Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy

Definitions

• RITs radionuclide-based immunotherapies
• 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.
• 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 795667431 are 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.
• the cancer is acute myeloid leukemia (AML).
• AML acute myeloid leukemia
• a method of treating cancer in a subject comprising administering to the subject an immunoconjugate according to Formula I as follows,
• 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.
• the cancer is a cancer that comprises cells expressing aITGB2.
• the antibody comprises an aITGB2 binding domain comprising: (1) a heavy chain variable region (V H ) comprising an HCDR1 sequence comprising ISYYYM, an HCDR2 sequence comprising SISSSSGYTY; and an HCDR3 sequence comprising GAM; and (2) a light chain variable region (V L ) comprising an LCDR1 sequence comprising SVSSA, an LCDR2 sequence comprising SASSLYS; and an LCDR3 sequence comprising FSSGSWAPI.
• V H heavy chain variable region
• V L light chain variable region
• 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 795667431 comprising FSSGSWAPI.
• VH heavy chain variable region
• VL light chain variable region
• the antibody comprises a VH comprising amino acid sequence SEQ ID NO:1 and a V L comprising amino acid sequence SEQ ID NO: 2.
• the cancer is acute myeloid leukemia (AML).
• the immunoconjugate comprises a structure according to Formula Ia as follows: (Ia).
• the immunoconjugate comprises a structure according to Formula Ib as follows: [0009]
• the chelator moiety X includes, but is not limited to, one of the following structures: KILPATRICK TOWNSEND 795667431
• 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.
• the immunoconjugate further comprises an alpha-emitting radionuclide, wherein the chelator moiety of the immunoconjugate chelates the alpha-emitting radionuclide.
• the alpha-emitting radionuclide is selected from the group consisting of 225 Ac, 134 Ce, 213 Bi, 224 Ra, 212 Pb, 227 Th, 223 Ra, 211 At, and 149 Tb.
• the alpha-emitting radionuclide is 225 Ac.
• the immunoconjugate comprises a structure according to Formula IIa as follows: KILPATRICK TOWNSEND 795667431
• 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.
• 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.
• the immunoconjugate comprises a structure according to Formula IId as follows: KILPATRICK TOWNSEND 795667431
• M is the alpha-emitting radionuclide.
• the alpha-emitting radionuclide is 225 Ac.
• a method of treating cancer in a subject comprising administering to the subject an immunoconjugate according to Formula IIa as follows: (IIa).
• M is an alpha-emitting radionuclide 225 Ac;
• 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.
• 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.
• the disclosure features an antibody composition comprising [ 225 Ac]Macropa-PEG4-7065 or comprising [ 89 Zr]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.
• the composition comprising [ 225 Ac]Macropa-PEG 4 -706 is a pharmaceutical composition for the treatment of cancer that 7 KILPATRICK TOWNSEND 795667431 expresses aITGB2, e.g., AML.
• the composition comprising i[ 89 Zr]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.
• FIG.1A-F Schematic representation of radioimmunoconjugates
• A Conjugation of aITGB2 targeting 7065 antibody with DFO*-NCS followed by radiolabeling with 89 Zr(C2O4)2
• B Conjugation of IgG (non-specific binding) antibody with DFO*-NCS followed by radiolabeling with 89 Zr(C2O4)2
• C Conjugation of 7065 antibody with Macropa-PEG4-TFP followed by radiolabeling with 225 Ac(NO3)3 and 134 CeCl3
• D Conjugation of IgG antibody with Macropa-PEG4-TFP followed by radiolabeling with 225 Ac(NO3)3
• E Conjugation of anti-CD33 antibody (Lintuzumab) with DOTA-NCS followed by radiolabeling with 225 Ac(NO3)3.
• FIG.2A-F [ 89 Zr]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)
• D Cell binding assay to measure the percentage cell-associated activity of [ 89 Zr]DFO*-7065 using different AML cell lines
• FIG.3A-Q [ 89 Zr]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) [ 89 Zr]DFO*-7065 in Nomo-1-Luc model, (C) [ 89 Zr]DFO*-7065 + 25fold excess of 7065 in Nomo-1-Luc model (D) [ 89 Zr]DFO*-IgG in Nomo-1-Luc model (E) [ 89 Zr]DFO*- 7065 in Nomo-1-ITGB2 KO-Luc model (F) Ex vivo BLI and PET/CT fusion images of KILPATRICK TOWNSEND 795667431 [ 89 Zr]DFO*-7065 in Nomo-1-Luc, BLI and PET/CT fusion images of [ 89 Zr]DFO*-7065 +25fold excess of 7065 in Nomo-1 ITGB2-KO-Luc, BLI and PET/CT fusion images of [ 89 Zr]DFO*-IgG in Nomo-1-Luc and BLI and PET/CT fusion images of [
• FIG.4A-H [ 225 Ac]Macropa-PEG 4 -7065 demonstrates high therapeutic efficacy as compare to controls and [ 134 Ce/ 225 Ac]Macropa-PEG 4 -7065 demonstrates favorable tumoral and whole animal biodistribution.
• A Colony formation assay [ 225 Ac]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 [ 225 Ac]Macropa-PEG 4 -7065 in Nomo-1 and Nomo-1 ITGB2 KO cell line and with [ 225 Ac]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 [ 134 Ce]Macropa-PEG 4 -7065 in Nomo-1-Luc bearing mouse at 24 h, 48 h, 96 h, 168 h post injection.
• FIG.5A-I [ 225 Ac]Macropa-PEG4-7065 based radiopharmaceutical therapy for the effective treatment of AML
• A Schematic showing the workflow for the therapy study
• FIG.6A-G [ 225 Ac]Macropa-PEG 4 -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 [ 225 Ac]Macropa-PEG4-7065 and [ 225 Ac]DOTA-anti-CD33 respectively has significantly less CD45+ tumor population.
• the term “integrin beta 2,” “integrin 2 ” 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.
• ITGB2 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 (geno
• ITGB2 can bind to a number of alpha chains and thus can from KILPATRICK TOWNSEND 795667431 multiple 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.
• integrin ITGAL/ITGB2 which is a receptor for ICAM1, ICAM2, ICAM3 and ICAM4, and is also a receptor for the secreted form of ubi
• anti-ITGB2 antibody “ITGB2 specific antibody,” “ITGB2 antibody,” and “anti-Itg ” are used synonymously herein to refer to an antibody that specifically binds to Itg
• 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.
• an “anti-ITGB2 binding domain” as used herein refers to an antigen binding domain comprising a V H and a V L region of an anti-ITGB2 antibody as described herein, which antigen binding domain binds to active conformation ITGB2.
• 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.
• the “variable region” contains the antigen-binding region of the antibody (or its KILPATRICK TOWNSEND 795667431 functional equivalent) and is important in specificity and affinity of binding.
• 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).
• 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 (V L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.
• 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.
• hypervariable region 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.
• CDR complementarily determining region
• 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.
• 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 795667431 Biol. 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.
• 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).
• 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).
• CDRl amino acid positions 24 to 34
• CDR2 amino acid positions 50 to 56
• CDR3 amino acid positions 89 to 97
• an amino acid’s Kabat number is not necessarily the same as its linear amino acid number.
• 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.
• the IgG is an IgG1, IgG2, IgG3 or IgG4.
• the subunit KILPATRICK TOWNSEND 795667431 structures and three-dimensional configurations of different classes of immunoglobulins are well known. Different isotypes have different effector functions.
• 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 to one of two clearly distinct types, called kappa ( ) and lambda ( ), based on the amino acid sequences of their constant domains.
• 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 V H -C H 1 by a disulfide bond.
• the F(ab)’ 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)’ 2 dimer into an Fab’ monomer.
• the Fab’ monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993).
• 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.
• 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).
• a “humanized” antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans.
• the non-human CDR regions can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts.
• some, most or all of the amino acids outside the KILPATRICK TOWNSEND 795667431 CDR domains are replaced with amino acids corresponding to the human immunoglobulin germline, while amino acids within one or more CDR regions are unchanged.
• 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.
• 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.
• 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.
• an antibody that specifically binds ITGB2 typically binds to ITGB2 with at least a 2-fold greater affinity than a non-ITGB2 target.
• an antibody that specifically binds activated ITGB2 typically binds to aITGB2 with at least a 2-fold greater affinity than an inactive form of ITGB2.
• an antibody binds to aITGB2 with a K D that is at least 100-fold greater than its affinity for inactive ITGB2.
• ELISA enzyme-linked immunosorbent assay
• 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.
• an antibody that binds to the antigen has a dissociation constant (Kd) of 1 M, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM (e.g., 10 7 M or less, e.g., from 10 7 M to 10 13 M, e.g. from 10 9 M to 10 13 M).
• Kd dissociation constant
• 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 795667431 epitope 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).
• chimeric antibody 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.
• recombinant human antibody 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.
• the recombinant human antibodies have been subjected to in vivo somatic hypermutation.
• the amino acid sequences of the V H and V L regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.
• the term “valent” as used herein denotes the presence of a specified number of binding sites in an antigen binding molecule.
• bispecific antibodies 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”).
• 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).
• the invention relates to bispecific bivalent antibodies, having one binding site for each antigen they specifically bind to.
• 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 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.
• 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) 4,6,8,12 linker with malemide and N-hydroxysuccinamide (NHS) functional groups (Mal-PEGn-NHS, wherein n is 4, 6, 8, 12).
• 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.
• 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.
• 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.
• 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.
• 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.
• each codon in a nucleic acid 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.
• 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.
• 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.
• 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.
• a polynucleotide e.g., a cDNA or mRNA
• a polynucleotide may include either or KILPATRICK TOWNSEND 795667431 both 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.).
• uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.
• charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
• a reference to a nucleic acid sequence encompasses its complement unless otherwise specified.
• 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.
• the term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
• 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.
• nucleic acids or two or more polypeptides
• identity refers 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.
• KILPATRICK TOWNSEND 795667431 definition 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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).
• nucleic acid or protein 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.
• 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.
• 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.
• 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.
• a therapeutically effective amount refers to that amount of the therapeutic agent sufficient to ameliorate a disorder, as described above.
• 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.
• 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 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.
• dose refers to the amount of active ingredient given to an individual at each administration.
• 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).
• dose can be modified depending on the above factors or based on therapeutic progress.
• dosage form refers to the particular format of the pharmaceutical, and depends on the route of administration.
• a dosage form can be in a liquid, e.g., a saline solution for injection.
• Subject “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).
• 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.
• 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.
• KILPATRICK TOWNSEND 795667431 for 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.
• 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.
• 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 K D and IC 50 values ⁇ 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.
• compositions and methods related to radioimmunoconjugates for the treatment of cancer 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).
• 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).
• 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”).
• 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.
• 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.
• 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) (PEG n ) linker.
• aITGB2 activated form or active conformation of ITGB2
• PEG n poly(ethylene glycol) linker
• 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 795667431 and, 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.
• PEG linkers PEG4-12
• radioimmunotherapy methods comprising administering the disclosed radioimmunoconjugates to treat cancers, such as AML.
• aITGB2 Bind Activated Integrin 2
• therapeutic conjugates e.g., radioimmunotherapy reagents, that comprise an anti-ITGB2 antibody that binds the active conformation of ITGB2 (aITGB2).
• the anti-ITGB2 antibody binds to aITGB2 with a KD of less than about 10 nM.
• the anti-ITGB2 antibody is internalized by cancer cells.
• 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.
• the anti-ITGB2 antibody is internalized by leukemia cells, e.g., acute myeloid leukemia (AML) cells.
• AML acute myeloid leukemia
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the VH region e.g., of the scFv
• the VL region e.g., of the scFv
• the linker comprises 1-4 tandem repeats of a Gly4Ser.
• 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.
• 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.
• the anti-ITGB2 antibody disclosed herein comprises an immunoglobulin constant region (e.g., an Fc region).
• 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.
• the immunoglobulin constant region e.g., the Fc region
• 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.
• 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 e.g., a single chain antibody
• scFv single chain antibody
• 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.
• 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.
• antibody is a recombinant antibody (or antigen binding fragment thereof) that specifically binds aITGB2.
• antibody or antigen binding fragment or variant thereof is a monoclonal antibody.
• antibody or antigen binding fragment or variant thereof is a human antibody, a murine antibody, a humanized antibody, or a chimeric antibody.
• 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.
• 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 795667431 full length antibody.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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]
• the antibody (or antigen binding fragment thereof) is derived from non-human (e.g. rabbit or mouse) antibodies.
• the humanized form of the non-human antibody contains a minimal non-human sequence to maintain original antigenic specificity.
• 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.
• donor antibody such as rat, rabbit, or mouse donor having the desired specificity, affinity, avidity, binding kinetics, and/or capacity.
• one or more framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues of the donor antibody.
• 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.
• 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.
• the chelator can be Macropa.NH2, which was developed by Thiele et al., (Thiele NA et al.
• 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).
• DOTA 1,4,7,10-Tetraazacyclododecane-l,4,7,10-tetraacetic acid; tetraxetan
• DOTP 1,4,7,10-Tetraazacyclododecane- l,4,7,10-tetra(methylene phosphonic) acid
• 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).
• the chelator can also be NOTA (2,2 ,2”-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid).
• the chelator can include, but is not limited to, the following: isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (NCS-DTPA) (see, e.g., PCT Publication No.
• NCS-DTPA isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
• 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).
• 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
• D03A 1,
• the chelator can be directly or indirectly coupled to the antibody.
• the chelator can be coupled to the antibody by any chemical reaction that will bind the chelator KILPATRICK TOWNSEND 795667431 and 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.
• the chelator is attached to the antibody through a PEG linker.
• each chelator carries one radionuclide.
• 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.
• 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).
• radionuclides examples include, but are not limited to, 225 Ac, 67 Cu, 177 Lu, 213 Bi, 90 Y, 188 Re, 47 Sc, 227 Th, 212 Ph , lll In, 124 I, 131 I, 134 Ce, 213 Bi, 89 Zr, 211 At, 212 B, and 186 Re.
• Other suitable radionuclide suitable for use in the radioimmunoconjugates disclosed herein will be known to those skilled in the art.
• 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: 225 Ac, 134 Ce, 213 Bi, 224 Ra, 212 Pb, 227 Th, 223 Ra, 211 At, and 149 Tb.
• the radionuclide is Actinium-225 ( 225 Ac), an alpha particle emitter.
• Use of 225 Ac 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 unique ability to produce a total of 4 and 2 particles in its decay chain. E.
• 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 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.
• the leukemia is AML.
• the terms “subject,” patient,” and “animal” include all members of the animal kingdom.
• the subject is a mammal.
• the subject is a human being.
• the subject is a patient having a disease, such as a cancer, e.g., an aITGB2-expressing cancer, such as AML.
• 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.
• 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.
• 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.
• compositions are preferably manufactured under GMP conditions.
• 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.
• Dosage unit form 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.
• 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.
• 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 795667431 can readily be determined by one of ordinary skill in the art.
• the dosages for administration can range from, for example, about 1 ng to about 10,000 mg, about 5 ng to about 9,500 mg, about 10 ng to about 9,000 mg, about 20 ng to about 8,500 mg, about 30 ng to about 7,500 mg, about 40 ng to about 7,000 mg, about 50 ng to about 6,500 mg, about 100 ng to about 6,000 mg, about 200 ng to about 5,500 mg, about 300 ng to about 5,000 mg, about 400 ng to about 4,500 mg, about 500 ng to about 4,000 mg, about 1 ⁇ g to about 3,500 mg, about 5 ⁇ g to about 3,000 mg, about 10 ⁇ g to about 2,600 mg, about 20 ⁇ g to about 2,575 mg, about 30 ⁇ g to about 2,550 mg, about 40 ⁇ g to about 2,500 mg, about 50 ⁇ g to about 2,475 mg, about 100 ⁇ g to about 2,450 mg, about 200 ⁇ g to about 2,425 mg, about 300 ⁇ g to about 2,000, about 400 ⁇ g to about 1,175 mg
• 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.
• 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).
• the active compound e.g., antibody
• the active compound 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]
• the anti-ITGB2 antibody or immune effector cells comprising the antibody are administered with an antineoplastic agent or a chemotherapeutic agent.
• 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 (DaurismoTM).
• venetoclax Venclexta®
• azacitidine Vidaza, Onureg®
• cytarabine daunorubicin
• idarubicin mitoxantron
• 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, fo
• paclitaxel and doxetaxel 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
• 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.
• an antibody such as an antibody that binds aITGB2
• PEGylated linker to produce an antibody-chelator conjugate (ACC)
• ACC antibody-chelator conjugate
• 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.
• the chelator When the chelator is to be coupled or attached to a lysine residue on the antibody, the following exemplary reaction scheme can be used: .
• the chelator When the chelator is to be coupled or attached to a cysteine residue on the antibody, the following exemplary reaction scheme can be used: .
• radioimmunoconjugates 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.
• 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.
• 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.
• 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.
• Embodiments of the disclosure include, but are not limited to, embodiments listed below: Embodiment 1.
• a method of treating cancer in a subject 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 795667431 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 (V L ) comprising an LCDR1 sequence comprising SVSSA, an LCDR2 sequence comprising SASSLYS; and an LCDR3 sequence comprising FSSGSWAPI.
• VH heavy chain variable region
• V L light chain variable region
• 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 795667431
• Embodiment 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 795667431 the 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 795667431
• Embodiment 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 of 225 Ac, 134 Ce, 213 Bi, 224 Ra, 212 Pb, 227 Th, 223 Ra, 211 At, and 149 T.
• Embodiment 16 The method of Embodiment 15, wherein the alpha-emitting radionuclide is 225 Ac.
• 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 795667431 and M is the alpha-emitting radionuclide, and subscript p is 0 or 1.
• Embodiment 19 The method of Embodiment 14, wherein the immunoconjugate comprises a and M is the alpha-emitting radionuclide, and subscript p is 0 or 1.
• Embodiment 20 The method of Embodiment 14, wherein the immunoconjugate comprises a and M is the alpha-emitting radionuclide.
• Embodiment 21 The method of any one of Embodiments 17-20, wherein the alpha-emitting radionuclide is 225 Ac.
• 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 795667431 wherein M is an alpha-emitting radionuclide 225 Ac; A is an antibody that specifically binds to activated integrin beta-2 (aITGB2), wherein the antibody comprises a V H comprising amino acid sequence SEQ ID NO: 1 and a V L 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.
• aITGB2 activated integrin beta-2
• 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 SASS
• 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
• 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.
• Embodiment 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 of 225 Ac, 134 Ce, 213 Bi, 224 Ra, 212 Pb, 227 Th, Embodiment 39: The immunoconjugate of Embodiment 38, wherein the alpha-emitting radionuclide is 225 Ac.
• 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 795667431
• 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 is 225 Ac.
• Embodiment 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 radionuclide 225 Ac; A is an antibody that specifically binds to activated integrin beta-2 (aITGB2), wherein the antibody comprises a V H comprising amino acid sequence SEQ ID NO: 1 and a V L 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.
• Formula IIa, (IIa) wherein M is an alpha-emitting radionuclide 225 Ac
• A is an antibody that specifically binds to activated integrin beta-2 (aITGB2), wherein the antibody comprises a V H comprising amino acid sequence SEQ ID NO: 1 and a V L comprising amino acid sequence SEQ
• Embodiment 47 A pharmaceutical composition comprising the immunoconjugate of Embodiment 46 and a pharmaceutically acceptable excipient.
• Materials 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 795667431 demonstrated 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).
• the anti-CD33 biosimilar, Lintuzumab was purchased from Creative Biolabs and certified for purity and molecular weight.
• Radiopharmaceuticals Synthesis of [ 225 Ac]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 ( 225 Ac) was obtained from U.S. Department of Energy from the 229 Th generator decay pathway. See van Cleve, S. et al. J Med Imag Rad Sci 50 (Supplement), S11-S12 (2019). [ 89 Zr]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.
• the 7065 antibody was incubated with 5 equivalents of DFO*-NCS (1.27 L) at 37°C for 45 minutes.
• 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.
• the 7065 antibody underwent buffer exchange through three cycles with 0.1 M Na CO /NaHCO buffer at pH 9.5 by centrifugation at 10,000 rpm for eight minutes, achieving a final concentration around 15 mg/ml.
• the bifunctional chelator Macropa-PEG4-TFP was prepared as previously described 21 . It was dissolved in DMSO (1 mg/30 L). The 7065 antibody was incubated with 7.5 equivalents of Macropa-PEG 4 -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-PEG 4 -TFP was carried out similarly to the conjugation of the 7065 antibody.
• the resulting conjugate, Macropa-PEG 4 -7065 and Macropa-PEG 4 -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 procedure 1 .
• Radiolabeling with 89 Zr(C 2 O 4 ) 2 , 134 CeCl 3 and 225 Ac(NO 3 ) 3 [0129]
• 89 Zr-oxalate was purchased from 3D Imaging (Little Rock, AK). 3-5 ⁇ L of 89 Zr-oxalate (37 MBq) was mixed with equal amount of 1 M Na 2 CO 3 (3-5 ⁇ L) and 200 ⁇ L of 1M NH 4 OAc, followed by the addition of 130 ⁇ g of DFO*-7065 (7.2 mg/ml, pre-dissolved in 0.25 M NaOAc) and the mixture was incubated at 37 C for 1 hour.
• 134 CeCl 3 (100 ⁇ L, 111 MBq) was mixed with 200 ⁇ l KILPATRICK TOWNSEND 795667431 of 2M NH4OAc (pH 8), 200 ⁇ g of Macropa-PEG4-7065 (15.87 mg/ml) was added, and the reaction mixture was incubated at 37 C for 1 hour.
• the reaction yield was analyzed by iTLC SG (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.
• 225 Ac(NO3)3 was purchased from the Department of Energy Isotope Production Program and produced at Oak Ridge National Laboratory via the 229 Th generator route 23 . 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
• the reaction mixture was passed through YM30K centrifugation filter with three washes and final radioimmunoconjugate purity was analyzed by ITLC-SG.
• labelling of Macropa-PEG 4 -IgG similar procedure was followed as for radiolabeling of Macropa-PEG 4 -7065.
• radiolabeling of DOTA-anti-CD33 with 225 Ac an aliquot of 4 ⁇ L (approx. 1.11 MBq) of Ac-225 was added to a vial.
• [ 89 Zr]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.
• the radiopharmaceuticals [ 89 Zr]DFO*-7065 and [ 225 Ac]Macropa-PEG4-7065 were analyzed using size exclusion chromatography.
• the LabLogic Logi-CHROM HPLC instrument was used, and the column used was a BioSepTM SEC-s3000290 ⁇ with dimensions of 300 ⁇ 7.8 mm.
• 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 ⁇ g/ml streptomycin in a humidified incubator at 37°C and 5% CO 2 . 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 nucleofection of Cas9 ribonuclease protein complex.
• Cell-associated activity percentage was calculated by cell pallet activity/9.25 KBq [ 89 Zr]DFO*-7065 activity.
• Saturation Binding Assay The kd value of [ 89 Zr]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 ⁇ L PBS, with 15 vials total for each cell line. 20 ⁇ L of 0.1% milk in PBS was added to block the non-specific binding.
• 1 nM (0.099 ng; 0.66 ⁇ l) solution of [ 89 Zr]DFO*-7065 (original solution concentration is 433.3 nM) was prepared in 100 ⁇ l PBS and 1% nonfat milk (20 ⁇ l, 0.1 mg/ml) and added to each vial containing cells. The total volume of the solution was 220 ⁇ l.
• the cells were incubated with [ 89 Zr]DFO*-7065 solution for 1 h, 4 h and 24 h at 37 C. After incubation, the cells were centrifuged and washed with PBS twice.
• 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 [ 225 Ac]Macropa-PEG 4 -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).
• NSG stock number 005557
• NRG stock number from Jackson Lab 007799
• mice 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 10 5 to 10 7 photons/sec/cm 2 /steradian, mice were injected with [ 89 Zr]DFO*-7065 followed by PET imaging and biodistribution study.
• In vivo PET Imaging [0152] General PET Imaging method.
• the animals were imaged at day 1, day 2, and day 4 post injection of all the radiotracer.
• ⁇ PET/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 795667431 simultaneously.
• 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.
• FOV field of view
• CT data were reconstructed using the vendor-provided cone-beam filtered back projection (FBP) with a cosine filter.
• FBP cone-beam filtered back projection
• 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.
• mice were injected with 100 ⁇ l 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.
• Dosimetry calculations were performed by determining time- integrated activity coefficients and applying curve-fitting techniques within the EXM module of OLINDA/EXM Version 1.1 27 . For this analysis, the digital mouse phantom provided in OLINDA Version 2.0 was used.
• the treatment arms included: 1) saline control (mice injected with 100 ⁇ L of saline + 0.5 mg non-specific binding IgG), 2) 9.25 KBq of [ 225 Ac]Macropa- PEG4-IgG, 3) 9.25 KBq [ 225 Ac]DOTA-anti-CD33, and 4) 9.25 KBq of [ 225 Ac]Macropa-PEG4- 7065.
• An additional fractionated dose treatment involving a total of three doses of 9.25 KBq of [ 225 Ac]Macropa-PEG4-7065, was also included in the study. The fractionated doses of [ 225 Ac]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.
• 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 ⁇ m were prepared for H & E staining at Comparative Pathology Laboratory, School of Veterinary Medicine, University of California Davis.
• Immunostaining of cells was performed as per the instructions from the antibody vendor unless stated otherwise.
• Cells were resuspended at 1 ⁇ 106 cells/100 ⁇ l of FACS buffer with 5 ⁇ g of human Fc Block (Biolegend, 422302) added. Cells were incubated at 22 °C for 10–15 min, then 3 ⁇ g 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 70 ⁇ m 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+.
• Toxicity study in healthy NRG, Black C57BL/6 and humanized mice [0159] Acute toxicity in black mice. The toxicity of [ 225 Ac]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 [ 225 Ac]DOTA-anti-CD33, 9.25 KBq of [ 225 Ac]Macropa-PEG4-7065, and 9.25 KBq of [ 225 Ac]Macropa-PEG4-IgG.
• the mice were monitored, and their body condition score and body weights are recorded at every alternate day.
• peripheral blood was withdrawn by submandibular vein and blood parameters was analyzed by Hemavet 950/950FS Multi-Species Analyzer.
• 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 795667431 blood 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 10 minutes at 4 C.
• the blood and serum samples were sent to 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.
• Cohorts included saline, 4.62 KBq single dose, 4.62 KBq ⁇ Ci 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 [ 225 Ac]Macropa-PEG4-7065 and [ 225 Ac]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.
• Treatment groups included saline control, 4.62 KBq dose of [ 225 Ac]DOTA-anti-CD33, 4.62 KBq of [ 225 Ac]Macropa-PEG 4 -7066.
• the mice were monitored, and their body condition score and body weights are recorded at every alternate day.
• peripheral blood was withdrawn by submandibular vein and blood parameters was analyzed by Hemavet 950/950FS Multi-Species Analyzer.
• 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 minutes to separate the serum from the clotted blood.
• the serum samples were separated from the clots by centrifugation at 10,000 rpm for 10 minutes at 4 C.
• the blood and serum samples were sent to 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.
• 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 cells 20 .
• mice were euthanized when the hCD45 cell population reached approximately 50–90% and when 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.
• DFO* chelator which has stable binding to 89 Zr, and lower non-specific bone KILPATRICK TOWNSEND 795667431 uptake compared to the more commonly used DFO 28 .
• 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.
• SEC analysis showed no aggregation of the [ 89 Zr]DFO*-7065 after the labeling and purification process.
• This bifunctional chelator employes the efficient Macropa chelator 30 , 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.
• Macropa-PEG 4 -7065 FIG.1C
• Macropa-PEG4-IgG FIG.1D
• the anti-CD33 antibody, Lintuzumab was conjugated to DOTA-NCS (FIG.
• We also developed a 134 Ce labeled version of Macropa-PEG4-7065 to directly image this conjugate. Macropa-PEG4-7065 was radiolabeled with 134 CeCl 3 , with a radiochemical yield of 75 0.32% (n 3 syntheses) KILPATRICK TOWNSEND 795667431 with 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 [ 225 Ac]Macropa-PEG 4 -7065 revealed an immunoreactive fraction of 81.35 0.93%.
• We also performed immunoreactivity assay with [ 225 Ac]DOTA-anti-CD33 with CD33 recombinant protein demonstrated the immunoreactive fraction of 77.04 3.17%.
• the stability of [ 225 Ac]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]
• 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).
• 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.
• Nomo-1 and HL-60 exhibited the highest cell-binding percentages for [ 89 Zr]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 [ 89 Zr]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.
• K d The dissociation constant (K d ) of [ 89 Zr]DFO*-7065 for aITGB2-expressing cell lines was determined through a saturation binding assay, yielding a K d value 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) .
• Example 3 PET/CT imaging and biodistribution analysis of [89Zr]DFO*-7065 in disseminated AML models reveal high aITGB2-targeted uptake [0169]
• 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 [ 89 Zr]DFO*-7065 in the bone marrow of the Nomo-1 disseminated model, with spatial colocalization of bioluminescence (BLI) and PET signals (FIG. 3B).
• 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 [ 89 Zr]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 [ 89 Zr]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.
• 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).
• [ 89 Zr]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 [ 89 Zr]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 [ 89 Zr]DFO*-7065 uptake by PET/CT. Similar findings were seen in biodistribution studies (FIG. 3N, 3O). The tumor-to-blood (Tumor/Blood) ratio (FIG.
• PET/CT imaging and biodistribution studies were performed using [ 18 F]-FDG in Nomo-1 and THP-1 disseminated models. PET/CT imaging revealed some [ 18 F]-FDG uptake in tumors (data not shown), which corresponded to areas identified by bioluminescence imaging (BLI).
• KILPATRICK TOWNSEND 795667431 the 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 [ 18 F]-FDG accumulation, while kidneys and heart exhibited higher uptake consistent with their roles in [ 18 F]-FDG metabolism and excretion.
• the Kd value for [ 225 Ac]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 795667431 line. After validating promising therapeutic efficacy in vitro in AML cell lines, we evaluated the biodistribution and dosimetry of [ 225 Ac/ 134 Ce]Macropa-PEG 4 -7065 in vivo in an AML disseminated model.
• 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).
• liver or kidney function was observed in saline and single or fractioned dose injected dose of 4.62 KBq or 9.25 KBq of [ 225 Ac]Macropa-PEG 4 -7065.
• H & E analysis of dose limiting organs showed that in case of single and fractionated dose of 4.62 KBq of [ 225 Ac]Macropa-PEG 4 -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.
• Example 6 – [225Ac]Macropa-PEG4-7065 demonstrates high therapeutic efficacy and prolonged survival in Nomo-1 disseminated AML model
• a treatment study was designed to evaluate the therapeutic efficacy of [ 225 Ac]Macropa-PEG 4 -7065 (FIG. 5A), with groups including vehicle control, non-targeting [ 225 Ac]Macropa-PEG 4 -IgG, the previously evaluated leukemia RIT [ 225 Ac]DOTA-anti-CD33, and [ 225 Ac]Macropa-PEG 4 -7065.
• An additional group received three fractionated doses of [ 225 Ac]Macropa-PEG 4 -7065.
• the Kaplan-Meier survival curve showed that the median survival time for mice of 43 days for the vehicle group, 50 days for [ 225 Ac]Macropa-PEG 4 -IgG, and 43 days for the [ 225 Ac]DOTA-anti-CD33 group.
• the median survival was undefined and significantly higher than negative as well as positive control (FIG.5D).
• 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 [ 225 Ac]Macropa-PEG4-7065 groups (FIG. 5E, 5F), saline, [ 225 Ac]DOTA-anti-CD33 (FIG. 5G) [ 225 Ac]Macropa-PEG4-IgG (FIG. 5H), saline + IgG injected groups (FIG.
• Radioligand therapy 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. 37 Unlike conventional chemotherapy or CAR-T cell therapy 38 , 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 strategy 39 .
• [0184] The imaging capability of [ 89 Zr]DFO*-7065 was compared to that of [ 18 F]FDG which is a gold standard of care for most of the hematological malignancies in Nomo-1 and THP-1 disseminated AML models.
• [ 18 F]FDG demonstrate moderate uptake in tumorous organs (bone marrow and liver) and lower tumor to background ration as compared to [ 89 Zr]DFO*- 7065 (FIG. 3).
• [ 89 Zr]DFO*-7065 specifically binds aITB2, and can be employed for detecting AML in preclinical models.
• [ 225 Ac]Macropa- PEG 4 -7065 exhibited higher therapeutic efficacy as compared to [ 225 Ac]DOTA-antiCD33 with a significant improvement in median time period survival compared to negative and positive control.
• PDX patient- derived xenograft
• KILPATRICK TOWNSEND 795667431 Summary of example results [0188] [ 89 Zr]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 [ 18 F]FDG. Radioimmunotherapy agent [ 225 Ac]Macropa-PEG4-7065 was successfully prepared and demonstrated improved safety profile and promising therapeutic in disseminated models of AML. Overall, [ 89 Zr]DFO*-7065 showed great potential for imaging and theraonstic companion biomarker and [ 225 Ac]Macropa- PEG 4 -7065 shows promising therapeutic efficacy.
• KILPATRICK TOWNSEND 795667431 (4) Magee, G.; Ragon, B. K. Allogeneic Hematopoietic Cell Transplantation in Acute Myeloid Leukemia. Best Practice & Research Clinical Haematology 2023, 36 (2), 101466. https://doi.org/10.1016/j.beha.2023.101466. (5) Liu, Y.; Wang, S.; Schubert, M.-L.; Lauk, A.; Yao, H.; Blank, M.
• KILPATRICK TOWNSEND 795667431 Nix, M. A., Mandal, K., Geng, H., Paranjape, N., Lin, Y. H. T., Rivera, J. M., ... & Wiita, A. P. (2021).
• KILPATRICK TOWNSEND 795667431 (31) Ludwig, D. L.; Garg, R.; Allen, K.; Dadachova, E.; Patel, V. Radiolabeling of DOTA- Conjugated Lintuzumab with 225Ac: Comparison of 229Th-Produced and High-Energy Proton Accelerator-Produced 225Ac. (32) Rosenblat, T. L.; McDevitt, M. R.; Carrasquillo, J. A.; Pandit-Taskar, N.; Frattini, M.

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