose (Gy) with radiation weighting factors.

[0156] Therapy Study in Nomo-1 disseminated model. One million Nomo-1 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 five 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 μL of saline + 0.5 mg non-specific binding IgG), 2) 9.25 KBq of [225Ac]Macropa- PEG4-IgG, 3) 9.25 KBq [225Ac]DOTA-anti-CD33, and 4) 9.25 KBq of [225Ac]Macropa-PEG4- 7065. An additional fractionated dose treatment, involving a total of three doses of 9.25 KBq of [225Ac]Macropa-PEG4-7065, was also included in the study. The fractionated doses of [225Ac]Macropa-PEG4-7065 were administered on days 0, 28, and 56, respectively. 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. After euthanasia, bone marrow and spleen were harvested and used for flow cytometry analysis of the ITGB2 population in relapsed mice after treatment. On day 150, the study was terminated, and for the remaining mice from two cohorts, tissues were harvested and analyzed for toxicity. Dose limiting organs including kidney, liver, lungs, spleen, heart and bone were extracted and fixed in 10% formalin. Histologic analysis was carried out to examine the microscopic features of the tissues. For Hematoxylin and Eosin (H&E) staining, the tissues were fixed in formalin, processed through EtOH gradient (30% to 70%), and embedded in paraffin. Tissue sections with a thickness of 4 μm were prepared for H & E staining at Comparative Pathology Laboratory, School of Veterinary Medicine, University of California Davis. KILPATRICK TOWNSEND 795667431

[0157] Additionally, organs, bone marrow, and spleen from three relapsed mice in each group (saline, 9.25 KBq [225Ac]Macropa-PEG4-IgG, and 9.25 KBq [225Ac]Macropa-PEG4- 7065) were utilized for proteomics studies.

[0158] FACS Study to analyze the ITGB2+ tumor population in relapsed mice. Spleens and bone marrows were harvested from mice at humane endpoints. Samples were spun down at 400g for 5 minutes and resuspended in 1X Ammonium Chloride (ACK) Lysis, then gently mixed on a rocker at room temperature for 10 minutes. Samples were then spun at 500g for 5 minutes and ACK lysis was decanted into ethanol. ACK Lysis steps were repeated until minimal RBC was visible in the cell pellet. Cell pellets were then resuspended in 5mL of Bulk Lyse Wash Solution (PBS + 0.1% Sodium Azide + 0.5% Bovine Serum Album (BSA)) and run through a 70μm filter. Cells were resuspended in the FACS Buffer (PBS + 2%FBS + 2mM EDTA) and proceeded to FACS staining protocol. Immunostaining of cells was performed as per the instructions from the antibody vendor unless stated otherwise. Cells were resuspendedat 1 × 106 cells / 100 μl of FACS buffer with 5μg of human Fc Block (Biolegend, 422302) added.Cells were incubated at 22 °C for 10–15 min, then 3μg of CD45-APC (Biolegend 368512)antibody was added. Cells were incubated at 4°C for 30-45 minutes protected from light then washed three times with FACS buffer and filtered through a 70μm filter. All samples were gated on FSC-A / SSC-A for lymphocyte population, then single cells gated in SSC-A / SSC-H, and tumor cells were gated on CD45+. Cells were sorted on a BD Biosciences FACSAria Fusion 2 Cell Sorter using FMO and unstained controls to establish gates. Toxicity study in healthy NRG, Black C57BL / 6 and humanized mice

[0159] Acute toxicity in black mice. The toxicity of [225Ac]Macropa-PEG4-7065 was evaluated in healthy Black C57BL / 6 mice aged 5-6 weeks (Jackson Laboratory). For acute toxicity, the mice were divided into four groups (n=5 mice per group). Treatment groups included saline control, 9.25 KBq dose of [225Ac]DOTA-anti-CD33, 9.25 KBq of [225Ac]Macropa-PEG4-7065, and 9.25 KBq of [225Ac]Macropa-PEG4-IgG. The mice were monitored, and their body condition score and body weights are recorded at every alternate day. At day 7, peripheral blood was withdrawn by submandibular vein and blood parameters was analyzed by Hemavet 950 / 950FS Multi-Species Analyzer. At day 28, mice were sacrificed, blood was collected through cardiac puncture and stored in EDTA coated tubes to prevent coagulation and blood parameters were analyzed. Serum samples were obtained by allowed the KILPATRICK TOWNSEND 795667431blood containing vials to sit at 4 C for 30 minutes to separate the serum from the clotted blood.The serum samples were separated from the clots by centrifugation at 10,000 rpm for 10minutes at 4 C. The blood and serum samples were sent to the pathology laboratory atComparative Pathology Laboratory, School of Veterinary Medicine, University of California Davis for analysis where blood cell counts, and organ function testing was performed.

[0160] Chronic Toxicity in healthy NRG mice. The toxicity of [225Ac]Macropa-PEG4-7065 was evaluated in NRG mice aged 5-6 weeks. For the chronic toxicity, the mice were divided into five groups (n=5 mice per group). Cohorts included saline, 4.62 KBq single dose, 4.62 KBq μCi fractionated dose, 9.25 KBq single dose and 9.25 KBq fractionated dose. Fractionated doses were injected every four weeks (day 0, day 28 and day 56). Body condition score and body weights are recorded at every alternate day till day 100. At day 100 mice were sacrificed, and blood parameters and organ function test were performed as the procedure described previously.

[0161] Toxicity analysis in humanized mice NOD.Cg-Prkdc Il2rg Tg (SV40 / HTLVIL3,CSF2)10-7Jic / JicTac Engrafted, CD34+ huHSCs female. The toxicity of [225Ac]Macropa-PEG4-7065 and [225Ac]DOTA-antiCD33 was evaluated in NOG EXL huCD34+ engrafted female mice and compared with healthy ones (age range 12 weeks post engraftment of hCD34+ cells). The NOD.Cg-Prkdc Il2rg Tg (SV40 / HTLVIL3, CSF2)10- 7Jic / JicTac Engrafted, CD34+ huHSCs female were purchased from Taconic Bioscience. For the acute toxicity, the mice were divided into three groups (n=4 mice per group). Treatment groups included saline control, 4.62 KBq dose of [225Ac]DOTA-anti-CD33, 4.62 KBq of [225Ac]Macropa-PEG4-7066. The mice were monitored, and their body condition score and body weights are recorded at every alternate day. At day 7, peripheral blood was withdrawn by submandibular vein and blood parameters was analyzed by Hemavet 950 / 950FS Multi-Species Analyzer. At day 28, mice were scarified, blood was collected through cardiac puncture and stored in EDTA coated tubes to prevent coagulation and blood parameters were analyzed.Serum samples were obtained by allowed the blood containing vials to sit at 4 C for 30 minutesto separate the serum from the clotted blood. The serum samples were separated from the clotsby centrifugation at 10,000 rpm for 10 minutes at 4 C. The blood and serum samples were sentto the pathology laboratory at Comparative Pathology Laboratory, School of Veterinary Medicine, University of California Davis for analysis where blood cell counts, and organ function testing was performed. KILPATRICK TOWNSEND 795667431

[0162] H& E and Necropsy Analysis. After euthanasia, dose limiting organs including kidney, liver, lungs, spleen, heart and bone were extracted and fixed in 10% formalin. Histologic analysis was carried out to examine the microscopic features of the tissues. For Hematoxylin and Eosin (H&E) staining of dose-limiting organs, the tissues were fixed in formalin, processed through EtOH gradient (30% to 70%), and embedded in paraffin. Tissue sections with a thickness of 4 μm were prepared for H & E staining at University of California Davis.

[0163] Therapy study in disseminated Patient Derived Xenograft model. NSG-SGM3 humanized mice (stock number 703321) were purchased from Jackson Laboratory. Mice were pre-conditioned with 6.25 mg / kg dose of busulfan (intravenously) for two days. One day after pre-conditioning NSG-SGM3 mice were injected intravenously with 1 million PDX B cells20. Following 10 days after inoculation with PDX cells, peripheral blood was withdrawn, and flow analysis was performed to monitor the PDX engraftment in the animal’s blood. On day 11 after inoculation, mice were randomized in three groups (n=5 per group). Treatment arms included 4.62 KBq dose of [225Ac]Macropa-PEG4-7065, 4.62 KBq dose of [225Ac]DOTA-anti-CD33, and saline vehicle control. The body condition score, mobility and body weight were recorded at every alternate day. Tumor burden was monitored by collecting peripheral blood via thesubmandibular vein, followed by flow cytometric analysis to quantify human CD45 cells.Mice were euthanized when the hCD45 cell population reached approximately 50–90% andwhen clinical symptoms such as reduced mobility or hunching were observed. Statistical Significance

[0164] All data were expressed as mean SD. Data was analyzed using GraphPad Prism 8 and a P value 0.05 was considered statically significant. Two-way ANOVA was used for calculation of biodistribution values and tumor to background ratio values. The log-rank sum test was used for survival analysis. Example 1 – Design and synthesis of radioconjugates targeting aITGB2

[0165] IgG 7065 was selected for imaging and therapy studies based on selectivity for aITGB2, and low binding to healthy tissues and bone marrow progenitors. For imaging studies, we selected the DFO* chelator, which has stable binding to89Zr, and lower non-specific bone KILPATRICK TOWNSEND 795667431uptake compared to the more commonly used DFO28. This property is particularly important in the study of preclinical disseminated models of hematologic malignancies, where tumor cells often localize to the bones. DFO*-7065 and DFO*-IgG were synthesized by conjugation of DFO*-NCS with 7065 and non-targeting IgG (FIG 1A-B), achieving antibody-to-chelator ratios of 0.46 and 0.36. Following radiolabeling with Zr-oxalate, [89Zr]DFO*-7065 was obtained with a radiochemical yield of 80.2 0.19 % yield (n = 6 syntheses), with a final specific activity of 5.75 mCi / mg with a radiochemical purity of greater than 98%. SEC analysis showed no aggregation of the [89Zr]DFO*-7065 after the labeling and purification process. [89Zr]DFO*-IgG was obtained with a radiochemical yield of 86.2 0.34 % yield (n = 5 syntheses), with a final specific activity of 5.75 mCi / mg with a radiochemical purity of greater than 98%. A LINDMO assay with [89Zr]DFO*-7065 in Nomo-1 cells, revealed an immunoreactive fraction of 87.8 ± 0.65%. Over 99% of the radioimmunoconjugate remained stable in both saline and human serum for seven days at 37°C.

[0166] For225Ac therapy studies, we employed a bifunctional chelator, Macropa-PEG4-TFP21,29. This bifunctional chelator employes the efficient Macropa chelator30, together with a pegylated linker, which results in efficient radiolabeling, high tumor uptake, and rapid, renal clearance of metabolic fragments, resulting in high tumor uptake and lower background tissue uptake, resulting in improved therapeutic outcomes compared to conventional DOTA-based labeling strategies. Utilizing optimized labeling protocols, Macropa-PEG4-7065 (FIG.1C) and Macropa-PEG4-IgG (FIG.1D) were obtained with antibody-to-chelator ratios of 1.61 and 0.43, respectively, as determined by MALDI-MS spectrometry. The anti-CD33 antibody, Lintuzumab, was conjugated to DOTA-NCS (FIG. 1E) following a previously reported method, yielding an antibody-to-chelator ratio of 13.42. [225Ac]Macropa-PEG4-7065 and [225Ac]Macropa-PEG4-IgG were obtained with a radiochemical yield of 55 1.76 % and 65 0.06 % yield (n=4 syntheses each) with radiochemical purity exceeding 97%, and 98%, with a specific activity of 0.25 mCi / mg and 0.5 mCi / mg respectively. In addition, SEC analysis for [225Ac]Macropa-PEG4-7065 showed no aggregation of [225Ac]Macropa-PEG4-7065 after the labeling and purification process. [225Ac]DOTA-anti-CD33 was obtained with a radiochemical yield of 40 0.45 % yield (n = 4 syntheses) with a radiochemical purity of more than 98% after centrifugal filtration, with a specific activity of 0.166 mCi / mg. We also developed a134Ce labeled version of Macropa-PEG4-7065 to directly image this conjugate. Macropa-PEG4-7065 was radiolabeled with134CeCl3, with a radiochemical yield of 75 0.32% (n = 3 syntheses) KILPATRICK TOWNSEND 795667431with radiochemical purity of greater than 95% after secular equilibrium, with a specific activity of 5 mCi / mg. Magnetic bead immunoreactivity assay performed with ITGB2 recombinant protein with [225Ac]Macropa-PEG4-7065 revealed an immunoreactive fraction of 81.35 0.93%. We also performed immunoreactivity assay with [225Ac]DOTA-anti-CD33 with CD33 recombinant protein demonstrated the immunoreactive fraction of 77.04 3.17%. The stability of [225Ac]Macropa-PEG4-7065 in saline and human serum was greater than 95% in saline and human serum up to seven days. Overall, the radiosynthesis of the agents was robust, reproducible, with excellent purity, stability, specific activity, and retention of immunoreactivity. Example 2 – aITGB2 is highly expressed in AML cell lines and patient samples

[0167] We performed flow cytometry to measure the expression of aITGB2 on AML cell lines, demonstrating heterogeneous but generally high aITGB2 expression in all AML cell lines. Nomo-1, THP-1, HL-60 (Nomo-1 HL-60 THP-1 MV411) have comparable and higher aITGB2 expression followed by MV411 and all cell lines have greater aITGB2 expression compared to the Nomo-1 KO cell line, where aITGB2 was knocked out (FIG.2A). We also used flow cytometry to examine the expression of aITGB2 in 15 primary patient samples of AML from the time of initial clinical diagnosis, including a number of common AML genetic abnormalities. These include mutations in FLT3 (10 samples), DNMT3A (7), NPM1 (5), and TET2 (5), and chromosomal abnormalities +8 (6), +5 (3), 11q23 MLL rearrangement (2), -7 (2), and -5 (1). We found a similarly variable expression profile in these samples, with a mean of 62.3 ± 25.4% of AML blasts per patient found to express aITGB2 (FIG. 2B). As an aITGB2-targeting therapy would likely be evaluated for clinical translation in a relapsed and / or refractory AML patient population, frequently with azacytidine and / or venetoclax pre-treatment, we sought to characterize aITGB2 expression in this population as well. We used flow cytometry to evaluate the percent expression and median fluorescence intensity in matched AML samples from 7 patients, taken at initial diagnosis and following refractory disease or relapse (r / r) after at least one round of azacitidine and / or venetoclax. Notably, we saw no mean decrease in percent expression of aITGB2 and a moderate, albeit non-statistically significant, 1.4-fold increase in median fluorescence intensity in r / r samples compared to those collected at diagnosis (FIG. 2C). No significant differences were seen in KILPATRICK TOWNSEND 795667431aITGB2 expression between the major genetic subtypes of AML between the primary samples evaluated. [89Zr]DFO*-7065 demonstrates high binding to AML cell lines

[0168] After analysis expression of aITGB2 in cells as well as in PDX samples, we performed cell-binding study to evaluate the binding of [89Zr]DFO*-7065 to different AML cell lines. Nomo-1 and HL-60 exhibited the highest cell-binding percentages for [89Zr]DFO*- 7065, followed by THP-1 and MV411, consistent with the higher expression of aITGB2 detected by flow cytometry in the Nomo-1, HL-60, and THP-1 cell lines compared to MV411 and the Nomo-1 ITGB2 KO cell line. The binding percentage of [89Zr]DFO*-7065 was approximately 3.5-fold lower in the Nomo-1 ITGB2 KO cell line compared to the Nomo-1 WT cell line. Both Nomo-1 and Nomo-1 ITGB2 KO cells express Fc receptors. To block Fc receptor-mediated binding in the Nomo-1 ITGB2 KO cells, 10 fold excess of cold IgG was added, significantly reducing the binding of [89Zr]DFO*-7065 to the Nomo-1 ITGB2 KO cell line (FIG.2D). The dissociation constant (Kd) of [89Zr]DFO*-7065 for aITGB2-expressing cell lines was determined through a saturation binding assay, yielding a Kdvalue of 2.09 ± 0.02 nM, 0.86 ± 0.46 nM, 1.19 ± 0.10 nM, 56.46 ± 0.23 nM and 127.9 ± 0.46 nM in the Nomo-1, HL-60, THP-1, MV411 and Nomo-1 ITGB2 KO cell lines respectively (FIG. 2E) . In internalization assays, the membrane-bound fraction of [89Zr]DFO*-7065 was 0.5% for Nomo- 1 and 0.65% for HL-60, while the internalized fraction was 52.85 ± 5.41% and 79.63 ± 3.60 % of total administered activity for Nomo-1 and HL-60, respectively (FIG. 2F). These findings support the notion that aITGB2 is highly but variably expressed in AML cell lines and that [89Zr]DFO*-7065 binds selectively to the activated form of aITGB2 in these cells. Example 3 – PET / CT imaging and biodistribution analysis of [89Zr]DFO*-7065 in disseminated AML models reveal high aITGB2-targeted uptake

[0169] Next, we evaluated the imaging properties of [89Zr]DFO*-7065 in disseminated AML models. The experimental workflow included the inoculation of AML cells, bioluminescence and PET imaging followed by ex vivo biodistribution and imaging studies (FIG. 3A). The PET / CT imaging demonstrated high uptake of [89Zr]DFO*-7065 in the bone marrow of the Nomo-1 disseminated model, with spatial colocalization of bioluminescence (BLI) and PET signals (FIG. 3B). To confirm the specificity of [89Zr]DFO*-7065, we conducted control KILPATRICK TOWNSEND 795667431experiments, including imaging with [89Zr]DFO*-7065 in the Nomo-1 ITGB2 KO model (FIG. 3C), imaging with a non-specific [89Zr]-labeled antibody ([89Zr]DFO*-IgG; FIG. 3D), and a blocking experiment using a 25-fold excess of unlabeled 7065 (FIG. 3E). All three control studies showed significantly reduced uptake in tumor lesions, with no overlap between the BLI and PET / CT signals, confirming the specificity of [89Zr]DFO*-7065. Segmentation of multi- time point PET imaging revealed increasing gradual accumulation of the radiopharmaceutical in the tumor in NOMO-1, but not control studies, while activity cleared out of the blood and other organs over time. Region of interest drawn on femur, liver, kidney and blood pool at day1, day2, day4 showed higher %ID / g of [89Zr]DFO*-7065 at day 4 in femur and excretion by liver. Ex vivo BLI and PET images demonstrated high concordance of signal for NOMO-1, but not the control studies (FIG. 3F). Ex vivo biodistribution studies showed higher uptake of [89Zr]DFO*-7065 in the bone marrow compared to negative controls (FIG.3G). The tumor-to- blood (Tumor / Blood) ratio (FIG. 3H) and tumor-to-muscle (Tumor / Muscle) ratio (FIG. 3I) were significantly higher in the Nomo-1 model compared to the control models.

[0170] We extended this evaluation to other AML disseminated models, including MV411, THP-1, and HL-60. BLI revealed that leukemic cells accumulated variably among the models, with NOMO-1 and HL-60 localizing primarily to bone marrow, THP-1 to liver, and MV411 to both liver and bone marrow (Fig 3J – L). In all cases, [89Zr]DFO*-7065 revealed concordant localization with the tumor associated BLI signal. Ex vivo PET / CT and BLI analyses of the bone marrow in all disseminated models further confirmed the colocalization of signals in these regions (FIG.3M), validating the imaging capabilities of [89Zr]DFO*-7065 for AML detection. Ex vivo PET / CT and BLI analysis revealed that spleen and lungs in all three AML models (MV411, THP-1, HL-60) had high tumor cells accumulation by BLI and concordant [89Zr]DFO*-7065 uptake by PET / CT. Similar findings were seen in biodistribution studies (FIG. 3N, 3O). The tumor-to-blood (Tumor / Blood) ratio (FIG. 3P) and tumor-to-muscle (Tumor / Muscle) ratio (FIG.3Q) were high in all three AML models, indicating a strong tumor signal with low background, and supporting the further evaluation of therapeutic radiopharmaceuticals.

[0171] In order to compare against current standard of care imaging methods, PET / CT imaging and biodistribution studies were performed using [18F]-FDG in Nomo-1 and THP-1 disseminated models. PET / CT imaging revealed some [18F]-FDG uptake in tumors (data not shown), which corresponded to areas identified by bioluminescence imaging (BLI). However, KILPATRICK TOWNSEND 795667431the uptake in tumor sites, particularly in the femur and liver (primary sites of disease), was significantly lower than in other background organs, including the heart, kidneys, and brain. Regions of interest (ROIs) drawn for the femur and liver demonstrated reduced [18F]-FDG accumulation, while kidneys and heart exhibited higher uptake consistent with their roles in [18F]-FDG metabolism and excretion. Ex vivo biodistribution studies corroborated these findings, showing a lower %IA / g of [18F]-FDG in bone marrow tumors compared to other organs, and lower Tumor / Blood ratio and Tumor / Muscle ratio compared to [89Zr]DFO*-7065. Example 4 – [225Ac]Macropa-PEG4-7065 demonstrates aITGB2 dependent cell killing in vitro and favorable tumoral and whole animal biodistribution in vivo

[0172] After evaluating the imaging capability of [89Zr]DFO*-7065, we analyzed the therapeutic efficacy of [225Ac]Macropa-PEG4-7065 targeting the activated conformation of ITGB2. In clonogenic survival assays, dose-dependent reduction in cell proliferation or colonies was observed after treatment with [225Ac]Macropa-PEG4-7065 in Nomo-1 cells (IC50 of 90.05 0.03 pCi / ml) with less efficacy observed in Nomo-1 ITGB2 KO cells (3.08 0.90 nCi / ml) (FIG.4A). In the highly aITGB2 expressing HL-60 cell line the IC50 was 80.3 0.03 pCi / ml and in the moderately expressing MV411 cell line the IC50 was 10.61 4.74 nCi / ml. These findings recapitulate the trends seen in flow cytometry (Fig 2A) and cell binding (Fig 2D) studies, linking the expression of aITGB2 to therapeutic efficacy.

[0173] Similarly, in the cytotoxicity assay, the dose-dependent reduction in cell viability was observed following treatment with [225Ac]Macropa-PEG4-7065 and [225Ac]DOTA-antiCD33 in Nomo-1 cells. [225Ac]Macropa-PEG4-7065 had a significantly lower IC5023.8 pCi / ml 0.02 compared to [225Ac]DOTA-antiCD33 with a IC50 value of 0.40 0.25 nCi / ml (p 0.05) (FIG.4B). In contrast, IC50 value for [225Ac]Macropa-PEG4-7065 in Nomo-1 ITGB2 KO cells was markedly higher 4.42 2.99 nCi / ml, demonstrating that aITGB2 is necessary for a strong therapeutic effect.

[0174] Next, we performed saturation binding assay and compared the kd value of [225Ac]Macropa-PEG4-7065 against the widely studied [225Ac]DOTA-anti-CD33 in the Nomo- 1 cells.31,32. The Kd value for [225Ac]Macropa-PEG4-7065 was 13.01 ± 7.01 nM with a receptor density (Bmax) of 5.45 fmol / mg in a saturation binding assay. The Kd value for [225Ac]DOTA- antiCD33 was slightly higher at 63.03 ± 5.33 nM with a Bmax of 0.33 fmol / mg. Additionally, flow cytometry was used to compare the expression of CD33 and aITGB2 in the Nomo-1 cell 69 KILPATRICK TOWNSEND 795667431line. After validating promising therapeutic efficacy in vitro in AML cell lines, we evaluated the biodistribution and dosimetry of [225Ac / 134Ce]Macropa-PEG4-7065 in vivo in an AML disseminated model. First, we performed PET / CT imaging and biodistribution analysis utilizing [134Ce]Macropa-PEG4-7065 as a surrogate to monitor the uptake by PET / CT and biodistribution analysis of [225Ac]Macropa-PEG4-7065. As expected, BLI and PET / CT imaging demonstrated high, gradually increasing, and concordant uptake of [134Ce]Macropa- PEG4-7065 in the bone marrow over 7 days, with spatial colocalization of bioluminescence (BLI) and PET signals. Ex vivo PET / CT and BLI showed the uptake of [134Ce]Macropa- PEG4-7065 in femur, liver, and spleen with matching areas of uptake (FIG. 4C). The ex vivo Biodistribution studies performed 7 days post injection of [134Ce]Macropa-PEG4-7065 and [225Ac]Macropa-PEG4-7065 showed that the uptake of both the radioimmunoconjugates in bone marrow and other organs are approximately similar except in liver (FIG.4D). In addition, we performed a multi-time point ex vivo biodistribution for [225Ac]Macropa-PEG4-7065 in Nomo-1 disseminated model, which revealed high and sustained uptake in bone marrow tumor (FIG.4E). In contrast, uptake in other organs was initially high, but washed out at delayed time points. Tumor to blood ratio at days 1, 2, 4 and 7 were 6.99 7.08, 19.19 15.2, 52.89 34.99 and 106.95 61.77. Tumor to muscle ratio at days 1, 2, 4 and 7 were 20 15.17, 20.18 8.66, 57.37 35.76, 82.33 49.93 (FIG. 4F and FIG. 4G). Dosimetry analysis revealed that tumor infiltrated organs including bone marrow, liver, and spleen and lungs tumor showed high tumor absorbed dose, with low uptake in non-tumor bearing organs (FIG. 4H). These results suggest that [225Ac]Macropa-PEG4-7065 exhibits high specificity and potent cytotoxicity against AML cell lines, and a favorable biodistribution, suggesting feasibility for therapeutic studies. Example 5 – [225Ac]Macropa-PEG4-7065 demonstrates a favorable toxicity profile

[0175] Initial acute toxicity studies of 9.25 KBq of [225Ac]Macropa-PEG4-7065 were carried out at day 7 and day 28 in healthy black C57BJ / 6J mice and compared with vehicle group injected with saline, 9.25 KBq of [225Ac]Macropa-PEG4-IgG and 9.25 KBq of [225Ac]DOTA- anti-CD33 injected cohorts. Peripheral blood was withdrawn through submandibular vein, and blood parameters were recorded. White blood cells showed a moderate decrease in cell count in [225Ac]Macropa-PEG4-7065 and [225Ac]Macropa-PEG4-IgG injected cohort, but not as much depletion as the [225Ac]DOTA-anti-CD33 injected group, with similar trends observed for KILPATRICK TOWNSEND 795667431granulocytes, and lymphocytes. Other blood parameters including hemoglobin, and Platelet counts were similar between groups.

[0176] A chronic toxicity study of [225Ac]Macropa-PEG4-7065 was performed in NRG mice with endpoint at day 100 post administration. No significant difference in any blood parameters, liver or kidney function was observed in saline and single or fractioned dose injected dose of 4.62 KBq or 9.25 KBq of [225Ac]Macropa-PEG4-7065. H & E analysis of dose limiting organs showed that in case of single and fractionated dose of 4.62 KBq of [225Ac]Macropa-PEG4-7065 injected groups all organs were normal except in kidney there is minimal renal focal tubular damage was observed in one out of five mice. Similarly, in case of single and fractionated dose of 9.25 KBq of [225Ac]Macropa-PEG4-7065 injected groups, minimal damage to kidney was observed with renal injury in 1 / 5 mice (single dose) and 4 / 5 (fractionated dose) mice respectively, presumably due to redistribution of daughter213Bi into the kidney, as previously reported33. Taken together, these findings suggest that [225Ac]Macropa-PEG4-7065 demonstrates the expected toxicity profile for a225Ac-IgG, with transient hematologic abnormalities and delayed nephrotoxicity, with no additional marrow toxicity due to “on-target, off tumor” granulocyte targeting. Example 6 – [225Ac]Macropa-PEG4-7065 demonstrates high therapeutic efficacy and prolonged survival in Nomo-1 disseminated AML model

[0177] A treatment study was designed to evaluate the therapeutic efficacy of [225Ac]Macropa-PEG4-7065 (FIG. 5A), with groups including vehicle control, non-targeting [225Ac]Macropa-PEG4-IgG, the previously evaluated leukemia RIT [225Ac]DOTA-anti-CD33, and [225Ac]Macropa-PEG4-7065. An additional group received three fractionated doses of [225Ac]Macropa-PEG4-7065. In the vehicle group as well as the [225Ac]Macropa-PEG4-IgG- injected group, a continuous increase in tumor burden was observed on BLI, with all mice reaching the endpoint at day 43 and day 50, respectively (FIG.5B). In the [225Ac]DOTA-anti- CD33 cohort, there was an initial decrease in tumor burden during the first week, followed by a continuous increase, with all mice reaching the endpoint at day 43. In contrast, the single- dose and fractionated-dose groups of [225Ac]Macropa-PEG4-7065 demonstrated a significant decrease in tumor burden. No weight loss was observed in any of the cohort, except one injected with a total three fractionated doses of 9.25 KBq (a non-statistically significant trend with KILPATRICK TOWNSEND 795667431approximate 5% body weight loss) (FIG.5C). In the single-dose [225Ac]Macropa-PEG4-7065 injected group, two mice relapsed, showing high tumor burden, and were euthanized at days 57 and 71, respectively. However, the remaining animals 6 out of 8 in this group survived until the end of the study, which was terminated on day 150. The Kaplan-Meier survival curve showed that the median survival time for mice of 43 days for the vehicle group, 50 days for [225Ac]Macropa-PEG4-IgG, and 43 days for the [225Ac]DOTA-anti-CD33 group. In contrast, for both the single-dose and fractionated-dose [225Ac]Macropa-PEG4-7065 injected groups, more than 50% of the animals survived; therefore, the median survival was undefined and significantly higher than negative as well as positive control (FIG.5D).

[0178] To monitor aITGB2 expression in relapsed tumors and investigate potential resistance to treatment, bone marrow and spleen samples were harvested from mice in all groups when they approached the endpoint. CD33 and CD45-positive cells were isolated from the bone marrow and spleen, and aITGB2 expression in the CD33 / CD45-positive tumor population was analyzed using flow cytometry (FIG. 5E). Interestingly, aITGB2 expression remained high (greater than 95%) in the CD33 / CD45-positive tumor population across all cohorts, including single- or fractionated-dose [225Ac]Macropa-PEG4-7065 groups (FIG. 5E, 5F), saline, [225Ac]DOTA-anti-CD33 (FIG. 5G) [225Ac]Macropa-PEG4-IgG (FIG. 5H), saline + IgG injected groups (FIG. 5I These results suggest that [225Ac]Macropa-PEG4-7065 demonstrates promising therapeutic efficacy compared to both the negative and positive controls, while retaining high aITGB2 expression in the tumor population. Example 7 – [225Ac]Macropa-PEG4-7065 is an effective treatment in a patient derived xenograft model of AML

[0179] We next evaluated the therapeutic efficacy of [225Ac]Macropa-PEG4-7065 in a disseminated patient derived xenograft model. Humanized NRG-SGM3 mice (n=5 per group) were engrafted with monocytic leukemia Patient derived Xenografts obtained from PRoXe35. Following preconditioning with busulfan and inoculation of PDX cells, treatment was initiated 10 days following xenograft inoculation with treatment arms including vehicle control, [225Ac]DOTA-anti-CD33, and [225Ac]Macropa-PEG4-7065 (FIG. 6A). Therapeutic outcomes included tumor measurements with blood sampling and FACS analysis human CD45+ cells, in addition to routine body weight and body condition score assessment. KILPATRICK TOWNSEND 795667431

[0180] Results are shown in FIGS.6B-G. By day 30, high tumor burden was observed in thevehicle (saline) group, with ~90% hCD45 blasts detected by flow cytometry, with much lowerburden in the [225Ac]DOTA-anti-CD33 cohort. In contrast, tumor burden was significantlylower in the [225Ac]Macropa-PEG4-7065 cohort, with less than 2% hCD45 blasts. By day 40,all mice in the [225Ac]DOTA-anti-CD33 injected cohorts exhibited high circulating tumor burden, reaching endpoint by day 43. In contrast, in the [225Ac]Macropa-PEG4-7065 cohort,two mice had more than 10% hCD45 blasts, while the remaining three mice had tumor burdensbelow 5%. By day 52, all mice in the 4.62 KBq [225Ac]Macropa-PEG4-7065 group eventually relapsed, two mice reaching the end point on day 60 and remaining mouse died on day 67. No body weight loss was seen at early time points following [225Ac]Macropa-PEG4-7065, while loss at later time points is likely due to tumor relapse (Fig 6F). Average overall survival was 30 days, 43 days, and 67 days in the saline, [225Ac]DOTA-anti-CD33, and [225Ac]Macropa- PEG4-7065 injected group. General concepts supported by the examples detailed above.

[0181] Radioligand therapy (RLT) is a promising therapy for AML that delivers radioactive isotopes directly to leukemia cells via specific antibodies. By binding to AML-associated markers, RLT delivers localized radiation that selectively destroys cancer cells inducing cytotoxic effects while minimizing damage to surrounding healthy tissues.37Unlike conventional chemotherapy or CAR-T cell therapy38, RLT is not reliant on immune activation and is less susceptible to antigen loss, making it a viable option for patients with relapsed or refractory AML. Its ability to penetrate deep into the bone marrow and target widely dispersed leukemic cells provides a broader and potentially more effective treatment strategy39.

[0182] In previous studies, AML exhibited only limited responses to RLT with no FDA approved products39have emerged. Early studies focused on the beta-particle emitters Iodine- 131 [131I]40and Yttrium-90 [90Y]41conjugated to anti-CD33 (M195 and huM195) antibodies, demonstrating potential in reducing leukemic burden. However, their therapeutic impact was modest particularly in patients with high tumor burdens. Additionally, the use of beta-emitting isotopes like Iodine-131 posed challenges due to nonspecific cytotoxicity, leading to prolonged myelosuppression and necessitating hematopoietic stem cell transplantation in some cases42. These limitations have prompted the exploration of alternative strategies, such as conjugating KILPATRICK TOWNSEND 795667431huM195 (officially named as Lintuzumab) with alpha-emitting isotopes like Bismuth-213 and Actinium-225, which offer more potent and targeted cytotoxic effects with potentially reduced collateral damage to healthy tissues,43.

[0183] The experimental data detailed above provided a systemic evaluation of aITGB2- targeted PET / CT imaging with [89Zr]DFO*-7065 and radioligand therapy with [225Ac]Macropa-PEG4-7065 in disseminated models of AML. This is the first study that explored the potential of aITGB2 as a radioimmunotherapy target for the diagnosis and treatment of AML. Flow cytometry indicated high but variable expression of aITGB2, and cell binding and cytotoxicity studies indicated concordant high binding, internalization, and cytotoxic efficacy (Fig 2). PET imaging revealed high specific uptake of [89Zr]DFO*-7065 in all disseminated AML models (Nomo-1, MV411, THP-1, HL-60). Relevant control studies including aITGB2 knock out, non-specific IgG imaging and blocking demonstrated that the uptake of [89Zr]DFO*-7065 is specific to aITGB2 target and not dominated by non-specific accumulation.

[0184] The imaging capability of [89Zr]DFO*-7065 was compared to that of [18F]FDG which is a gold standard of care for most of the hematological malignancies in Nomo-1 and THP-1 disseminated AML models. [18F]FDG demonstrate moderate uptake in tumorous organs (bone marrow and liver) and lower tumor to background ration as compared to [89Zr]DFO*- 7065 (FIG. 3). Collectively, these results demonstrated that [89Zr]DFO*-7065 specifically binds aITB2, and can be employed for detecting AML in preclinical models. While molecular imaging is not commonly employed in management of leukemia in the clinic, this agent could find future use for dosimetry analysis to advance a companion radiopharmaceutical therapy or as a biomarker for selection for patients for subsequent aITGB2 targeted therapy.

[0185] [225Ac]-Lintuzumab, alpha-emitting radioimmunotherapy agent directed CD33- positive AML cells, has been evaluated in both preclinical and clinical studies. Preclinical research demonstrated its potent cytotoxic effects due to alpha-particle–induced DNA damage, making it an effective option for eliminating leukemia cells, even in cases resistant to conventional therapies like venetoclax44. In Phase 1 trials, [225Ac]-Lintuzumab was well- tolerated at doses up to 111 kBq / kg, with promising anti-leukemic activity, including significant reductions in peripheral and bone marrow blasts, and some patients achieving a morphologic leukemia-free state45. However, higher doses were associated with dose-limiting KILPATRICK TOWNSEND 795667431toxicities such as prolonged myelosuppression, sepsis, and delayed hematopoietic recovery46. While its targeted approach offers advantages over beta-emitting therapies by minimizing damage to surrounding tissues, challenges remain, including the need for better patient selection, optimizing dosing regimens, and mitigating toxicity. Another limitation of current radioligand therapies in AML is the associated damage to marrow due to higher expression of explored targets (CD33 CD123 and CLL-1) on normal myeloid cells and progenitors. As aITGB2 has low expression on normal hematopoietic stem cells, targeting this integrin could avoid complications and could provide benefits to the patients.

[0186] We compared the therapeutic efficacy of [225Ac]-Macropa-PEG4-7065 with [225Ac]DOTA-antiCD33 in pre-clinical disseminated model of AML. Firstly, we observed that [225Ac]Macropa-PEG4-7065 exhibited a better radiochemical yield and higher specific activity compared to [225Ac]DOTA-anti-CD33. Our results also demonstrated that [225Ac]Macropa- PEG4-7065 exhibited higher therapeutic efficacy as compared to [225Ac]DOTA-antiCD33 with a significant improvement in median time period survival compared to negative and positive control. We also compared the therapeutic efficacy of [225Ac]-Macropa-PEG4-7065 with [225Ac]DOTA-antiCD33 in disseminated patient- derived xenograft (PDX) model using humanized mice. [225Ac]Macropa-PEG4-7065 again showed therapeutic efficacy over [225Ac]DOTA-antiCD33 with a notable increase in median survival.

[0187] The experimental data also demonstrated that aITGB2 exhibits lower expression on normal immune cells (myeloid cells, NK cells and T cells) as compared to CD33. To validate this, we performed PET / CT imaging using [89Zr]DFO*-7065 and [89Zr]DFO*-anti-CD33 in humanized NOG-EXL mice which closely replicate the human immune system and compared the binding of both probe with human immune cells. This study showed that [89Zr]DFO*-7065 has minimal background binding to human immune cells as compared to [89Zr]DFO*-anti- CD33 potentially explaining the increased toxicity and myelosuppression associated [225Ac]DOTA-anti-CD33. We compared the toxicity profile of [225Ac]Macropa-PEG4-7065 with [225Ac]DOTA-anti-CD33 in humanized NOG EXL mice which demonstrated that [225Ac]- DOTA-anti-CD33 significantly depleted the huCD34+ hematopoietic stem and progenitor cells, whereas [225Ac]Macropa-PEG4-7065 had a markedly more favorable safety profile. KILPATRICK TOWNSEND 795667431Summary of example results

[0188] [89Zr]DFO*-7065 was tested as a aITGB2-targeted immunoPET imaging probe for AML in multiple disseminated AML models, with higher tumorous uptake and lower background as compared to negative controls and [18F]FDG. Radioimmunotherapy agent [225Ac]Macropa-PEG4-7065 was successfully prepared and demonstrated improved safety profile and promising therapeutic in disseminated models of AML. Overall, [89Zr]DFO*-7065 showed great potential for imaging and theraonstic companion biomarker and [225Ac]Macropa- PEG4-7065 shows promising therapeutic efficacy.

[0189] All publications, issued patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0190] It is to be understood that this disclosure is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure, which will be limited only by the appended claims. References cited by number in Technical Section Examples. (1) Key Statistics for Acute Myeloid Leukemia (AML). https: / / www.cancer.org / cancer / types / acute-myeloid-leukemia / about / key-statistics.html (accessed 2024-12-31). (2) Lai, C.; Doucette, K.; Norsworthy, K. Recent Drug Approvals for Acute Myeloid Leukemia. Journal of Hematology & Oncology 2019, 12 (1), 100. https: / / doi.org / 10.1186 / s13045-019-0774-x. (3) Roloff, G. W.; Odenike, O.; Bajel, A.; Wei, A. H.; Foley, N.; Uy, G. L. Contemporary Approach to Acute Myeloid Leukemia Therapy in 2022. 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Claims

WHAT IS CLAIMED IS:

1. A method of treating cancer in a subject, the method comprising administering to the subject an immunoconjugate according to Formula I,wherein, X is a chelator moiety; Y is selected from the group consisting of -O- and -NR-; Z is a moiety selected from the group consisting of:wherein A is an antibody that specifically binds to activated integrin beta-2 (aITGB2); and further wherein the antibody comprises an aITGB2 binding domain comprising: a heavy chain variable region (VH) comprising an HCDR1 sequence comprising ISYYYM, an HCDR2 sequence comprising SISSSSGYTY; and an HCDR3 sequence comprising GAM; and a light chain variable region (VL) comprising an LCDR1 sequence comprising SVSSA, an LCDR2 sequence comprising SASSLYS; and an LCDR3 sequence comprising FSSGSWAPI. subscript m is 3 or 5; subscript n is 4, 6, 8, 10, 12, 14, or 16; and R selected from the group consisting of H, OH, and a negative charge; and wherein the cancer is a cancer that comprises cells expressing aITGB2.

2. The method of claim 1, wherein the cancer is acute myeloid leukemia (AML).

3. The method of claim 1 or 2, wherein the antibody comprises a VHcomprising an amino acid sequence that comprises at least 95% identity to SEQ ID NO: 1 KILPATRICK TOWNSEND 795667431and a VL comprising an amino acid sequence that comprises at least 95% identity to SEQ ID NO:

2.

4. The method of claim 3, wherein the antibody comprises a VHcomprising amino acid sequence SEQ ID NO: 1 and / or a VLcomprising amino acid sequence SEQ ID NO:

2.

5. The method of any one of claims 1-4, wherein the immunoconjugate comprises a structure according to Formula Ia:(Ia).

6. The method of any one of claims 1-4, wherein the immunoconjugate comprises a structure according to Formula Ib:The method of any one of claims 1-6, wherein the chelator moiety X is selected from the group consisting of:KILPATRICK TOWNSEND 7956674318. The method of any one of claims 1-7, wherein: the chelator moiety X is: ;subscript m is 3.

9. The method of any one of claims 1-7, wherein: the chelator moiety X is:; Y is -NR-; and subscript m is 3.

10. The method of any one of claims 1-7, wherein: the chelator moiety X is: KILPATRICK TOWNSEND 795667431; Y is -NR-; and subscript m is 3.

11. The method of any one of claims 1-7, wherein: the chelator moiety X is:Y is -NR-; and subscript m is 5.

12. The method of any one of claims 1-11, wherein subscript n is 4, 6, 8, or 12.

13. The method of any one of claims 1-12, wherein subscript n is 4 or 8.

14. The method of any one of claims 1-13, wherein the immunoconjugate further comprises an alpha-emitting radionuclide, wherein the chelator moiety of the immunoconjugate chelates the alpha-emitting radionuclide.

15. The method of claim 14, wherein the alpha-emitting radionuclide is selected from the group consisting of225Ac,134Ce,213Bi,224Ra,212Pb,227Th,223Ra,211At, andKILPATRICK TOWNSEND 79566743116. The method of claim 15, wherein the alpha-emitting radionuclide is 17. The method of claim 14, wherein the immunoconjugate comprises a structure according to Formula IIa:(IIa), and M is the alpha-emitting radionuclide.

18. The method of claim 14, wherein the immunoconjugate comprises a structure according to Formula IIb:

19. The method of claim 14, wherein the immunoconjugate comprises a structure according to Formula IIc: KILPATRICK TOWNSEND 795667431and M is the alpha-emitting radionuclide, and subscript p is 0 or 1.

20. The method of claim 14, wherein the immunoconjugate comprises a structure according to Formula IId:and M is the alpha-emitting radionuclide.

21. The method of any one of claims 17-20, wherein the alpha-emitting radionuclide is225Ac.

22. A method of treating cancer in a subject, the method comprising administering to the subject an immunoconjugate according to Formula IIa,KILPATRICK TOWNSEND 795667431M is an alpha-emitting radionuclide225Ac; A is an antibody that specifically binds to activated integrin beta-2 (aITGB2), wherein the antibody comprises a VH comprising amino acid sequence SEQ ID NO: 1 and a VLcomprising amino acid sequence SEQ ID NO: 2 ; and subscript n is 4; and wherein the cancer is a cancer that comprises cells expressing aITGB2.The method of claim 22, wherein the cancer is AML.

24. The method of any one of claims 1-23, wherein the immunoconjugate is administered with a pharmaceutically acceptable excipient.

25. An immunoconjugate according to Formula I,wherein, X is a chelator moiety; Y is selected from the group consisting of -O- and -NR-; Z is a moiety selected from the group consisting of:wherein A is an antibody that specifically binds to activated integrin beta-2 (aITGB2); and further wherein the antibody comprises an aITGB2 binding domain comprising: a heavy chain variable region (VH) comprising an HCDR1 sequence comprising ISYYYM, an HCDR2 sequence comprising SISSSSGYTY; and an HCDR3 sequence comprising GAM; and a light chain variable region (VL) comprising an LCDR1 sequence comprising SVSSA, an LCDR2 sequence comprising SASSLYS; and an LCDR3 sequence comprising FSSGSWAPI. subscript m is 3 or 5; KILPATRICK TOWNSEND 795667431subscript n is 4, 6, 8, 10, 12, 14, or 16; and R selected from the group consisting of H, OH, and a negative charge.

26. The immunoconjugate of claim 25, wherein the antibody comprises a VHcomprising at least 95% identity to SEQ ID NO: 1 and a VLcomprises an amino acid sequence comprising at least 95% identity to SEQ ID NO:

2.

27. The immunoconjugate of claim 26, wherein the antibody comprises a VHcomprising amino acid sequence SEQ ID NO: 1 and / or a VLcomprising amino acid sequence SEQ ID NO:

2.

28. The immunoconjugate of claim 25, 26, or 27, comprising a structure according to Formula Ia:(Ia).

29. The immunoconjugate of claim 25, 26, or 27, comprising a structure according to Formula Ib:

30. The immunoconjugate of any one of claims 25-29, wherein the chelator moiety X is selected from the group consisting of:KILPATRICK TOWNSEND 79566743131. The immunoconjugate of any one of claims 25-30, wherein: the chelator moiety X is:Y is -O-; and subscript m is 3.

32. The immunoconjugate of any one of claims 25-30, wherein: the chelator moiety X is: KILPATRICK TOWNSEND 795667431Y is -NR-; and subscript m is 3.

33. The immunoconjugate of any one of claims 25-30, wherein: the chelator moiety X is:Y is -NR-; and subscript m is 3.

34. The immunoconjugate of any one of claims 25-30, wherein: the chelator moiety X is:; Y is -NR-; and subscript m is 5. KILPATRICK TOWNSEND 79566743135. The immunoconjugate of any one of claims 25-34, wherein subscript n is 4, 6, 8, or 12.

36. The immunoconjugate of claim 35, wherein subscript n is 4 or 8.

37. The immunoconjugate of any one of claims 25-36, wherein the immunoconjugate further comprises an alpha-emitting radionuclide, wherein the chelator moiety of the immunoconjugate chelates the alpha-emitting radionuclide.

38. The immunoconjugate of claim 37 wherein the alpha-emitting radionuclide is selected from the group consisting of225Ac,134Ce,213Bi,224Ra,212Pb,227Th,223Ra,211At, and149T.

39. The immunoconjugate of claim 38, wherein the alpha-emitting radionuclide is225Ac.

40. The immunoconjugate of claim 37, wherein the immunoconjugate comprises a structure according to Formula IIa:(IIa), and M is the alpha-emitting radionuclide.

41. The immunoconjugate of claim 37, wherein the immunoconjugate comprises a structure according to Formula IIb: KILPATRICK TOWNSEND 795667431and M is the alpha-emitting radionuclide, and subscript p is 0 or 1.

42. The immunoconjugate of claim 37, wherein the immunoconjugate comprises a structure according to Formula IIc:and M is the alpha-emitting radionuclide, and subscript p is 0 or 1.

43. The immunoconjugate of claim 37, wherein the immunoconjugateand M is the alpha-emitting radionuclide.

44. The immunoconjugate of any one of claims 40-43, wherein the alpha- emitting radionuclide is225Ac. KILPATRICK TOWNSEND 79566743145. A pharmaceutical composition comprising the immunoconjugate of any one of claims 25-44 and a pharmaceutically acceptable excipient.

46. An immunoconjugate according to Formula IIa,(IIa), wherein M is an alpha-emitting radionuclide225Ac; A is an antibody that specifically binds to activated integrin beta-2 (aITGB2), wherein the antibody comprises a VH comprising amino acid sequence SEQ ID NO: 1 and a VL comprising amino acid sequence SEQ ID NO: 2 ; and subscript n is 4; and wherein the cancer is a cancer that comprises cells expressing aITGB2.

47. A pharmaceutical composition comprising the immunoconjugate of claim 46 and a pharmaceutically acceptable excipient. KILPATRICK TOWNSEND 795667431

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