Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
  • A61K51/1096—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies radioimmunotoxins, i.e. conjugates being structurally as defined in A61K51/1093, and including a radioactive nucleus for use in radiotherapeutic applications
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
  • A61K51/103—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants against receptors for growth factors or receptors for growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
  • A61K51/1051—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from breast, e.g. the antibody being herceptin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
  • A61K51/1054—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from lung
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
  • A61K51/1057—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from liver or pancreas
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
  • A61K51/106—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from kidney or bladder
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
  • A61K51/1063—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from stomach or intestines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
  • A61K51/1069—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from blood cells, e.g. the cancer being a myeloma
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/10—Drugs for disorders of the urinary system of the bladder
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/77—Internalization into the cell

Definitions

• Fibroblast growth factors and their receptors (FGFRs) play critical roles during embryonic development, tissue homeostasis and metabolism.
• FGFs Fibroblast growth factors
• FGFRs Fibroblast growth factors
• FGF1-14, FGF16-23 FGF1-14, FGF16-23
• FGF1- 4 FGF receptors with tyrosine kinase domain
• FGFRs consist of an extracellular ligand binding region, with two or three immunoglobulin-like domains (IgDl-3), a single-pass transmembrane region, and a cytoplasmic, split tyrosine kinase domain.
• FGFRs are overexpressed in many cancer types, often due to mutations that confer constitutive activation.
• Aberrantly activated FGFRs have been implicated in specific human malignancies.
• the t(4; 14) (pl6.3;q32) chromosomal translocation occurs in about 15-20% of multiple myeloma patients, leading to overexpression of FGFR3 and correlates with shorter overall survival.
• FGFR3 is implicated in conferring chemoresistance to myeloma cell lines in culture, consistent with the poor clinical response of t(4; 14)+ patients to conventional chemotherapy.
• FGFR3-TACC3 transforming acidic coiled-coil 3 oncogenic fusions have also been observed in a subset of glioblastomas and other cancers, and early data suggests that such tumors may be sensitive to FGFR inhibition. Additionally, genomic alterations that activate FGFR3 are frequent in bladder cancer, including metastatic bladder urothelial carcinoma. [0005] FGFR3 has thus been proposed as a potential therapeutic target for cancer.
• the present disclosure relates to radioimmunoconjugates that target FGFR3 (e.g., human FGFR3, including wild type and/or mutant FGFR3), pharmaceutical compositions thereof, and methods of treating cancer using such pharmaceutical compositions.
• target FGFR3 e.g., human FGFR3, including wild type and/or mutant FGFR3
• provided radioimmunoconjugates exhibit an increased excretion rate (e.g., after being administered to a mammal) compared to some currently known radiotherapeutics, while still maintaining therapeutic efficacy.
• a faster excretion may limit off-target toxicities by limiting the amount of time that the radioimmunoconjugate stays in a subject.
• provided immunoconjugates exhibit reduced off-target toxicities.
• radioimmunoconjugate comprising the following structure: A-L-B (Formula I-a) wherein A is a chelating moiety or metal complex thereof, wherein B is an FGFR3 targeting moiety, and wherein L is a linker.
• A is a metal complex of a chelating moiety.
• the metal complex comprises a radionuclide.
• the radionuclide is an alpha emitter, e.g., an alpha emitter selected from the group consisting of Astatine-211 ( 211 At), Bismuth-212 ( 212 Bi), Bismuth-213 ( 213 Bi), Actinium-225 ( 225 Ac), Radium-223 ( 223 Ra), Lead-212 ( 212 Pb), Thorium-227 ( 227 Th), and Terbium-149 ( 149 Tb), or a progeny thereof.
• the radionuclide is 225 Ac or a progeny thereof.
• the FGFR3 targeting moiety is at least 100 kDa in size, e.g., at least 150 kDa in size, at least 200 kDa in size, at least 250 kDa in size, or at least 300 kDa in size.
• the FGFR3 targeting moiety is capable of binding to human FGFR3.
• the FGFR3 targeting moiety is capable of binding to wild type FGFR3.
• the FGFR3 targeting moiety is capable of binding to a mutant FGFR3.
• FGFR3 targeting moiety is capable of binding to both wild type and a mutant FGFR3.
• the mutant FGFR3 comprises a point mutation, e.g., a point mutation is associated with cancer.
• the point mutant is selected from the group consisting of FGFR3Y375C, FGFR3R248C, FGFR3S249C, FGFR3G372C, FGFR3K652E, FGFR3K652Q, FGFR3K652M, and combinations thereof.
• the mutant FGFR3 comprises an FGFR3 fusion.
• the FGFR3 fusion is selected from the group consisting of FGFR3-TACC3, FGFR3-CAMK2A, FGFR3-JAKMOP1, FGFR3-TNIP2, FGFR3-WHSC 1 , FGFR3- BAIAP2L1, and combinations thereof.
• the FGFR3 targeting moiety comprises an antibody or antigen-binding fragment thereof, e.g., a humanized antibody or antigen-binding fragment thereof.
• the antibody or antigen-binding fragment thereof comprises at least one complementarity determining region (CDR) selected from the group consisting of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 or 4, or an amino acid sequence differing in 1 or 2 amino acids from SEQ ID NO: 3 or 4; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence differing in 1 or 2 amino acids therefrom; or CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence differing in 1 or 2 amino acids therefrom.
• CDR-H1 compris
• the antibody or antigen-binding fragment thereof comprises at least two CDRs selected from the group consisting of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 or 4, or an amino acid sequence differing in 1 or 2 amino acids from SEQ ID NO: 3 or 4; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence differing in 1 or 2 amino acids therefrom; or CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence differing in 1 or 2 amino acids therefrom.
• CDR-H1 comprising the amino acid sequence of S
• the antibody or antigen-binding fragment thereof comprises at least three CDRs selected from the group consisting of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 or 4, or an amino acid sequence differing in 1 or 2 amino acids from SEQ ID NO: 3 or 4; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence differing in 1 or 2 amino acids therefrom; or CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence differing in 1 or 2 amino acids therefrom.
• CDR-H1 comprising the amino acid sequence of S
• the antibody or antigen-binding fragment thereof comprises at least four CDRs selected from the group consisting of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 or 4, or an amino acid sequence differing in 1 or 2 amino acids from SEQ ID NO: 3 or 4; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence differing in 1 or 2 amino acids therefrom; or CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence differing in 1 or 2 amino acids therefrom.
• CDR-H1 comprising the amino acid sequence of S
• the antibody or antigen-binding fragment thereof comprises at least five CDRs selected from the group consisting of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 or 4, or an amino acid sequence differing in 1 or 2 amino acids from SEQ ID NO: 3 or 4; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence differing in 1 or 2 amino acids therefrom; or CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence differing in 1 or 2 amino acids therefrom.
• CDR-H1 comprising the amino acid sequence of S
• the antibody or antigen-binding fragment thereof comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 or 4, or an amino acid sequence differing in 1 or 2 amino acids from SEQ ID NO: 3 or 4; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence differing in 1 or 2 amino acids therefrom; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence differing in 1 or 2 amino acids therefrom.
• the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable domain comprising at least one CDR selected from the group consisting of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence differing in 1 or 2 amino acids therefrom; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 or 4, or an amino acid sequence differing in 1 or 2 amino acids from SEQ ID NO: 3 or 4; and (ii) a light chain variable domain comprising at least one CDR selected from the group consisting of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence differing in 1 or 2 amino acids therefrom; and CDR-
• the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable domain comprising at least one CDR selected from the group consisting of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 or 4; and (ii) a light chain variable domain comprising at least one CDR selected from the group consisting of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7.
• the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable domain comprising at least two CDRs selected from the group consisting of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence differing in 1 or 2 amino acids therefrom; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 or 4, or an amino acid sequence differing in 1 or 2 amino acids from SEQ ID NO: 3 or 4; and (ii) a light chain variable domain comprising at least two CDRs selected from the group consisting of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence differing in 1 or 2 amino acids therefrom; and C
• the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable domain comprising at least two CDRs selected from the group consisting of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 or 4; and (ii) a light chain variable domain comprising at least two CDRs selected from the group consisting of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7.
• the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable domain comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence differing in 1 or 2 amino acids therefrom; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 or 4, or an amino acid sequence differing in 1 or 2 amino acids from SEQ ID NO: 3 or 4; and (ii) a light chain variable domain comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence differing in 1 or 2 amino acids therefrom; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence differing in 1 or 2 amino acids therefrom; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence differing in 1
• the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable domain comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 or 4; and (ii) a light chain variable domain comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7.
• the antibody or antigen-binding fragment thereof comprises:(i) a heavy chain variable domain having an amino acid sequence with at least 85% identity with the amino acid sequence of SEQ ID NO: 8; and (ii) a light chain variable domain having an amino acid sequence with at least 85% identity with the amino acid sequence of SEQ ID NO: 9. [0031] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable domain having an amino acid sequence with at least 90% identity with the amino acid sequence of SEQ ID NO: 8; and (ii) a light chain variable domain having an amino acid sequence with at least 90% identity with the amino acid sequence of SEQ ID NO: 9.
• the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable domain having an amino acid sequence with at least 95% identity with the amino acid sequence of SEQ ID NO: 8; and (ii) a light chain variable domain having an amino acid sequence with at least 95% identity with the amino acid sequence of SEQ ID NO: 9. [0033] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 8; and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 9. [0034] In some embodiments, the antibody is MFGR1877S (vofatamab).
• the proportion of radiation excreted by the intestinal routes, renal route, or both routes is at least 2-fold greater than the proportion of radiation excreted by the same route(s) by a comparable mammal that has been administered a reference radioimmunoconjugate.
• the proportion of radiation excreted by the intestinal routes, renal route, or both routes is at least 3-fold greater than the proportion of radiation excreted by the same route(s) by a comparable mammal that has been administered a reference radioimmunoconjugate.
• A-L- is a metal complex of a compound selected from the group consisting of: (iii) [0038] In some embodiments, A-L- is a metal complex of: . [0039] In some embodiments, A-L- is a metal complex of , and the metal complex comprises a radionuclide, such as an alpha emitter (e.g., Astatine-211 ( 211 At), Bismuth-212 ( 212 Bi), Bismuth-213 ( 213 Bi), Actinium-225 ( 225 Ac), Radium-223 ( 223 Ra), Lead-212 ( 212 Pb), Thorium-227 ( 227 Th), and Terbium-149 ( 149 Tb), or a progeny thereof).
• an alpha emitter e.g., Astatine-211 ( 211 At), Bismuth-212 ( 212 Bi), Bismuth-213 ( 213 Bi), Actinium-225 ( 225 Ac), Radium-223 ( 223 Ra), Lead-212 ( 212 Pb), Thorium-2
• the FGFR3 targeting moiety is an antibody or antigen- binding fragment thereof (e.g., a humanized antibody or antigen-binding fragment thereof).
• A-L- is a metal complex of , the metal complex comprises 225 Ac or a progeny thereof, and the FGFR3 targeting moiety is MFGR1877S (vofatamab) or an antigen-binding fragment thereof. In some embodiments, the FGFR3 targeting moiety is MFGR1877S (vofatamab).
• the radioimmunoconjugate comprising the following structure: , wherein is MFGR1877S (vofatamab), wherein the amine group NH– attached to the antibody shown above is from a lysine unit that is part of the antibody.
• MFGR1877S vofatamab
• the amine group NH– attached to the antibody shown above is from a lysine unit that is part of the antibody.
• provided are pharmaceutical compositions comprising a radioimmunoconjugate as described herein and a pharmaceutically acceptable carrier.
• methods of treating cancer comprising administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of a radioimmunoconjugate as described herein.
• the subject is a mammal, e.g., a human.
• the cancer is a solid tumor cancer.
• the solid tumor cancer is adrenocortical carcinoma, bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial adenocarcinoma, Ewing’s sarcoma, gallbladder carcinoma, glioma, head and neck cancer, liver cancer, lung cancer, neuroblastoma, neuroendocrine cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, salivary adenoid cystic cancer, or spermatocytic seminoma.
• the solid tumor cancer is bladder cancer.
• the solid tumor cancer is glioma. In some embodiments, the solid tumor cancer is neuroblastoma. In some embodiments, the solid tumor cancer is pancreatic cancer. In some embodiments, the solid tumor cancer is breast cancer. In some embodiments, the solid tumor cancer is head and neck cancer. In some embodiments, the solid tumor cancer is live cancer. In some embodiments, the solid tumor cancer is lung cancer. [0046] In some embodiments, the cancer is a non-solid tumor cancer. In some embodiments, the cancer is a liquid cancer or hematologic cancer, e.g., a myeloma (e.g., multiple myeloma), a leukemia, or a lymphoma.
• a myeloma e.g., multiple myeloma
• leukemia e.g., multiple myeloma
• the pharmaceutical composition is administered systemically.
• the pharmaceutical composition is administered parenterally, e.g., intravenously, intraarterially, intraperitoneally, subcutaneously, or intradermally.
• the pharmaceutical composition is administered enterically, e.g., trans-gastrointestinally or orally.
• the pharmaceutical composition is administered locally, e.g., by peritumoral injection or by intratumoral injection.
• FIG.1A is a schematic depicting the general structure of a conjugate comprising a chelate, a linker, and a targeting moiety.
• FIG.1B is a schematic depicting the structure of [ 225 Ac]-DOTA-anti-FGFR3, an exemplary radioimmunoconjugate disclosed herein.
• FIG.2 is a schematic depicting the synthesis of the bifunctional chelate, 4- ⁇ [11-oxo-11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl ⁇ -2-[4,7,10-tris(carboxymethyl)- 1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound B). Synthesis of Compound B is described in Example 2.
• FIG.3 is a schematic depicting the synthesis of the bifunctional chelate, 4- ⁇ [2- (2- ⁇ 2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy ⁇ ethoxy)ethyl]carbamoyl ⁇ -2- [4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound C). Synthesis of Compound C is described in Example 4.
• FIGs.4A-4C are binding curves for unlabeled DOTA-anti-FGFR3 binding to RT4 (FIG.4A), RT112 (FIG.4B), and HepG2 (FIG.4C) FGFR3-positive tumor cells. See Example 16.
• FIG.5 shows a plot representing the results of biodistribution studies in mice bearing RT4 (bladder cancer) xenograft tumors and injected with [ 177 Lu]-DOTA-anti-FGFR3.
• FIG.6A shows a plot representing the results of biodistribution studies in mice bearing RT112 (bladder cancer) xenograft tumors and injected with [ 177 Lu]-DOTA-anti- FGFR3.
• % ID/g is plotted on the x-and is shown for blood, intestine, kidney and adrenal glands, liver and gall bladder, lung, spleen, skin, bladder, urine, and tumor at 4, 24, 48, and 96 hours. See Example 18.
• FIG.6B shows a plot representing the results of biodistribution studies in mice bearing RT112 (bladder cancer) xenograft tumors and injected with [ 177 Lu]-DOTA-anti- FGFR3 after a pre-dose with cold anti-FGFR3.
• % ID/g is plotted on the x-axis and is shown for blood, intestine, kidney and adrenal glands, liver and gall bladder, lung, spleen, skin, bladder, urine, and tumor at 4, 24, 48, and 96 hours.
• Mice received a pre-dose of 100 ⁇ g cold (non-radioactive, un-conjugated) anti-FGFR3 antibody 3 hours before receiving [ 177 Lu]- DOTA-anti-FGFR3. See Example 18.
• FIGs.7A-7C show plots representing the results of biodistribution studies in mice bearing RT112 (bladder cancer) xenograft tumors and co-dosed with cold anti-FGFR3 and [ 177 Lu]-DOTA-anti-FGFR3.
• % ID/g is plotted on the x-axis and is shown for blood, intestine, kidney, liver, lung, spleen, skin, bladder, urine, and tumor at 24 and 96 hours.
• FIGs.8A-8B show plots representing the results of biodistribution studies in mice bearing RT112 (bladder cancer) xenograft tumors and co-dosed with cold anti-FGFR3 and either [ 177 Lu]-DOTA-anti-FGFR3 (FIG.8A) or [ 111 In]-DOTA-anti-FGFR3 (FIG.8B).
• FIGs.9A and 9B are plots showing relative tumor volumes (FIG.9A) and relative body weights (FIG.9B) in mice who, at the beginning of the experiment, bore RT112 xenograft tumors. Relative tumor volumes (FIG.9A) and relative body weights (FIG.
• FIG.10A and 10B are plots showing relative tumor volumes (FIG.10A) and relative body weights (FIG.10B) in mice who, at the beginning of the experiment, bore RT112 xenograft tumors. Relative tumor volumes (FIG.10A) and relative body weights (FIG.10B) are shown at various timepoints after treatment with [ 225 Ac]-DOTA-anti-FGFR3.
• Radioimmunoconjugates are designed to target a protein or receptor that is upregulated in a disease state to deliver a radioactive payload to damage and kill cells of interest (radioimmunotherapy).
• the process of delivering such a payload, via radioactive decay, produces an alpha, beta, or gamma particle or Auger electron that can cause direct effects to DNA (such as single or double stranded DNA breaks) or indirect effects such as by- stander or crossfire effects.
• Radioimmunoconjugates typically contain a biological targeting moiety (e.g., an antibody or antigen binding fragment thereof that is capable of specifically binding to human FGFR3), a radioisotope, and a molecule that links the two. Conjugates are formed when a bifunctional chelate is appended to the biological targeting molecule so that structural alterations are minimal while maintaining target affinity. Once radiolabelled, the final radioimmunoconjugate is formed.
• Bifunctional chelates structurally contain a chelate, the linker, and a targeting moiety, e.g., an antibody or antigen-binding fragment thereof (FIG.1). When developing new bifunctional chelates, most efforts focus around the chelating portion of the molecule.
• Radioimmunoconjugates do not need to block a receptor, as needed with a therapeutic antibody, or release the cytotoxic payload intracellularly, as required with an antibody drug conjugate, in order to have therapeutic efficacy.
• the emission of the toxic particle is an event that occurs as a result of first-order (radioactive) decay and can occur at random anywhere inside the body after administration. Once the emission occurs, damage could occur to surrounding cells within the range of the emission leading to the potential of off-target toxicity. Therefore, limiting exposure of these emissions to normal tissue is the key to developing new drugs.
• One potential method for reducing off-target exposure is to remove the radioactivity more effectively from the body (e.g., from normal tissue in the body).
• Cleavable linkers have been taken on different meaning as it relates to radioimmunoconjugates.
• Cornelissen, et al. has described cleavable linkers as those by which the bifunctional conjugate attaches to the biologic targeting agent through a reduced cysteine, whereas others have described the use of enzyme- cleavable systems that require the co-administration of the radioimmunoconjugate with a cleaving agent/enzyme to release [Mol Cancer Ther.2013, 12(11), 2472-2482; Methods Mol Biol.2009, 539, 191-211; Bioconjug Chem.2003, 14(5), 927-33].
• the present disclosure provides, among other things, radioimmunoconjugates that are more effectively eliminated from the body after catabolism and/or metabolism, while maintain therapeutic efficacy.
• Disclosed immunoconjugates may, in some embodiments, achieve a reduction of total body radioactivity, for example, by increasing the extent of excretion of the catabolic/metabolic products while maintaining the pharmacokinetics of the intact molecule when compared to known bifunctional chelates.
• radioimmunoconjugates achieve reduced radioactivity in the human body while maintaining on-target activity.
• antibody refers to a polypeptide whose amino acid sequence includes immunoglobulins and fragments thereof which specifically bind to a designated antigen, or fragments thereof.
• Antibodies in accordance with the present invention may be of any type (e.g., IgA, IgD, IgE, IgG, or IgM) or subtype (e.g., IgA1, IgA2, IgG1, IgG2, IgG3, or IgG4).
• a characteristic sequence or portion of an antibody may include amino acids found in one or more regions of an antibody (e.g., variable region, hypervariable region, constant region, heavy chain, light chain, and combinations thereof).
• a characteristic sequence or portion of an antibody may include one or more polypeptide chains, and may include sequence elements found in the same polypeptide chain or in different polypeptide chains.
• antigen-binding fragment refers to a portion of an antibody that retains the binding characteristics of the parent antibody.
• bind or binding of a targeting moiety means an at least temporary interaction or association with or to a target molecule, e.g., to human FGFR3 and/or mutant FGFR3, e.g., as described herein.
• bifunctional chelate or bifunctional conjugate refers to a compound that comprises a chelate or metal complex thereof, a linker, and a targeting moiety e.g., an antibody or antigen-binding fragment thereof.
• cancer refers to any cancer caused by the proliferation of malignant neoplastic cells, such as tumors, neoplasms, carcinomas, sarcomas, leukemias, and lymphomas.
• a “solid tumor cancer” is a cancer comprising an abnormal mass of tissue, e.g., sarcomas, carcinomas, and lymphomas.
• a “hematological cancer” or “liquid cancer,” as used interchangeably herein, is a cancer present in a body fluid, e.g., lymphomas and leukemias.
• chelate refers to an organic compound or portion thereof that can be bonded to a central metal or radiometal atom at two or more points.
• conjugate refers to a molecule that contains a chelating group or metal complex thereof, a linker group, and which optionally contains a targeting moiety, e.g., an antibody or antigen-binding fragment thereof.
• compound is meant to include all stereoisomers, geometric isomers, and tautomers of the structures depicted.
• the compounds recited or described herein can be asymmetric (e.g., having one or more stereocenters).
• detection agent refers to a molecule or atom which is useful in diagnosing a disease by locating the cells containing the antigen.
• Various methods of labeling polypeptides with detection agents are known in the art.
• detection agents include, but are not limited to, radioisotopes and radionuclides, dyes (such as with the biotin-streptavidin complex), contrast agents, luminescent agents (e.g., fluorescein isothiocyanate or FITC, rhodamine, lanthanide phosphors, cyanine, and near IR dyes), and magnetic agents, such as gadolinium chelates.
• radioisotopes and radionuclides include, but are not limited to, radioisotopes and radionuclides, dyes (such as with the biotin-streptavidin complex), contrast agents, luminescent agents (e.g., fluorescein isothiocyanate or FITC, rhodamine, lanthanide phosphors, cyanine, and near IR dyes), and magnetic agents, such as gadolinium chelates.
• luminescent agents e.g., fluorescein isothio
• the term “radionuclide,” refers to an atom capable of undergoing radioactive decay (e.g., 3 H, 14 C, 15 N, 18 F, 35 S, 47 Sc, 55 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 75 Br, 76 Br , 77 Br , 89 Zr, 86 Y, 87 Y, 90 Y, 97 Ru, 99 Tc, 99m Tc 105 Rh, 109 Pd, 111 In, 123 I, 124 I, 125 I, 131 I, 149 Pm, 149 Tb, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 198 Au, 199 Au, 203 Pb, 211 At, 212 Pb , 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227 Th, 229 Th, 66 Ga, 67 Ga, 68 Ga, 82 Rb, 117m Sn, 201 Tl).
• radioactive decay e.
• radioactive nuclide may also be used to describe a radionuclide.
• Radionuclides may be used as detection agents, as described herein.
• the radionuclide may be an alpha-emitting radionuclide.
• an “effective amount” of an agent e.g., any of the foregoing conjugates, as used herein, is that amount sufficient to effect beneficial or desired results, such as clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied.
• an “effective amount” may be an amount sufficient to cure or at least partially arrest the symptoms of the disorder and its complications, and/or to substantially improve at least one symptom associated with the disease or a medical condition.
• an agent or compound that decreases, prevents, delays, suppresses, or arrests any symptom of the disease or condition would be therapeutically effective.
• a therapeutically effective amount of an agent or compound is not required to cure a disease or condition but may, for example, provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, such that the disease or condition symptoms are ameliorated, or such that the term of the disease or condition is changed.
• the disease or condition may become less severe and/or recovery is accelerated in an individual.
• An effective amount may be administered by administering a single dose or multiple (e.g., at least two, at least three, at least four, at least five, or at least six) doses.
• a targeting moiety such as an antibody (or antigen-binding fragment thereof), nanobody, affibody, or a consensus sequence from Fibronectin type III domain.
• the immunoconjugate comprises an average of at least 0.10 conjugates per targeting moiety (e.g., an average of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 4, 5, or 8 conjugates per targeting moiety).
• the term “radioconjugate,” as used herein, refers to any conjugate that includes a radioisotope or radionuclide, such as any of the radioisotopes or radionuclides described herein.
• radioimmunoconjugate refers to any immunoconjugate that includes a radioisotope or radionuclide, such as any of the radioisotopes or radionuclides described herein.
• radiationoimmunotherapy refers a method of using a radioimmunoconjugate to produce a therapeutic effect.
• radioimmunotherapy may include administration of a radioimmunoconjugate to a subject in need thereof, wherein administration of the radioimmunoconjugate produces a therapeutic effect in the subject.
• radioimmunotherapy may include administration of a radioimmunoconjugate to a cell, wherein administration of the radioimmunoconjugate kills the cell.
• radioimmunotherapy involves the selective killing of a cell
• the cell is a cancer cell in a subject having cancer.
• pharmaceutical composition represents a composition containing a radioimunoconjugate described herein formulated with a pharmaceutically acceptable excipient.
• the pharmaceutical composition is manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal.
• compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gelcap, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); or in any other formulation described herein.
• Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, radioprotectants, sorbents, suspending or dispersing agents, sweeteners, or waters of hydration.
• excipients include, but are not limited to: ascorbic acid, histidine, phosphate buffer, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid,
• salts represent those salts of the compounds described here that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, or allergic response.
• Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting the free base group with a suitable organic acid.
• the compounds of the invention may have ionizable groups so as to be capable of preparation as pharmaceutically acceptable salts.
• These salts may be acid addition salts involving inorganic or organic acids or the salts may, in the case of acidic forms of the compounds of the invention be prepared from inorganic or organic bases.
• the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases.
• Suitable pharmaceutically acceptable acids and bases are well-known in the art, such as hydrochloric, sulphuric, hydrobromic, acetic, lactic, citric, or tartaric acids for forming acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines for forming basic salts.
• Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate
• alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.
• polypeptide refers to a string of at least two amino acids attached to one another by a peptide bond.
• a polypeptide may include at least 3-5 amino acids, each of which is attached to others by way of at least one peptide bond.
• polypeptides can include one or more “non-natural” amino acids or other entities that nonetheless are capable of integrating into a polypeptide chain.
• a polypeptide may be glycosylated, e.g., a polypeptide may contain one or more covalently linked sugar moieties.
• a single “polypeptide” e.g., an antibody polypeptide
• subject is meant a human or non-human animal (e.g., a mammal).
• substantially identical is meant a polypeptide sequence that has the same polypeptide sequence, respectively, as a reference sequence, or has a specified percentage of amino acid residues, respectively, that are the same at the corresponding location within a reference sequence when the two sequences are optimally aligned.
• an amino acid sequence that is “substantially identical” to a reference sequence has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the reference amino acid sequence.
• the length of comparison sequences will generally be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90, 100, 150, 200, 250, 300, or 350 contiguous amino acids (e.g., a full- length sequence).
• Sequence identity may be measured using sequence analysis software on the default setting (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Such software may match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.
• sequence analysis software e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705
• Such software may match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.
• targeting moiety refers to any molecule or any part of a molecule that is capable of binding to a given target.
• FGFR3 targeting moiety refers to a targeting moiety that is capable of binding to an FGFR3 molecule, e.g., a human FGFR3, e.g. a wild type or mutant FGFR3.
• FGFR3 targeting moiety refers to a targeting moiety that is capable of binding to an FGFR3 molecule, e.g., a human FGFR3, e.g. a wild type or mutant FGFR3.
• “to treat” a condition or “treatment” of the condition is an approach for obtaining beneficial or desired results, such as clinical results.
• Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease, disorder, or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition; and remission (whether partial or total), whether detectable or undetectable.
• “Palliating” a disease, disorder, or condition means that the extent and/or undesirable clinical manifestations of the disease, disorder, or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment.
• Radioimmunoconjugates having structure of Formula I-a: A-L-B Formula I-a wherein A is a chelating moiety or metal complex thereof, wherein B is a FGFR3 targeting moiety, and wherein L is a linker.
• the radioimmunoconjugate has or comprises the structure shown in Formula II: , wherein B is the FGFR3 targeting moiety.
• A-L- is a metal complex of a compound selected from the group consisting of
• the radioimmunoconjugate comprises a chelating moiety or metal complex thereof, which metal complex may comprise a radionuclide.
• the average ratio or median ratio of the chelating moiety to the FGFR3 targeting moiety is eight or less, seven or less, six or less, five or less, four or less, three or less, two or less, or about one.
• the average ratio or median ratio of the chelating moiety to the FGFR3 targeting moiety is about one.
• the proportion of radiation (of the total amount of radiation that is administered) that is excreted by the intestinal route, the renal route, or both is greater than the proportion of radiation excreted by a comparable mammal that has been administered a reference radioimmunoconjugate.
• reference immunoconjugate it is meant a known radioimmunoconjugate that differs from a radioimmunoconjugate described herein at least by (1) having a different linker; (2) having a targeting moiety of a different size and/or (3) lacking a targeting moiety.
• the reference radioimmunoconjugate is selected from the group consisting of [ 90 Y]-ibritumomab tiuxetan (Zevalin ( 90 Y)) and [ 111 In]- ibritumomab tiuxetan (Zevalin ( 111 In)).
• the proportion of radiation excreted by a given route or set of routes is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% greater than the proportion of radiation excreted by the same route(s) by a comparable mammal that has been administered a reference radioimmunoconjugate.
• the proportion of radiation excreted is at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5 fold, at least 4-fold, at least 4.5 fold, at least 5 fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than proportion of radiation excreted by a comparable mammal that has been administered a reference radioimmunoconjugate.
• the extent of excretion can be measured by methods known in the art, e.g., by measuring radioactivity in urine and/or feces and/or by measuring total body radioactivity over a period time. See also, e.g., International Patent Publication WO 2018/024869.
• the extent of excretion is measured at a time period of at least or about 12 hours after administration, at least or about 24 hours after administration, at least or about 2 days after administration, at least or about 3 days after administration, at least or about 4 days after administration, at least or about 5 days after administration, at least or about 6 days after administration, or at least or about 7 days, after administration.
• the radioimmunoconjugate after a radioimmunoconjugate has been administered to a mammal, the radioimmunoconjugate exhibits decreased off-target binding effects (e.g., toxicities) as compared to a reference conjugate (e.g., a reference immunoconjugate such as a reference radioimmunoconjugate).
• Targeting moieties include any molecule or any part of a molecule that is capable of binding to a given target, e.g., FGFR3.
• the targeting moiety comprises a protein or polypeptide.
• the targeting moiety is selected from the group consisting of antibodies or antigen binding fragments thereof, nanobodies, affibodies, and consensus sequences from Fibronectin type III domains (e.g., Centyrins or Adnectins).
• a moiety is both a targeting and a therapeutic moiety, i.e., the moiety is capable of binding to a given target and also confers a therapeutic benefit.
• the targeting moiety comprises a small molecule.
• the targeting moiety has a molecular weight of at least 50 kDa, at least 75 kDa, at least 100 kDa, at least 125 kDa, at least 150 kDa, at least 175 kDa, at least 200 kDa, at least 225 kDa, at least 250 kDa, at least 275 kDa, or at least 300 kDa.
• the targeting moiety is capable of binding to FGFR3, e.g., wild type and/or mutant FGFR3. In some embodiments, the targeting moiety is capable of binding to human FGFR3, e.g., wild type and/or mutant human FGFR3. [00105] In some embodiments, the targeting moiety is capable of binding specifically to FGFR3 (e.g., is capable of binding to FGFR3 while exhibiting comparatively little or no binding to other kinases such as other FGFR proteins).
• the targeting moiety is capable of binding to an extracellular region of FGFR3, e.g., the IgD1 region, the IgD2 region, the IgD3 region, the linker region between IgD1 and IgD2, the linker region between IgD2 and IgD3, or the extracellular juxtamembrane domain.
• the targeting moiety is capable of binding to the linker region between IgD2 and IgD3.
• the targeting moiety is capable of binding to the extracellular juxtamembrane domain.
• the targeting moiety is capable of binding to the IIIb isoform of FGFR3.
• the targeting moiety is capable of binding to the IIIc isoform of FGFR3. In some embodiments, the targeting moiety is capable of binding to both the IIIb and IIIc isoforms of FGFR3. [00108] In some embodiments, the targeting moiety is capable of binding to a mutant FGFR3, e.g., a mutant human FGFR3. Some FGFR3 mutations give rise to an unpaired cysteine, which may lead to ligand-independent receptor dimerization and/or constitutive activation. In some embodiments, the mutant FGFR3 is an activated mutant and/or is associated with cancer.
• the targeting moiety is capable of binding to wild type FGFR3 and at least one mutant FGFR3 associated with cancer.
• the mutant FGFR3 comprises a mutation in an extracellular region of FGFR3.
• the mutant FGFR3 comprises a mutation in the linker region between IgD2 and IgD3 and/or in the extracellular juxtamembrane region of FGFR3.
• the mutant FGFR3 comprises a mutation in an intracellular region of FGFR3, e.g., a kinase domain, of FGFR3.
• the mutant FGFR3 comprises a point mutation.
• Non- limiting examples of FGFR3 point mutants associated with cancer include FGFR3 Y375C , FGFR3 R248C , FGFR3 S249C , FGFR3 G372C , FGFR3 K652E , FGFR3 K652Q , FGFR3 K652M , and combinations thereof.
• the mutant FGFR3 is ligand-dependent (e.g., FGFR3 G372C or FGFR3 Y375C ).
• the mutant FGFR3 is constitutively active (e.g., FGFR3 R248C or FGFR3 S249C ).
• the mutant FGFR3 is both ligand-dependent and constitutively active (e.g., FGFR3 K652E ).
• the mutant FGFR3 comprises an FGFR3 fusion, e.g., a constitutively activated and/or oncogenic fusion, such as a fusion that arises from a translocation.
• FGFR3-TACC3, FGFR3-CAMK2A, FGFR3-JAKMOP1, FGFR3-TNIP2, FGFR3-WHSC1, and FGFR3-BAIAP2L1 (also known as FGFR3-IRTKS) fusions have been associated with cancer.
• the mutant FGFR3 is an amplifying mutation, e.g., comprising increased copy numbers and/or resulting in higher expression relative to a wild type FGFR3.
• the targeting moiety inhibits FGFR3.
• inhibits it is meant that the targeting moiety at least partially inhibits one or more functions of FGFR3 (e.g., human FGFR3).
• the targeting moiety at least partially inhibits one or more functions of wild type FGFR3, e.g., wild type human FGFR3.
• the targeting moiety at least partially inhibits one or more functions of a mutant FGFR3, e.g., mutant human FGFR3.
• targeting moiety blocks ligand binding to FGFR3 and/or receptor dimerization of FGFR3.
• a targeting moiety that blocks ligand binding competes with FGF ligands for interaction with the IIIb and/or the IIIc isoforms of FGFR3.
• the targeting moiety impairs signaling downstream of the FGFR3 receptor, e.g., results in decreased phosphorylation and/or protein or transcript levels of one or more downstream mediators of FGFR3 such as FRS2 ⁇ , AKT, and p44/42 MAPK.
• Antibodies typically comprise two identical light polypeptide chains and two identical heavy polypeptide chains linked together by disulfide bonds.
• the first domain located at the amino terminus of each chain is variable in amino acid sequence, providing the antibody-binding specificities of each individual antibody. These are known as variable heavy (VH) and variable light (VL) regions.
• the other domains of each chain are relatively invariant in amino acid sequence and are known as constant heavy (CH) and constant light (CL) regions.
• Light chains typically comprise one variable region (VL) and one constant region (CL).
• An IgG heavy chain includes a variable region (VH), a first constant region (CH1), a hinge region, a second constant region (CH2), and a third constant region (CH3).
• Antibodies suitable for use with the present disclosure can include, for example, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and antigen- binding fragments of any of the above.
• the antibody or antigen- binding fragment thereof is humanized.
• the antibody or antigen- binding fragment thereof is chimeric.
• Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.
• antigen binding fragment of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen.
• binding fragments encompassed within the term “antigen binding fragment” of an antibody include a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv fragment, a dAb fragment (Ward et al., (1989) Nature 341:544-546), and an isolated complementarity determining region (CDR).
• an “antigen binding fragment” comprises a heavy chain variable region and a light chain variable region.
• Antibodies or antigen-binding fragments described herein can be produced by any method known in the art for the synthesis of antibodies (See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Brinkman et al., 1995, J. Immunol. Methods 182:41-50; WO 92/22324; WO 98/46645). Chimeric antibodies can be produced using the methods described in, e.g., Morrison, 1985, Science 229:1202, and humanized antibodies by methods described in, e.g., U.S. Pat. No. 6,180,370.
• Additional antibodies described herein are bispecific antibodies and multivalent antibodies, as described in, e.g., Segal et al., J. Immunol. Methods 248:1-6 (2001); and Tutt et al., J. Immunol.147: 60 (1991), or any of the molecules described herein.
• “Avimer” relates to a multimeric binding protein or peptide engineered using, for example, in vitro exon shuffling and phage display. Multiple binding domains are linked, resulting in greater affinity and specificity compared to single epitope immunoglobin domains.
• Nanobodies are antibody fragments consisting of a single monomeric variable antibody domain.
• Nanobodies may also be referred to as single-domain antibodies. Like antibodies, nanobodies are capable of binding selectively to a specific antigen. Nanobodies may be heavy-chain variable domains or light chain domains. Nanobodies may occur naturally or be the product of biological engineering. Nanobodies may be biologically engineered by site-directed mutagenesis or mutagenic screening (e.g., phage display, yeast display, bacterial display, mRNA display, ribosome display).“Affibodies” are polypeptides or proteins engineered to bind to a specific antigen. As such, affibodies may be considered to mimic certain functions of antibodies.
• Affibodies may be engineered variants of the B-domain in the immunoglobulin- binding region of staphylococcal protein A.
• Affibodies may be engineered variants of the Z- domain, a B-domain that has lower affinity for the Fab region.
• Affibodies may be biologically engineered by site-directed mutagenesis or mutagenic screening (e.g., phage display, yeast display, bacterial display, mRNA display, ribosome display).
• site-directed mutagenesis or mutagenic screening e.g., phage display, yeast display, bacterial display, mRNA display, ribosome display.
• Diabodies are antibody fragments with two antigen- binding sites that may be bivalent or bispecific. See for example Hudson et al., (2003). Single-chain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all, or a portion of the light chain variable domain of an antibody.
• Antibody fragments can be made by various techniques including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant hosts (e.g., E. coli or phage) as described herein.
• the antibody or antigen-binding fragment thereof is a multispecific, e.g. bispecific.
• Multispecific antibodies include monoclonal antibodies (or antigen-binding fragments thereof) that have binding specificities for at least two different sites.
• amino acid sequence variants of antibodies or antigen- binding fragments thereof are contemplated; e.g., variants that are capable of binding to human FGFR3 and/or a mutant FGFR3 (such as a mutant FGFR3 associated with cancer). For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody or antigen-binding fragment thereof.
• Amino acid sequence variants of an antibody or antigen-binding fragment thereof may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or antigen- binding fragment thereof, or by peptide synthesis.
• the antibody or antigen binding fragment thereof is an inhibitory antibody (also called “antagonistic antibody”) or antigen-binding fragment thereof, e.g., the antibody or antigen binding fragment thereof at least partially inhibits one or more functions of the target molecule (e.g., FGFR3) as explained further herein.
• an inhibitory antibody also called “antagonistic antibody”
• antigen-binding fragment thereof e.g., the antibody or antigen binding fragment thereof at least partially inhibits one or more functions of the target molecule (e.g., FGFR3) as explained further herein.
• Non-limiting examples of inhibitory antibodies include humanized monoclonal antibodies such as MFGR1877S (CAS No.1312305-12-6; Genentech) (a human monoclonal antibody also known as vofatamab, and whose lyophilized form is also known as B-701 or R3Mab); PRO-001 (Prochon); PRO-007 (Fibron); IMC-D11 (Imclone); and AV-370 (Aveo Pharmaceuticals).
• MFGR1877S CAS No.1312305-12-6; Genentech
• vofatamab whose lyophilized form is also known as B-701 or R3Mab
• PRO-001 Prochon
• PRO-007 Fibron
• IMC-D11 Imclone
• AV-370 Ado Pharmaceuticals
• the antibody or antigen binding fragment thereof is an agonistic antibody (also known as stimulatory antibody).
• the antibody or antigen biding fragment thereof is neither agonistic or antagonistic, or has not been characterized as either agonistic or antagonistic.
• Additional known FGFR3 antibodies include, for example, mouse monoclonal antibodies such as, for example, 1G6, 6G1, and 15B2 from Genentech (See, e.g., US8,410,250), B9 (Sc-13121) (Santa Cruz Biotechnology), MAB766 (clone 136334) (R&D systems), MAB7661 (clone 136318) (R&D systems), and OTI1B10 (OriGene); rabbit polyclonal antibodies such as, for example, ab10651 (Abcam); and rabbit monoclonal antibodies such as C51F2 (catalog number 4574) (Cell Signaling Technology).
• mouse monoclonal antibodies such as, for example, 1G6, 6G1, and 15B2 from Genentech (See, e.g., US8,410,250), B9 (Sc-13121) (Santa Cruz Biotechnology), MAB766 (clone 136334) (R&D systems), MAB7661 (clone 136318) (
• the antibody or antigen- binding fragment thereof comprises specific heavy chain complementarity determining regions CDR-H1, CDR-H2 and/or CDR-H3 as described herein.
• the complementarity determining regions (CDRs) of the antibody or antigen-binding fragment thereof are flanked by framework regions.
• a heavy or light chain of an antibody or antigen- binding fragment thereof containing three CDRs typically contains four framework regions.
• the heavy chain variable region of the FGFR3 antibody or antibody-binding fragment thereof comprises one, two, or three complementarity determining regions (CDRs) CDR-H1, CDR-H2, and/or CDR-H3, with amino acid sequences shown below,, or CDR region(s) having an amino acid sequence(s) differing in 1 or 2 amino acids therefrom:
• CDR-H1 GFTFTSTGIS (SEQ ID NO: 1)
• CDR-H2 GRIYPTSGSTNYADSV (SEQ ID NO: 2)
• the light chain variable region of the FGFR3 antibody or antibody-binding fragment thereof comprises one, two, or three complementarity determining regions (CDRs) CDR-L1, CDR-L2, and/or CDR-L3.
• the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain comprising: a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence differing in 1 or 2 amino acids therefrom, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence differing in 1 or 2 amino acids therefrom, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence as shown in SEQ ID NO: 3 or 4 or
• the antibody or antigen-binding fragment thereof has CDR sequences having amino acid sequences of SEQ ID NOs: 1, 2, 3, 5, 6, and 7 without any variation.
• the antibody or antigen-binding fragment thereof comprises heavy chain complementary determining regions CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 1, 2, and 3, and the chain complementarity determining regions CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs: 5, 6, and 7.
• the antibody or antigen-binding fragment thereof has CDR sequences having amino acid sequences of SEQ ID NOs: 1, 2, 4, 5, 6, and 7 without any variation.
• the antibody or antigen-binding fragment thereof comprises heavy chain complementary determining regions CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 1, 2, and 4, and the chain complementarity determining regions CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs: 5, 6, and 7.
• the heavy chain variable region of the FGFR3 antibody or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 9 or an amino acid sequence differing in 1, 2, 3, or 4 amino acids therefrom, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 8:
• the light chain variable region of the FGFR3 antibody or antigen-binding fragment thereof comprises an amino acid sequence of SEQ ID NO: 9 or an amino acid sequence differing in 1, 2, 3, or 4 amino acids therefrom, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 9: [00143]
• the FGFR3 targeting moiety is MFGR1877S (vofatamab) or an antigen-binding fragment thereof.
• the FGFR3 antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.
• the antibody or antigen-binding fragment thereof has a dissociation constant (Kd) of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM.
• the antibody or antigen-binding fragment thereof has a dissociation constant (Kd) of between 1 nM and 10 nM (inclusive of endpoints) or between 0.1 nM and 1 nM (inclusive of endpoints).
• Kd is measured by a radio-labeled antigen binding assay (Radioimmunoassay, RIA) performed with the Fab version of an antibody or antigen-binding fragment thereof of interest and its antigen.
• Kd is measured using surface plasmon resonance assays with immobilized antigen.
• the antibodies or antigen- binding fragments thereof are human monoclonal antibodies directed against an epitope of human FGFR3 as described herein.
• the antibody or antigen-binding fragment thereof may be any antibody or antigen-binding fragment thereof of natural and/or synthetic origin, e.g. an antibody of mammalian origin.
• the constant domain if present, is a human constant domain.
• the variable domain is a mammalian variable domain, e.g., a humanized or a human variable domain.
• antibodies used in accordance with this disclosure are monoclonal antibodies.
• antibodies are recombinant murine antibodies, chimeric, humanized or fully human antibodies, multispecific antibodies(e.g., bispecific antibodies), or antigen-binding fragments thereof.
• the antibody or antigen- binding fragment thereof is labelled, i.e. coupled to a labelling group.
• suitable labels include radioactive labels, fluorescent labels, suitable dye groups, enzyme labels, chromogenes, chemiluminescent groups, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter etc.
• one or more labels are covalently bound to the antibody or antigen-binding fragment thereof.
• Those labelled antibodies or antigen-binding fragments thereof may in particular be used in immunohistochemistry assays or for molecular imaging in vivo.
• the antibody or antigen- binding fragment thereof is further conjugated with an effector group, in particular, a therapeutic effector group such as a cytotoxic agent or a radioactive group agent.
• an effector group in particular, a therapeutic effector group such as a cytotoxic agent or a radioactive group agent.
• Polypeptides include, for example, any of a variety of hematologic agents (including, for instance, erythropoietin, blood-clotting factors, etc.), interferons, colony stimulating factors, antibodies, enzymes, and hormones. The identity of a particular polypeptide is not intended to limit the present disclosure, and any polypeptide of interest can be a polypeptide in the present methods.
• a reference polypeptide described herein can include a target-binding domain that is capable of binding to a target of interest (e.g., is capable of binding to an antigen, e.g., FGFR3).
• a polypeptide such as an antibody, can bind to a transmembrane polypeptide (e.g., receptor) or ligand (e.g., a growth factor).
• a transmembrane polypeptide e.g., receptor
• ligand e.g., a growth factor
• Modified polypeptides may be substantially identical to the corresponding reference polypeptide (e.g., the amino acid sequence of the modified polypeptide may have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of the reference polypeptide).
• the modification does not destroy significantly a desired biological activity (e.g., binding to FGFR3).
• the modification may reduce (e.g., by at least 5%, 10%, 20%, 25%, 35%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%), may have no effect, or may increase (e.g., by at least 5%, 10%, 25%, 50%, 100%, 200%, 500%, or 1000%) the biological activity of the original polypeptide.
• the modified polypeptide may have or may optimize a characteristic of a polypeptide, such as in vivo stability, bioavailability, toxicity, immunological activity, immunological identity, and conjugation properties.
• Modifications include those by natural processes, such as post-translational processing, or by chemical modification techniques known in the art.
• Modifications may occur anywhere in a polypeptide including the polypeptide backbone, the amino acid side chains and the amino- or carboxy-terminus.
• the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide, and a polypeptide may contain more than one type of modification.
• Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from post-translational natural processes or may be made synthetically.
• modifications include pegylation, acetylation, acylation, addition of acetomidomethyl (Acm) group, ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation, carboxyethylation, esterification, covalent attachment to flavin, covalent attachment to a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of drug, covalent attachment of a marker (e.g., fluorescent or radioactive), covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic
• a modified polypeptide can also include an amino acid insertion, deletion, or substitution, either conservative or non-conservative (e.g., D-amino acids, desamino acids) in the polypeptide sequence (e.g., where such changes do not substantially alter the biological activity of the polypeptide).
• conservative or non-conservative e.g., D-amino acids, desamino acids
• the addition of one or more cysteine residues to the amino or carboxy-terminus of a polypeptide herein can facilitate conjugation of these polypeptides by, e.g., disulfide bonding.
• a polypeptide can be modified to include a single cysteine residue at the amino-terminus or a single cysteine residue at the carboxy-terminus.
• Amino acid substitutions can be conservative (i.e., wherein a residue is replaced by another of the same general type or group) or non-conservative (i.e., wherein a residue is replaced by an amino acid of another type).
• a naturally occurring amino acid can be substituted for a non-naturally occurring amino acid (i.e., non-naturally occurring conservative amino acid substitution or a non-naturally occurring non-conservative amino acid substitution).
• Polypeptides made synthetically can include substitutions of amino acids not naturally encoded by DNA (e.g., non-naturally occurring or unnatural amino acid).
• non-naturally occurring amino acids include D-amino acids, N-protected amino acids, an amino acid having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, the omega amino acids of the formula NH 2 (CH 2 ) n COOH wherein n is 2-6, neutral nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N- methyl isoleucine, and norleucine.
• Phenylglycine may substitute for Trp, Tyr, or Phe; citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties.
• Analogs may be generated by substitutional mutagenesis and retain the biological activity of the original polypeptide. Examples of substitutions identified as “conservative substitutions” are shown in Table 1. If such substitutions result in a change not desired, then other type of substitutions, denominated “exemplary substitutions” in Table 1, or as further described herein in reference to amino acid classes, are introduced and the products screened. [00161] Table 1: Amino acid substitutions
• Substantial modifications in function or immunological identity are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, and/or (c) the bulk of the side chain.
• Chelating moiety or metal complex thereof Chelating moieties [00163]
• suitable chelating moieties include, but are not limited to, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (1R,4R,7R,10R)- ⁇ , ⁇ ’, ⁇ ”, ⁇ ’”-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA (1,4,7,10- tetraazacyclododecane-1,4,7,10-tetra propionic acid), DO3AM-acetic acid (2-(4,7,10-tris(2- amino-2-oxoethyl)-1,4,7,10-t
• radioimmunoconjugates comprise a metal complex of a chelating moiety.
• chelating groups may be used in metal chelate combinations with metals, such as manganese, iron, and gadolinium and isotopes (e.g., isotopes in the general energy range of 60 to 10,000 keV), such as any of the radioisotopes and radionuclides discussed herein.
• chelating moieties are useful as detection agents, and radioimmunoconjugates comprising such detectable chelating moieties can therefore be used as diagnostic or theranostic agents.
• the metal complex comprises a radionuclide.
• suitable radioisotopes and radionuclides include, but are not limited to, 3 H, 14 C, 15 N, 18 F, 35 S, 47 Sc, 55 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 66 Ga, 67 Ga, 67 Cu, 68 Ga, 75 Br, 76 Br , 77 Br , 82 Rb, 89 Zr, 86 Y, 87 Y, 90 Y, 97 Ru, 99 Tc, 99m Tc, 105 Rh, 109 Pd, 111 In, 123 I, 124 I, 125 I, 131 I, 149 Pm, 149 Tb, 153 Sm, 166 Ho, 177 Lu, 117m Sn, 186 Re, 188 Re, 198 Au, 199 Au, 201 Tl, 203 Pb, 211 At, 212 Pb , 212 Bi,
• the radionuclide is an alpha emitter, e.g., Astatine-211 ( 211 At), Bismuth-212 ( 212 Bi), Bismuth-213 ( 213 Bi), Actinium-225 ( 225 Ac), Radium-223 ( 223 Ra), Lead-212 ( 212 Pb), Thorium-227 ( 227 Th), or Terbium-149 ( 149 Tb), or a progeny thereof.
• the alpha-emitter is Actinium-225 ( 225 Ac), or a progeny thereof.
• the linker is as shown within the structure of Formula I- b, as that part of Formula I-b absent A and B: A-L 1 -(L 2 )n-B Formula I-b (A and B are as defined in Formula I-a.) [00169]
• L 1 is substituted C 1 -C 6 alkyl or substituted C 1 -C 6 heteroalkyl, the substituent comprising a heteroaryl group (e.g., six-membered nitrogen- containing heteroaryl).
• L 3 is substituted C 1 -C 50 alkyl or substituted C 1 -C 50 heteroalkyl, the substituent comprising a heteroaryl group (e.g., six-membered nitrogen- containing heteroaryl).
• A is a macrocyclic chelating moiety comprising one or more heteroaryl groups (e.g., six-membered nitrogen-containing heteroaryl).
• radioimmunoconjugates comprise a cross-linking group instead of or in addition to the targeting moiety (e.g., B in Formula I comprises a cross- linking group).
• a cross-linking group is a reactive group that is able to join two or more molecules by a covalent bond.
• Cross-linking groups may be used to attach the linker and chelating moiety to a therapeutic or targeting moiety.
• Cross-linking groups may also be used to attach the linker and chelating moiety to a target in vivo.
• the cross- linking group is an amino-reactive, methionine reactive or thiol-reactive cross-linking group, or a sortase-mediated coupling.
• the amino-reactive or thiol-reactive cross-linking group comprises an activated ester such as a hydroxysuccinimide ester, 2,3,5,6- tetrafluorophenol ester, 4-nitrophenol ester or an imidate, anhydride, thiol, disulfide, maleimide, azide, alkyne, strained alkyne, strained alkene, halogen, sulfonate, haloacetyl, amine, hydrazide, diazirine, phosphine, tetrazine, isothiocyanate, or oxaziridine.
• the sortase recognition sequence may comprise of a terminal glycine-glycine- glycine (GGG) and/or LPTXG amino acid sequence, where X is any amino acid.
• GGG terminal glycine-glycine- glycine
• LPTXG amino acid sequence where X is any amino acid.
• cross-linking groups is not limited to the specific constructs disclosed herein, but rather may include other known cross- linking groups.
• compositions may be formulated for any of a variety of routes of administration discussed herein (See, e.g., the “Administration and Dosage” subsection herein), Sustained release administration is contemplated, by such means as depot injections or erodible implants or components.
• compositions that include agents disclosed herein (e.g., radioimmunoconjugates) dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, e.g., water, buffered water, saline, or PBS, among others.
• an aqueous carrier e.g., water, buffered water, saline, or PBS
• pharmaceutical compositions contain pharmaceutically acceptable auxiliary substances to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, or detergents, among others.
• pharmaceutical compositions are formulated for oral delivery and may optionally contain inert ingredients such as binders or fillers for the formulation of a unit dosage form, such as a tablet or a capsule.
• compositions are formulated for local administration and may optionally contain inert ingredients such as solvents or emulsifiers for the formulation of a cream, an ointment, a gel, a paste, or an eye drop.
• provided pharmaceutical compositions are sterilized by conventional sterilization techniques, e.g., may be sterile filtered. Resulting aqueous solutions may be packaged for use as is, or lyophilized. Lyophilized preparations can be, for example, combined with a sterile aqueous carrier prior to administration.
• the pH of preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 6 and 7, such as 6 to 6.5.
• compositions in solid form may be packaged, for example, in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules.
• Pharmaceutical compositions in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.
• Methods of treatment comprising a subject a radioimmunoconjugate as disclosed herein.
• a therapy e.g., comprising a therapeutic agent is administered to a subject.
• the subject is a mammal, e.g., a human.
• the subject has cancer or is at risk of developing cancer.
• the subject may have been diagnosed with cancer.
• the cancer may be a primary cancer or a metastatic cancer.
• Subjects may have any stage of cancer, e.g., stage I, stage II, stage III, or stage IV with or without lymph node involvement and with or without metastases.
• Provided radioimmunoconjugates and compositions may prevent or reduce further growth of the cancer and/or otherwise ameliorate the cancer (e.g., prevent or reduce metastases).
• the subject does not have cancer but has been determined to be at risk of developing cancer, e.g., because of the presence of one or more risk factors such as environmental exposure, presence of one or more genetic mutations or variants, family history, etc. In some embodiments, the subject has not been diagnosed with cancer.
• the cancer is a solid tumor cancer, e.g., a sarcoma or carcinoma.
• the solid tumor cancer is adrenocortical carcinoma, bladder cancer (e.g., urothelial carcinoma), breast cancer, cervical cancer, colorectal cancer, endometrial adenocarcinoma, Ewing’s sarcoma, gallbladder carcinoma, glioma (e.g., glioblastoma mutiforme), head and neck cancer, liver cancer, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer, or adenocarcinoma of the lung), neuroblastoma, neuroendocrine cancer, pancreatic cancer (e.g., pancreatic exocrine carcinoma), prostate cancer, renal cell carcinoma, salivary adenoid cystic cancer, or spermatocytic seminoma.
• bladder cancer e.g., urothelial carcinoma
• breast cancer e.g., cervical cancer, colorectal cancer, endometrial adenocarcinoma, Ewing’s sarcoma
• the cancer is selected from the group consisting of bladder cancer, breast cancer, head and neck cancer, liver cancer, and lung cancer.
• the cancer is bladder cancer.
• the cancer is head and neck cancer.
• the cancer is liver cancer.
• the cancer is a non-solid tumor cancer, e.g., a liquid cancer or hematologic cancer.
• the cancer is a myeloma, e.g., multiple myeloma.
• the cancer is a leukemia, e.g., acute myeloid leukemia.
• the cancer is a lymphoma.
• Radioimmunoconjugates and pharmaceutical compositions thereof disclosed herein may be administered by any of a variety of routes of administration, including systemic and local routes of administration [00186]
• Systemic routes of administration include parenteral routes and enteral routes.
• radioimmunoconjugates or pharmaceutical compositions thereof are administered by a parenteral route, for example, intravenously, intraarterially, intraperitoneally, subcutaneously, or intradermally.
• radioimmunoconjugates or pharmaceutical compositions thereof are administered intravenously.
• radioimmunoconjugates or pharmaceutical compositions thereof are administered by an enteral route of administration, for example, trans-gastrointestinal, or orally.
• compositions can be administered for radiation treatment planning, diagnostic, and/or therapeutic treatments.
• the radioimmunoconjugate may be administered to a subject in a diagnostically effective dose and/or an amount effective to determine the therapeutically effective dose.
• pharmaceutical compositions may be administered to a subject (e.g., a human) already suffering from a condition (e.g., cancer) in an amount sufficient to cure or at least partially arrest the symptoms of the disorder and its complications.
• an amount adequate to accomplish this purpose is defined as a “therapeutically effective amount,” an amount of a compound sufficient to substantially improve at least one symptom associated with the disease or a medical condition.
• a therapeutically effective amount an amount of a compound sufficient to substantially improve at least one symptom associated with the disease or a medical condition.
• an agent or compound that decreases, prevents, delays, suppresses, or arrests any symptom of the disease or condition would be therapeutically effective.
• a therapeutically effective amount of an agent or compound is not required to cure a disease or condition but may, for example, provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, such that the disease or condition symptoms are ameliorated, or such that the term of the disease or condition is changed.
• the disease or condition may become less severe and/or recovery is accelerated in an individual.
• a subject is administered a first dose of a radioimmunoconjugate or composition in an amount effective for radiation treatment planning, then administered a second dose or set of doses of the radioimmunoconjugate or composition in a therapeutically effective amount.
• Effective amounts may depend on the severity of the disease or condition and other characteristics of the subject (e.g., weight).
• Therapeutically effective amounts of disclosed radioimmunoconjugates and compositions for subjects can be determined by the ordinarily-skilled artisan with consideration of individual differences (e.g., differences in age, weight, and the condition of the subject.
• disclosed radioimmunoconjugates exhibit an enhanced ability to target cancer cells.
• effective amount of disclosed radioimmunoconjugates are lower than (e.g., less than or equal to about 90%, 75%, 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of) the equivalent dose for a therapeutic effect of the unconjugated, and/or non-radiolabeled targeting moiety.
• Single or multiple administrations of pharmaceutical compositions disclosed herein including an effective amount can be carried out with dose levels and pattern being selected by the treating physician.
• Dose and administration schedule can be determined and adjusted based on the severity of the disease or condition in the subject, which may be monitored throughout the course of treatment according to the methods commonly practiced by clinicians or those described herein.
• the following specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Examples Example 1.
• General materials and methods [00193] Lutetium-177 can be obtained from Perkin Elmer as lutetium trichloride in a 0.05 N hydrochloric acid solution; indium-111, as a trichloride salt, can be obtained from Nordion; and actinium-225 can be obtained as actinium-225 trinitrate from Oak Ridge National Laboratories.
• Analytical HPLC-MS can be performed using a Waters Acquity HPLC-MS system comprised of a Waters Acquity Binary Solvent Manager, a Waters Acquity Sample Manager (samples cooled to 10 ⁇ C), a Water Acquity Column Manager (column temperature 30 ⁇ C), a Waters Acquity Photodiode Array Detector (monitoring at 254 nm and 214 nm), a Waters Acquity TQD with electrospray ionization and a Waters Acquity BEH C18, 2.1 ⁇ 50 (1.7 ⁇ m) column.
• a Waters Acquity HPLC-MS system comprised of a Waters Acquity Binary Solvent Manager, a Waters Acquity Sample Manager (samples cooled to 10 ⁇ C), a Water Acquity Column Manager (column temperature 30 ⁇ C), a Waters Acquity Photodiode Array Detector (monitoring at 254 nm and
• Preparative HPLC can be performed using a Waters HPLC system comprised of a Waters 1525 Binary HPLC pump, a Waters 2489 UV/Visible Detector (monitoring at 254 nm and 214 nm) and a Waters XBridge Prep phenyl or C1819 ⁇ 100 mm (5 ⁇ m) column.
• a Waters HPLC system comprised of a Waters 1525 Binary HPLC pump, a Waters 2489 UV/Visible Detector (monitoring at 254 nm and 214 nm) and a Waters XBridge Prep phenyl or C1819 ⁇ 100 mm (5 ⁇ m) column.
• Analytical Size Exclusion Chromatography can be performed using a Waters system comprised of a Waters 1525 Binary HPLC pump, a Waters 2489 UV/Visible Detector (monitoring at 280 nm), a Bioscan Flow Count radiodetector (FC-3300) and TOSOH TSKgel G3000SWxl, 7.8 ⁇ 300 mm column.
• MALDI-MS positive ion
• MALDI Bruker Ultraflextreme Spectrometer a MALDI Bruker Ultraflextreme Spectrometer.
• Radio thin-layer chromatography can be performed with Bioscan AR-2000 Imaging Scanner, and can be carried out on iTLC-SG glass microfiber chromatography paper (Agilent Technologies, SGI0001) plates using citrate buffer (0.1 M, pH 5.5).
• reaction is purified directly by Prep-HPLC using method 7 to provide Intermediate 2-B as a wax after concentration using a Biotage V10 Rapid Evaporator.
• Intermediate 2-B is dissolved in DCM / TFA (1.0 mL / 2.0 mL) and allowed to stir at room temperature for 24 hours.
• the reaction is concentrated by air stream and purified directly by Prep-HPLC using method 8 to yield Compound B as a clear wax after concentration. An aliquot is analyzed by HPLC-MS elution method 3.
• [ 225 Ac]-anti-FGFR3 conjugates are tested using the human UM-UC-1 bladder cell line, which expresses wild type FGFR3.
• UM-UC-1 cells are injected into immunocompromised mice. After the establishment of tumors, mice are administered an [ 225 Ac]-anti-FGFR3 conjugate, control (e.g., PBS buffer or other vehicle alone), or optionally unconjugated anti-FGFR3.
• control e.g., PBS buffer or other vehicle alone
• Tumor volume is monitored twice weekly using caliper measurements, and the results are compared across treatment groups. Survival is recorded.
• Example 7 Effects of [ 225 Ac]-anti-FGFR3 conjugates on tumor growth and survival in WT and mutant FGFR3 bladder cancer xenograft models [00221] While wild-type FGFR3 is overexpressed in certain cancers, some tumors are associated with mutant FGFR3. In this Example, [ 225 Ac]-anti-FGFR3 conjugates are tested using various human bladder cell lines that express either wild type or mutant FGFR3.
• RT112 bladder cancer cells which express WT FGFR3, are injected into nude (nu/nu) mice, and tumors are allowed to grow to a mean volume of ⁇ 100-150 mm 3 .
• Animals are dosed twice weekly with vehicle or with an [ 225 Ac]-anti-FGFR3 conjugate.
• a third set of animals are dosed with unconjugated anti-FGFR3.
• Tumors are measured twice weekly using a caliper, and tumor volume is calculated using the formula: ⁇ ⁇ 0.5 ⁇ ⁇ ⁇ ⁇ ⁇ wherein a and b are the length and width of the tumor, respectively.
• Tumor growth is compared across groups.
• tumor lysates are collected at 48 and 72 hour after treatment. Phosphorylation and total protein levels of FRS2 ⁇ , AKT, and p44/42 MAPK (downstream mediators of FGFR3 signaling) in tumor lysates are examined. [00226] Additionally, effects of [ 225 Ac]-anti-FGFR3 conjugates are studied in a Ba/F3- FGFR3 S249C allograft model.
• HBSS Hank’s Balanced Salt Solution
• Matrigel 1:1 v/v: BD Biosciences
• Tumors are measured twice weekly as a caliper, and tumor volume is calculated as described in Example 7.
• animals are randomly assigned to a treatment or control group.
• Each [ 225 Ac]-anti-FGFR3 conjugate may be tested in a separate treatment group.
• a control group may include mice administered HBSS or other vehicle.
• one or more treatment groups are included in which mice are administered unconjugated anti-FGFR3 (cold antibody). Mice in all groups are administered the relevant agents for their group twice weekly intraperitoneally. [00229] Tumor volume is monitored twice weekly using caliper measurements, and the results are compared across treatment groups. Survival is recorded. Greater inhibition of tumor growth and/or greater survival in [ 225 Ac]-anti-FGFR3 conjugate treatment groups indicates increased efficacy.
• [ 225 Ac]-anti-FGFR3 conjugates are tested in the MC38 mouse colon adenocarcinoma xenograft model.
• FGFR3-positive MC38 cells are expanded, and 1x10 6 MC38 cells are implanted subcutaneously into the flanks of female C57BL/6 mice that are 8 to 12 weeks of age.
• tumors reach an average size of 80-120 mm 3
• animals are pair matched and assigned to a treatment or control group.
• Each [ 225 Ac]-anti-FGFR3 conjugate may be tested in a separate treatment group.
• a control group may include mice administered phosphate-buffered saline (PBS).
• PBS phosphate-buffered saline
• one or more treatment groups are included in which mice are administered unconjugated anti-FGFR3 (cold antibody).
• Mice in all groups may be administered the relevant agents for their group according to a regular schedule, e.g., weekly, twice a week, or thrice per week, for one or more (e.g., 1, 2, or 3) weeks intravenously or intraperitoneally.
• Tumor volume is monitored twice weekly using caliper measurements, and the results are compared across treatment groups. Survival is recorded. Greater inhibition of tumor growth and/or greater survival in [ 225 Ac]-anti-FGFR3 conjugate treatment groups indicates increased efficacy.
• MC38 adenocarcinoma cells are implanted subcutaneously into the flanks of female C57BL/6 mice that are 8 to 12 weeks of age. When tumors reach an average size of 80-120 mm 3 , animals are pair matched and divided into treatment and control group.
• a control group of mice receive PBS, immunoconjugate treatment group(s) receive [ 225 Ac]- anti-FGFR3 conjugates, and optional antibody treatment group(s) receive unconjugated anti- FGFR3. All groups are administered according to the same route and dosing schedule: twice weekly intravenously.
• [ 225 Ac]-anti-FGFR3 conjugates are tested in two mouse lung cancer xenograft models: Madison 109 (M109) and Lewis Lung Carcinoma cells, both of which are FGFR3- positive.1x10 6 Lewis Lung Carcinoma tumor cells are implanted subcutaneously into flanks of female C57BL/6 mice that are 8 to 12 weeks of age. Additionally, 1x10 6 Madison 109 tumor cells are implanted subcutaneously into the flanks of CR female BALB/c mice that are 8 to 12 weeks of age. [00237] When tumors reach an average size of 100-200 mm 3 , animals are pair matched and treatment is initiated.
• Each [ 225 Ac]-anti-FGFR3 conjugate may be tested in a separate treatment group.
• a control group may include mice administered phosphate-buffered saline (PBS).
• PBS phosphate-buffered saline
• one or more treatment groups are included in which mice are administered unconjugated anti-FGFR3 (cold antibody).
• Mice in all groups may be administered (intravenously or intraperitoneally) the relevant agents for their group according to a regular schedule, e.g., weekly, twice a week, or thrice per week. In this example, mice are treated for one, two, or three weeks (see below).
• Tumors are measured using calipers twice weekly, and the results are compared across treatment groups.
• Example 12 and/or 13 is performed, except that mice are not sacrificed and are instead monitored for tumor growth and survival over a period of at least months. Enhanced survival in [ 225 Ac]-anti-FGFR3 conjugate treatment groups indicates enhanced therapeutic efficacy.
• Example 15. Effects of [ 225 Ac]-anti-FGFR3 conjugates on tumor growth and survival in a bladder cancer cell lines involving FGFR3 fusions [00241] [ 225 Ac]-anti-FGFR3 conjugates are tested in a tumor xenograft models based on one or more of the RT4, RT112, SW780, and UMUC-14 bladder cell lines, essentially as described in Example 8.
• RT4 and RT112 cells contain an FGFR3-TACC3 fusion
• SW780 cells contain an FGFR- BAIAP2L1 fusion
• UMUC-14 harbors an FGFR3 S249C .
• Example 16 Binding of DOTA-anti-FGFR3 conjugate to cancer cells expressing FGFR3 [00242] The present Example demonstrates binding of conjugated anti-FGFR3 to FGFR3-positive cancer cells at subnanomolar/picomolar Kd ranges.
• An unlabeled DOTA-anti-FGFR3 conjugate was synthesized using 1) a pure R enantiomer of Compound C (see Example 4) (that is, an R-enantiomer of a (2R)-2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]pentanedioic acid (R-DOTA-GA), connected through a PEG3 acid linker to a 2,3,5,6-tetrafluorophenol active ester) and 2) MFGR1877S (vofatamab), an anti-FGFR3 antibody.
• R-DOTA-GA an R-enantiomer of a (2R)-2-[4,7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]pentanedioic acid
• MFGR1877S vofatamab
• FIGs.4A, 4B, and 4C show the binding curves for RT4, RT112, and HepG2, respectively, and the corresponding binding affinities (Kd) are summarized in Table 2.
• Table 2 Binding affinities of anti-FGFR3 conjugate to FGFR3+ cancer cells Example 17.
• Doses contained about 23 microcuries ( ⁇ Ci) of activity on 2 ⁇ g (0.1 mg/kg) of antibody. Animals were euthanized at 4 h, 24 h, 48 h, 96 h, and 168 h after injection to determine levels of radioactivity in the blood, kidney, liver, lungs, spleen, skin, tumor, and tail (n 3 per time point). [00248] Results were expressed as the percentage injected dose per gram of tissue (% ID/g) and are depicted in FIG.5. [ 177 Lu]-DOTA-anti-FGFR3 cleared rapidly from the blood and demonstrated transient uptake in the liver, lungs, and spleen. Tumor uptake was about 5% ID/g at all time points.
• Example 18 In vivo biodistribution of [ 177 Lu]-DOTA-anti-FGFR3 conjugate after pre-dosing with cold anti-FGFR3 [00249] The present Example demonstrates uptake of presently disclosed radioimmunoconjugates in tumor cells with lower levels of uptake in normal tissues.
• a Balb/c nude / RT112 cell line xenograft mouse model was used to assess the in vivo biodistribution of [ 177 Lu]-DOTA-anti-FGFR3 after pre-dosing with cold (non- radiolabeled, unconjugated) anti-FGFR3 antibody.
• Groups of tumor-bearing mice were injected intravenously with [ 177 Lu]-DOTA- anti-FGFR3. Doses contained about 23 microcuries ( ⁇ Ci) of activity on 2 ⁇ g (0.1 mg/kg) of antibody.
• Pre-dosing with cold anti-FGFR3 reduced clearance of radioactivity from the blood, reduced uptake of [ 177 Lu]-DOTA-anti-FGFR3 in normal tissues, and increased uptake of [ 177 Lu]- DOTA-anti-FGFR3 in tumors.
• Example 19 In vivo biodistribution of radiolabeled anti-FGFR3 conjugates co-dosed with cold anti-FGFR3 [00253] The present Example demonstrates uptake of presently disclosed radioimmunoconjugates in tumor cells with lower levels of uptake in normal tissues. Moreover, the present Example demonstrates that DOTA-anti-FGFR3 conjugates labeled with different radionuclides exhibit similar biodistribution profiles.
• [ 111 In]-DOTA-anti-FGFR3 conjugate was synthesized using a pure R enantiomer of Compound C (see Example 4), MFGR1877S (vofatamab), and indium-111.
• a Balb/c nude / RT112 cell line xenograft mouse model was used to assess the in vivo biodistribution of [ 177 Lu]-DOTA-anti-FGFR3 conjugate and [ 111 In]-DOTA-anti- FGFR3 conjugates when co-dosed with cold anti-FGFR3.
• FIGs.8A and 8B show the results %ID/g in mice administered [ 177 Lu]-DOTA-anti-FGFR3 (FIG.8A) or [ 177 Lu]-DOTA-anti-FGFR3 (FIG.8B), each co-dosed with cold anti-FGFR3.
• Both [ 177 Lu]-DOTA-anti-FGFR3 and [ 111 In]-DOTA-anti-FGFR3 showed good tumor intake with about 34% - 37% ID/g at 96 h after dosing.
• Example 20 shows the results %ID/g in mice administered [ 177 Lu]-DOTA-anti-FGFR3 (FIG.8A) or [ 177 Lu]-DOTA-anti-FGFR3 (FIG.8B), each co-dosed with cold anti-FGFR3.
• Both [ 177 Lu]-DOTA-anti-FGFR3 and [ 111 In]-DOTA-anti-FGFR3 showed good tumor intake with about 34% - 37% ID/g at 96 h after
• a Balb/c nude / RT112 cell line xenograft mouse model was used to assess the in vivo activity of [ 225 Ac]-DOTA-anti-FGFR3 conjugate after pre-dosing with cold anti- FGFR3.
• Tumors were grown subcutaneously to about 150 mm 3 in volume.
• mice were injected intraperitoneally with 100 ⁇ g cold anti-FGFR3.
• Relative tumor volume (FIG.9A) and relative body weights (FIG.9B) were evaluated up to 28 days after administration.
• FIG.9A treatment with 200 nCi or 400 nCi [ 225 Ac]-DOTA-anti- FGFR3 significantly inhibited tumor growth.
• One mouse in the 400 nCi group lost 30% of its body weight and was sacrificed on Day 11.
• Example 21 Effects of [ 225 Ac]-DOTA-anti-FGFR3 conjugates on tumor growth and survival in a bladder cancer xenograft model
• the present Example demonstrates therapeutic efficacy of an [ 225 Ac]-DOTA- anti-FGFR3 conjugate in a bladder cancer model.
• a Balb/c nude / RT112 cell line xenograft mouse model was used to assess the in vivo activity of [ 225 Ac]-DOTA-anti-FGFR3 conjugate with co-dosing of cold anti-FGFR3.

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